A highly selective CHEF-type chemosensor for monitoring Zn²⁺ in aqueous solution and living cells

Abstract

A new simple quinolone-based chemosensor 1 was synthesized for Zn²⁺. 1 exhibited selective fluorescence enhancement in the presence of Zn²⁺ with a 1 : 1 stoichiometry in aqueous solution, which was reversible with the addition of ethylenediaminetetraacetic acid (EDTA). The detection limit (0.019 μM) of 1 for Zn²⁺ is far lower than the World Health Organization guideline (76 μM) in drinking water. 1 was successfully applied to quantify and image Zn²⁺ in water samples and living cells. The sensing mechanism of Zn²⁺ by 1 via the chelation-enhanced fluorescence (CHEF) effect was supported by theoretical calculations. Therefore, 1 could be used in a practical system for monitoring Zn²⁺ in aqueous solution.

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Introduction

Zinc ions are one of the most abundant metal ions present in living organism, owing to its rich coordination chemistry.^1–4 Although most of the Zn²⁺ ions in a cell are tightly bound to metalloproteins, free forms are also found throughout the cell. In the brain, 5–20% of the total Zn²⁺ is stored in presynaptic vesicles, and the highest intracellular free Zn²⁺ is found in the hippocampus.^5,6 Zn²⁺ modulates brain excitability and plays a key role in synaptic plasticity.^7,8 On the other hand, a deficiency of zinc causes an unbalanced metabolism, which in turn can induce retarded growth in children, brain disorders and high blood cholesterol, and also be implicated in various neurodegenerative disorders such as Alzheimer's disease, epilepsy, ischemic stroke, and infantile diarrhea.^9–15 In addition, an excess of zinc in the environment may reduce the soil microbial activity which results in a phytotoxic effect.^16–21 Therefore, selective detection and quantification for zinc ions is very important subject of increasing investigations.^22–24

A variety of traditional methods (e.g. inductively coupled plasma atomic emission spectrometry, atomic absorption spectroscopy, and electrochemical methods) have been developed for analyses of zinc in environmental samples and for diagnosis of their deficiencies in body tissue.^25–28 However, these methods require sophisticated instrumentations, tedious sample preparation procedures, and trained operators. By contrast, chemosensors are highly estimable means for the selective recognition of chemicals and biological species in environmental chemistry and biology.²⁹ The design of chemosensors with high selectivity and sensitivity for zinc, is currently of great importance as they allow nondestructive and prompt detection of metal ions by simple fluorescence enhancement (turn-on) responses.^22–24 Especially, the development of chemosensors that can discriminate Zn²⁺ from Cd²⁺ is still a huge challenge as they are in the same group of the periodic table and have similar properties.^30–33 Thus, low cost and easily-prepared Zn²⁺ fluorescence chemosensors are needed for convenience.^34–41

In view of this requirement and as part of our research effort devoted to zinc ion recognition, we have considered the combination of a quinoline moiety and a 2-(ethyl(pyridin-2-yl)amino)ethan-1-ol (2, see Scheme 1) unit.^22,42–45 In particular, we expected that the ethanol group would make water-solubility of 1 increase. Additionally, we theoretically expected that fluorescence enhancement was originated from the stabilization of quinoline group. Finally, we synthesized a new type of water-soluble chemosensor 1 (Scheme 1) by combining the quinolone group with 2, and tested its sensing properties towards various metal ions.

Scheme 1 Synthesis of 1.

Herein, we report a new chemosensor 1, composed of the quinoline and 2-(ethyl(pyridin-2-yl)amino)ethan-1-ol. 1 showed an intense fluorescence enhancement in the presence of zinc ions in aqueous solution, could distinguish Zn²⁺ from Cd²⁺, and sensed quantitatively Zn²⁺ in living cells for practical application. Additionally, the experimental data and the sensing mechanism were supported by theoretical calculations.

Experimental section

Materials and instrumentation

All the solvents and reagents (analytical grade and spectroscopic grade) were obtained commercially and used as received. NMR spectra were recorded on a Varian 400 spectrometer. Chemical shifts (δ) were reported in ppm, relative to tetramethylsilane Si(CH₃)₄. Absorption spectra were recorded at 25 °C using a Perkin Elmer model Lambda 2S UV-Vis spectrometer. The emission spectra were recorded on a Perkin-Elmer LS45 fluorescence spectrometer. Electrospray ionization mass spectra (ESI-MS) were collected on a Thermo Finnigan (San Jose, CA, USA) LCQTM Advantage MAX quadrupole ion trap instrument. Elemental analysis for carbon, nitrogen, and hydrogen was carried out by using a Flash EA 1112 elemental analyzer (thermo) in Organic Chemistry Research Center of Sogang University, Korea. 3-Chloro-N-(quinolin-8-yl)propanamide was obtained from the previous study.³⁴

Synthesis of sensor 1

2-Aminoethanol (0.97 mL, 10 mmol) and picolinaldehyde (0.61 mL, 10 mmol) were dissolved in ethanol (15 mL) and stirred for 3 h. Then, NaBH₄ (0.38 g, 10.2 mmol) was added, and the reaction solution was cooled in an ice bath. It was stirred for 2 h and the solvent was removed under reduced pressure to obtain colorless oil (2) (Scheme 1). Yield of 2 was 1.05 g (69%). The colorless oily residue (2) was dissolved in methylene chloride and then the solution was washed twice with water. Organic layer was dried over anhydrous Na₂SO₄ and the solvent was evaporated under vacuo. The resultant product 2 (0.30 g, 2 mmol), 3-chloro-N-(quinolin-8-yl)propanamide (0.49 g, 2.2 mmol) and triethylamine (TEA, 0.28 mL, 2 mmol) were dissolved in tetrahydrofuran (THF, 30 mL), stirred and refluxed for 1 day. The mixture was cooled down to room temperature and the solvent was removed under reduced pressure to obtain brown oil, which was purified by silica gel column chromatography (9 : 1 v/v CHCl₃–CH₃OH). Yield: 0.49 g (73%). ¹H NMR (400 MHz, DMSO-d₆, 25 °C): δ = 10.88 (s, 1H), 8.86 (d, J = 8.9 Hz, 1H), 8.65 (d, J = 8.7 Hz, 1H), 8.43 (d, J = 8.4 Hz, 1H), 8.40 (d, J = 8.4 Hz, 1H), 7.66–7.51 (m, 2H), 7.57 (d, J = 7.6 Hz, 1H), 7.53 (m, 2H), 7.49 (t, J = 7.1 Hz, 1H), 4.45 (t, J = 4.4 Hz, 1H), 3.89 (s, 2H), 3.58 (t, J = 3.6 Hz, 2H), 2.93 (t, J = 3.0 Hz, 2H), 2.49 (m, J = 2.9 Hz, 4H) ppm. ¹³C NMR (100 MHz, DMSO-d₆, 25 °C): δ = 171.7 (1C), 156.7 (2C), 147.1 (2C), 139.9 (1C), 138.8 (1C), 129.8 (1C), 129.0 (1C), 127.7 (1C), 127.5 (1C), 126.0 (2C), 124.8 (1C), 122.8 (2C), 121.8 (1C), 121.5 (1C), 110.2 (1C), 21.8 (1C) ppm. LRMS (ESI): m/z calcd for C₂₀H₂₂N₄O₂–H⁺ + Zn²⁺: 413.095; found 413.267. Elemental analysis calcd (%) for C₂₀H₂₂N₄O₂: C, 68.55; H, 6.33; N, 15.99; found: C, 68.65; H, 5.95; N, 15.75.

Fluorescence titration of 1 with Zn²⁺

Sensor 1 (0.5 mg, 0.0015 mmol) was dissolved in MeOH (0.5 mL) and 10 μL of the sensor 1 (3 mM) were diluted to 2.990 mL bis–tris buffer solution (10 mM, pH 7.0) to make the final concentration of 10 μM. Zn(NO₃)₂ (0.01 mmol) was dissolved in bis–tris buffer solution (1 mL) and 0.3–4.8 μL of the Zn²⁺ solution (10 mM) were added to the sensor 1 solution (10 μM, 3 mL) prepared above. After mixing them for a few seconds, fluorescence spectra were obtained at room temperature. Identical results were also obtained in pure water.

UV-Vis titration of 1 with Zn²⁺

Sensor 1 (0.5 mg, 0.0015 mmol) was dissolved in MeOH (0.5 mL) and 10 μL of the sensor 1 (3 mM) were diluted to 2.990 mL bis–tris buffer solution (10 mM, pH 7.0) to make the final concentration of 10 μM. Zn(NO₃)₂ (0.01 mmol) was dissolved in bis–tris buffer solution (1 mL) and 0.3–4.8 μL of the Zn²⁺ solution (10 mM) were transferred to separate sensor solutions (10 μM, 3 mL). After mixing them for a few seconds, UV-Vis spectra were taken at room temperature.

¹H NMR titration of 1 with Zn²⁺

Five NMR tubes of 1 (0.7 mg, 0.002 mmol) dissolved in D₂O/CD₃OD (9 : 1, v/v) were prepared, and five different equivalents (0, 0.3, 0.5, 1 and 1.5 equiv.) of zinc nitrate dissolved in D₂O (0.5 mL) were added separately to the solutions of 1. After shaking them for a few seconds, the ¹H NMR spectra were taken.

Methods of cell test of 1 with Zn²⁺

Human dermal fibroblast cells in low passage were cultured in FGM-2 medium (Lonza, Switzerland) supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin in the in vitro incubator with 5% CO₂ at 37 °C. Cells were seeded onto a 8 well plate (SPL Lifesciences, Korea) at a density of 2 × 10⁵ cells per well and then incubated at 37 °C for 4 h after addition of various concentrations (0–100 μM) of Zn(NO₃)₂ dissolved in MeOH. After washing with phosphate buffered saline (PBS) two times to remove the remaining Zn(NO₃)₂, the cells were incubated with 1 (20 μM) dissolved in MeOH at room temperature for 1 h. The cells were observed using a microscope (Olympus, Japan). The fluorescent images of the cells were obtained using a fluorescence microscope (Leica DMLB, Germany) at the excitation wavelength of 425 nm. The relative fluorescence intensity of fluorescence microscopy images was evaluated by Icy software.⁴⁶ The four cell-body regions in visible field (green light source) were randomly selected as the region of interest to determine the average fluorescence intensity.

Determination of Zn²⁺ in water samples

Fluorescence spectral measurement of water samples containing Zn²⁺ were carried by adding 20 μL of 3 mmol L⁻¹ stock solution of 1 and 0.60 mL of 50 mmol L⁻¹ bis–tris buffer stock solution to 2.38 mL sample solutions. After well mixed, the solutions were allowed to stand at 25 °C for 2 min before the test.

Method for theoretical calculations of 1 and 1–Zn²⁺ complex

All theoretical calculations were performed by using the Gaussian 03 suite.⁴⁶ The singlet ground states (S₀) of 1 and 1–Zn²⁺ complex were optimized by DFT methods with Becke's three parametrized Lee–Yang–Parr (B3LYP) exchange functional^47,48 with 6-31G** basis set.^49,50 In vibrational frequency calculations, there was no imaginary frequency for 1 and 1–Zn²⁺ complex, suggesting that the optimized 1 and 1–Zn²⁺ complex represented local minima.

Results and discussion

Synthesis of 1

The compound 2 (2-((pyridin-2-yl)methylamino)ethanol) was synthesized by condensing 2-aminoethanol and picolinaldehyde in ethanol (Scheme 1). Subsequently, the substitution reaction of 2 to 3-chloro-N-(quinolin-8-yl)propanamide afforded 1, which was characterized by ¹H NMR, ¹³C NMR, ESI-mass, spectrometry and elemental analysis.

Fluorescence and absorption spectroscopic studies of 1 toward Zn²⁺

The fluorescent behavior of 1 toward various metal ions was studied in bis–tris buffer solution (10 mM, pH 7.0). When excited at 355 nm, 1 exhibited a weak fluorescence emission (λ_max = 521 nm) compared to that (37-folds) in the presence of Zn²⁺ (Fig. 1). In contrast, upon addition of other metal ions such as Na⁺, K⁺, Mg²⁺, Ca²⁺, Al³⁺, Ga³⁺, Cr³⁺, Mn²⁺, Fe²⁺, Fe³⁺, Co²⁺, Ni²⁺, Cu²⁺, Zn²⁺, Ga³⁺ and Pb²⁺, no increase in intensity was observed. These results indicated that sensor 1 could be used as a fluorescence chemosensor for Zn²⁺. The selective fluorescence enhancement response to Zn²⁺ might be due to the effective coordination of Zn²⁺ with 1 over other metal ions, which might be attributed to the operation of CHEF (chelation enhanced fluorescence) mechanism.^31,51

Fig. 1 Fluorescence spectral changes of 1 (10 μM) in the presence of different metal ions (1.5 equiv.) such as Na⁺, K⁺, Mg²⁺, Ca²⁺, Al³⁺, Ga³⁺, Cr³⁺, Mn²⁺, Fe²⁺, Fe³⁺, Co²⁺, Ni²⁺, Cu²⁺, Zn²⁺, Ga³⁺ and Pb²⁺ with an excitation of 355 nm in buffer solution (10 mM bis–tris, pH 7.0).

To further investigate the chemosensing properties of 1, fluorescence titration of the sensor 1 with Zn²⁺ ion was performed. As shown in Fig. 2, the emission intensity of 1 at 521 nm steadily increased until the amount of Zn²⁺ reached 1.2 equiv. The photophysical properties of 1 were also examined using UV-Vis spectrometry. UV-Vis absorption spectrum of 1 showed two absorption bands at 237 and 302 nm (Fig. S1†). Upon the addition of Zn²⁺ ions to a solution of 1, the two bands have red-shifted to 253 and 351 nm, respectively. Meanwhile, three clear isosbestic points were observed at 243, 280 and 325 nm, suggesting that only one product was produced from the binding of 1 with Zn²⁺.

Fig. 2 Fluorescence spectral changes of 1 (10 μM) in the presence of different concentrations of Zn²⁺ ions in buffer solution (10 mM bis–tris, pH 7.0). Inset: fluorescence intensity at 521 nm versus the number of equiv. of Zn²⁺ added.

The Job plot⁵² showed a 1 : 1 complexation stoichiometry between 1 and Zn²⁺ (Fig. S2†), which was further confirmed by ESI-mass spectrometry analysis (Fig. 3). The positive-ion mass spectrum of 1 upon addition of 1 equiv. of Zn²⁺ showed the formation of the 1–H⁺ + Zn²⁺ [m/z: 413.267; calcd, 413.095]. Based on pK_a values (11.9 vs. 15.8) of the acetamide and ethanol moieties reported in the literatures,⁵³ we proposed that the proton of the amide moiety might be deprotonated when 1 coordinated to Zn²⁺ as shown in the Fig. 3. From the UV-Vis titration data, the association constant for 1 with Zn²⁺ was determined as 3.3 (±0.1) × 10⁷ M⁻¹ using Benesi–Hildebrand equation (Fig. S3†).⁵⁴ This value was within the range of those (1.0–1.0 × 10¹²) reported for Zn²⁺ sensing chemosensors.^55–58 The detection limit was estimated as 19 nM based on the 3σ/slope (Fig. S4†), which was about ten thousand folds lower than the WHO guideline (76 μM) for Zn²⁺ ions in drinking water.^35,59

Fig. 3 Positive-ion electrospray ionization mass spectrum of 1 (20 μM) upon addition of Zn(NO₃)₂ (1.5 equiv.).

To check the practical applicability of 1 as Zn²⁺ selective sensor, fluorescence competition experiments were carried out in the presence of Zn²⁺ mixed with various metal ions. When 1 was treated with 1.5 equiv. of Zn²⁺ in the presence of the same concentrations of other metal ions (Fig. 4), other background metal ions had no obvious interference with the detection of Zn²⁺ ion, except for Co²⁺ and Cu²⁺ which showed about 50% and 70% interferences, respectively. The interferences with Co²⁺ and Cu²⁺ might be due to the strong quenching properties to fluorescence.^60,61 Nevertheless, they still showed sufficient turn-on fluorescence. In the presence of excess other metal ions (15 equiv.), Hg²⁺ and Fe³⁺ interfered with some, and Co²⁺ and Cu²⁺ did completely (Fig. S5†). In particular, Cd²⁺ ion which is in the same group with Zn²⁺ didn't affect the emission intensity of 1–Zn²⁺. These results indicated that 1 could be a good Zn²⁺ sensor which could distinguish Zn²⁺ from Cd²⁺ commonly having similar properties.

Fig. 4 Competitive selectivity of 1 (10 μM) toward Zn²⁺ (1.5 equiv.) in the presence of other metal ions (1.5 equiv.) with an excitation of 355 nm in buffer solution (10 mM bis–tris, pH 7.0).

¹H NMR spectroscopic studies of 1 toward Zn²⁺

The ¹H NMR titration experiments were studied in D₂O/CD₃OD (9 : 1, v/v) to further examine the binding mode between 1 and Zn²⁺ ion (Fig. 5). Upon addition of Zn²⁺ to sensor 1, all of the aromatic and the aliphatic protons showed downfield shift. Moreover, ¹H NMR titration in CD₃CN showed that the proton signal of the amide moiety gradually disappeared according to the increase of Zn²⁺ (Fig. S6†). These chemical shifts suggested that the oxygen atom of the ethanol moiety and the four nitrogen atoms might coordinate to Zn ion. This coordinative behavior of potentially penta-dentate ligand 1 with a zinc ion was previously observed in the similar type of zinc complexes.³⁶ There was no shift in the position of proton signals on further addition of Zn²⁺ (>1.5 equiv.). Based on Job plot, ESI-mass spectrometry analysis, ¹H NMR titration, and the crystal structures of similar types of zinc complexes, we proposed the structure of 1–Zn²⁺ complex as shown in Scheme 2.

Fig. 5 ¹H NMR titration of 1 with Zn(NO₃)₂ in D₂O/CD₃OD solution (D₂O/CD₃OD, 9 : 1, v/v).

Scheme 2 Fluorescence enhancement mechanism and proposed structure of 1 in the presence of Zn²⁺.

pH effect of 1 toward Zn²⁺

To study the practical applicability of 1, the effects of pH on the fluorescence response of Zn²⁺ were investigated at pH ranging from 2 to 12 (Fig. 6). The fluorescence intensity of 1 in the presence of Zn²⁺ showed a significant response between pH 5 and 12. These results indicated that Zn²⁺ could be clearly detected by the fluorescence spectra measurement using 1 over the environmentally and physiologically relevant pH range (pH 7.0–8.4),⁶² especially for monitoring Zn²⁺ in water samples and living cells.

Fig. 6 Fluorescence intensity (at 521 nm) of 1 in the presence of Zn²⁺ at different pH values (2–12) in buffer solution (10 mM bis–tris, pH 7.0).

Reversible test of 1 toward Zn²⁺ by using EDTA

To examine the reversibility of sensor 1 toward Zn²⁺ in buffer solution, ethylenediaminetetraacetic acid (EDTA, 1.5 equiv.) was added to the complexed solution of sensor 1 and Zn²⁺. As shown in Fig. S7,† a fluorescence signal at 521 nm was immediately quenched. Upon addition of Zn²⁺ again, the fluorescence was recovered. The fluorescence emission changes were almost reversible even after several cycles with the sequentially alternative addition of Zn²⁺ and EDTA. These results indicated that sensor 1 could be recyclable simply through treatment with a proper reagent such as EDTA. Such reversibility and regeneration could be important for the fabrication of chemosensor to sense Zn²⁺.

Biological application for Zn²⁺

To further demonstrate the potential of 1 to monitor Zn²⁺ in living matrices, fluorescence imaging experiments were carried out in living cells (Fig. 7a). Adult human dermal fibroblasts were first incubated with various concentrations of aqueous Zn²⁺ solutions (0, 5, 10, 40, 80 and 100 μM) for 4 h and then exposed to 1 for 1 h before imaging. The experimental results showed that the fibroblast cells without either Zn²⁺ or 1 showed negligible intracellular fluorescence, while those cultured with both Zn²⁺ and 1 exhibited fluorescence. With an increase in Zn²⁺ concentration from 5 to 100 μM, the relative fluorescence intensity of the cells with 1 increased (Fig. 7b). These observations confirmed that 1 could be cell membrane permeable and thus successfully applied for the determination of Zn²⁺ in living cells.

Fig. 7 (a) Fluorescence images of fibroblasts cultured with Zn²⁺ and 1. Cells were exposed to 0 (A and G), 5 (B and H), 10 (C and I), 40 (D and J), 80 (E and K) and 100 μM (F and L) Zn(NO₃)₂ for 4 h and then later with 1 for 1 h. The top images (A–F) were observed with the light microscope and the bottom images (G–L) were taken with a fluorescence microscope. The scale bar is 50 μm. (b) Quantification (green light source) of mean fluorescence intensity in the bottom images (G–L).

Determination of zinc ion in water samples

In order to examine the practical properties of the chemosensor 1 in environmental samples, the chemosensor 1 was applied for the determination of Zn²⁺ in water samples, using a calibration curve of 1 toward Zn²⁺ (Fig. S8†). Tap water and artificial polluted water samples were chosen. As shown in Table 1, a suitable recovery and R.S.D. values of the water samples were obtained.

Table 1 Determination of Zn(II) in water samples

Sample	Zn(II) added (μmol L^−1)	Zn(II) found (μmol L^−1)	Recovery (%)	R.S.D. (n = 3) (%)
a Prepared by deionized water, 6.00 μmol L^−1 Zn(II), 10 μmol L^−1 Cd(II), Pb(II), Na(I), K(I), Ca(II), Mg(II). Conditions: [1] = 20 μmol L^−1 in 10 mM bis–tris buffer–MeOH solution (999 : 1, pH 7.0).
Tap water	0.00	0.00
	6.00	5.9	98.5	3.1
Water sample^a	0.00	5.8	97.3	4.2
	2.00	7.7	95.2	8.8

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Theoretical calculations

To obtain a deeper insight into the interaction of 1 with Zn²⁺, theoretical calculations were carried out in parallel to the experimental studies. We performed all theoretical calculations by a 1 : 1 stoichiometry, based on Job plot and ESI-mass analysis. As the proton of the amide group in 1 was deprotonated by Zn²⁺, we calculated the deprotonated product for 1–Zn²⁺ complex. Energy-minimized structures (S₀) for 1 and 1–Zn²⁺ complex were optimized by applying density functional theory (DFT/B3LYP/6-31G**). The significant structural properties of the energy-minimized structures were indicated in Fig. S9.† Based on the molecular orbitals (MOs) for the ground state of 1 and 1–Zn²⁺ complex (Fig. 8 and S10†), the chelation of Zn²⁺ with 1 rendered the HOMO to LUMO energy gap of 1 decrease, which is consistent with the red shift in the UV-Vis spectrum of 1–Zn²⁺ complex. Thus, the fluorescence enhancement of 1–Zn²⁺ complex could be explained by CHEF effect.^63,64

Fig. 8 Molecular orbital diagrams participating in the singlet state for the 1 and 1–Zn²⁺ complex.

Conclusion

We have developed a new ethyl alcohol-functionalized chemosensor 1, which detected Zn²⁺ ion in aqueous solution. 1 showed highly selective fluorescence response to Zn²⁺ with the detection limit at the nanomolar level (19 nM). Importantly, 1 can clearly distinguish Zn²⁺ from Cd²⁺, where such distinction of Zn²⁺ from Cd²⁺ is a well-known challenge. The binding of the sensor 1 and Zn²⁺ was chemically reversible with EDTA. In addition, 1 could be successfully applied to real samples with the satisfactory recovery and R.S.D. values. The living cell experiments demonstrated its value in the practical application. Moreover, the sensing mechanism of Zn²⁺ by 1 via CHEF effect was be explained by DFT calculations. Therefore, we believe that sensor 1 will be an excellent prototype for the feasible system for monitoring Zn²⁺ concentrations in biological and environmental environments.

Acknowledgements

Financial support from Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (NRF-2014R1A2A1A11051794, 2012001725 and 2012008875) are gratefully acknowledged. We thank Nano-Inorganic Laboratory, Department of Nano & Bio Chemistry, Kookmin University to access the Gaussian 03 program packages.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra05080d

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