A novel quinoline-based fluorescent sensor for imaging Copper (II) in living cells

2.1 Materials and methods

All the materials for synthesis were purchased from commercial suppliers and used without further purification. Solutions for spectra detection was HPLC reagent without fluorescent impurity. ¹H NMR spectra were taken on a Varian mercury-400 spectrometer with TMS as an internal standard and DMSO-d₆ as solvent. HRMS spectra was analysed on an Agilent 1290-micro TOF QII. Fluorescence spectra measurements were performed on a Hitachi F-4500 spectrofluorimeter. The pH measurements were made with Metteler-Toledo Instruments DELTE 320 pH. Cell experiment were applied on an inverted fluorescence microscope (Olympus IX-70) connected with a digital camera (Olympus, c-5050).

2.2 UV-vis and fluorometric analysis

The solution of Sensor 1 was prepared in DMF and the metal ion buffer solutions were using various cations dissolved in Tris-HCl buffer solution at pH 7.2. In titration and selectivity experiments, the test samples were prepared by placing appropriate amounts of ions into corresponding solution of sensor 1. For fluorescence measurements, excitation was provided at 325 nm, and emission was collected from 330 to 550 nm, both the excitation and emission slit widths were 5 and 5 nm, respectively.

2.3 Synthesis

2.3.2 Synthesis of sensor 1

(0.11 g, 0.5 mmol) intermediate 2 and (0.06 g, 0.5 mmol) acetylpyridine were dissolved in 10 mL DMF and kept stirring at 155°C for 8 h, after removed the solvent,the crude product was purified by column chromatography (CH₂Cl₂: CH₃OH = 2:1) on silica gel to obtain sensor 1 as white solid (0.10 g, ultimate yield 59%). ¹H NMR (300 MHz, DMSO-d₆) δ 1.07 (d, 3H, J 6.3 Hz, methyl-H), 4.61 (s, 2H, methene-H), 7.07 (d, 2H, J 3.0 Hz, Ph-H), 7.33 (d, 1H, J 6.2 Hz, imino-H), 7.55 (m, 4H, Ph-H), 8.02 (d, 2H, J 7.4 Hz, Ph-H), 8.75 (m, 2H, Ph-H). HRMS (FTMS + pESI) m/z, observed: 321.1; C₁₈H₁₆N₄O₂ [M]⁺ requires: 320.13. (Scheme 1).

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Scheme 1

Synthesis of Sensor 1.

3.1 Cu²⁺-titration and spectral responses

In order to investigate the identification characteristics of Sensor 1 toward Cu²⁺, titration experiments were adopted under Tris-HCl buffer solution (pH = 7.2). Sensor 1 revealed an absorption maximum at 319 nm as shown in Fig. 1 ([Cu²⁺] = 0-1×10^–5 mol L^–1), upon addition of Cu²⁺ buffer solution, the absorption band at 319 nm decreased and had a redshift to 325 nm until the first stoichiometry, so we take 325 nm as the excitation wavelength in fluorescence titration experiment. First we researched the stoichiometry between sensor and determinand, the job’s plot was used to discuss the issue, as shown in Fig. 2, we were informed that the maximum absorbance appeared at w = 50% of Sensor 1, that is to say, when the ratio between probe and Cu²⁺ became 1:1, the fluorescence intensity of Sensor 1 was reduced to the greatest extent, this situation indicated that Sensor 1 and Cu²⁺ formed a 1:1 stoichiometry complex. Moreover, we investigated the association constant of complex of Sensor 1 with Cu²⁺ from the Benesi-Hildebrand expression, as illustrated in Fig. 3, the measured absorbance [1/(A₀–A)] at 319 nm varied with 1/[Cu²⁺] in a linear relationship (R = 0.99847), and according to the linear equation, the association constant of complex of Sensor 1 with Cu²⁺ was calculated to be 3.179×10⁵ mol^–1 [19, 20]. In fluorescence titration experiment, the fluorescence emission of Sensor 1 toward Cu²⁺ was illustrated in Fig. 4, we could find that the independent Sensor 1 exhibited strong fluorescence at 399 nm in emission spectrum (water: DMF = 5:5, excited at 325 nm). Upon increasingly added Cu²⁺ into Sensor 1 solution, the fluorescence intensity at 399 nm gradually weakened until fluorescence quenched, the signal transformation was less than 10 s, and the fluorescent quenching process could be observed under the irradiation of a UV lamp. Furthermore, as shown in Fig. 5, the decreasing fluorescence intensity of Sensor 1 depend on the concentration of Cu²⁺ was in a linearship (R = 0.99717), so we considered that Sensor 1 could quantitative determine Cu²⁺, and the detecting limit could reach to 6.17×10^–7 mol L^–1 by calculation from the linear relation (based on DL = KSb₁/S,K = 3; Sb₁ is the standard deviation of the blank solution; S is the slope of the calibration curve).

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Fig. 1

UV-Vis absorption response of Sensor 1(10μmol/L) upon addition of different concentrations of Cu²⁺ (0-1×10^–5 mol L^–1) in Tris-HCl solution (DMF: H₂O = 5:5, pH = 7.2).

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Fig.2

Job’s plot for determining the stoichiometry of Sensor 1 and Cu²⁺ in Tris-HCl solution (DMF:H₂O = 5:5, pH = 7.2) [Cu²⁺] + [Sensor 1], the total concentration of Sensor 1 and Cu²⁺ was 1×10^–5 mol L^–1.

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Fig.3

Benesi Hilderbrand plot of Sensor 1 with Cu²⁺.

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Fig. 4

Fluorescence intensities of Sensor 1 (1×10^–5 mol L^–1) upon addition of different concentrations of Cu²⁺ (1×10^–6 mol L^–1—1×10^–5 mol L^–1) in Tris-HCl solution (DMF:H₂O = 5:5, pH = 7.2) (λ_ex = 325 nm).

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Fig.5

Normalized response of the fluorescence signal in different Cu²⁺ concentrations.

In order to explain the observed fluorescence quenching, two factors were taken into account. First, the strong fluorescence intensity of independent Sensor 1 might be attributed to radiational channel from the ΠΠ* state of the emission of the quinoline moiety in the excited state, the excited electrons directly transited to the ground state to emit fluorescence. Because of the special extranuclear structure of Cu²⁺, when Cu²⁺ extranuclear electrons coordinated with lone pair of acceptor moiety, the high energy level electrons transfered to the vacated orbit of fluorophore in the excited state, and blocked the electrons transfer to the ground state for emitting fluorescence (PET), this situation brought about a substantial decrease of the fluorescence intensity. Secondary, the coordination between Sensor 1 and Cu²⁺ caused its conformation restriction, which also quenched the fluorescence.

¹H NMR titration experiments were used for further verifying the configuration of Sensor 1-Cu²⁺. As illustrated in Fig. 6, upon addition of Cu²⁺ into Sensor 1 in DMSO-d₆, remarkable changes occured in the chemical shift of protons in Sensor 1,especially in the acceptor moiety. Concretely, the obvious change between δ 4.75 and δ 4.61 in the spectrum of Sensor 1 revealed that proton O influenced the process of Cu²⁺ bound with Sensor 1. The proton H_d and H_j,k indicated the downshift from δ 7.07 to δ 7.19 and δ 8.75 to δ 8.85 with Δδ up to 0.12 and 0.1 respectively. Meanwhile, the other aromatic protons demonstrated inappreciable downshift, so the above-mentioned experimental results provenly affirmed the conclusion that the nitrogen in quinoline and pyridine moiety were both related to the coordination between Sensor 1 and Cu²⁺.

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Fig.6

¹H NMR (300MHz) spectral changes of Sensor1 in DMSO-d₆ upon addition of Cu²⁺.

In order to further discuss the sensing ability of Sensor 1, selectivity experiments and competition experiments were applied to examine the detectability of Sensor 1 toward Cu²⁺. In selectivity experiments, the fluorescence response of Sensor 1 (1.0×10^–5 mol L^–1) was examined upon addition of various metal ions such as Na⁺, K⁺, Ca²⁺, Cd²⁺, Hg²⁺, Mg²⁺, Cu²⁺, Ba²⁺, Mn²⁺, Fe²⁺, Pb²⁺, Zn²⁺, Fe^3 +, Al^3 + (1.0×10^–5 mol L^–1) in DMF–Tris buffer solution [V(DMF)/V(H₂O) = 5:5, pH 7.2]. As demonstrated in the black bar of Fig. 7, only Cu²⁺ caused a remarkable flourescence quenching, the adjunction of other metal ions such as Na⁺, K⁺, Ca²⁺, Mg²⁺, Cd²⁺, Ba²⁺, Pb²⁺, Al^3 +, Fe²⁺ and Mn²⁺ did not affect the fluorescence intensity at 399 nm, besides some metal ions like Fe^3 +, Zn²⁺, Hg²⁺ had inappreciable effect to the fluorescence. These above results revealed the good selectivity of the Sensor 1 toward Cu²⁺ preliminarily, to further validate the selectivity, we utilized competition experiments in order to ulteriorly test the disturbance of other metal ions in detecting Cu²⁺. First, 1.5 equiv. Cu²⁺ was added in Sensor 1 solution to quench the fluorescence of Sensor 1, then blended 10 equiv. Na⁺, K⁺, Mg²⁺, Ca²⁺, Cd²⁺, Fe²⁺, Ba²⁺, Mn²⁺, Hg²⁺, Pb²⁺, Zn²⁺, Fe^3 + and Al^3 + into the fluorescence-quenched solution, respectively. Next, the fluorescence intensities of the mixed systems were measured once again. As illustrated in the red bar of Fig. 7, all the fluorescence intensities of the mixed systems at 399 nm were still quenching. So we could conclude that these common metal ions would not induce any distinct disturbance of Sensor 1 in Cu²⁺ detection. In essence, the specific recognition about Cu²⁺ of this sensor was depended on the special extranuclear structure of Cu²⁺, compared with these transition metal cations, the extranuclear electrons of Cu²⁺ exclusively coordinated with lone pair of acceptor moiet of Sensor 1. So the above fluorometric analysis had certified that Sensor 1 could be an excellent fluorescent sensor in Cu²⁺ detection.

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Fig.7

Fluorescence intensities of sensor 1 (1×10^–5 mol L^–1) in the presence of various metal ions (black bar) and competition experiment (red bar) in Tris-HCl solution (DMF:H₂O = 5:5, pH = 7.2) (λ_ex = 325 nm,λ_em = 399 nm).

As description above, Sensor 1 exhibit good sensitivity and selectivity towards Cu²⁺ over common metal ions, in order to expand its biological application, we explored the tracing ability of Sensor 1 for Cu²⁺ in living Human gastric cancer cells (SGC-7901). The fluorescence microscope images were obtained upon irradiation at 405 nm with a band path from 395 to 425 nm under identical exposure conditions. The living SGC-7901 cells were incubated with 1×10^–4 mol/L Sensor 1 in DMF solution for 30 min at 37 °C in 5% CO₂ atmosphere, after that, the phosphate buffered saline (PBS, pH = 7.4) was used to wash the incubated cells for 3 times. As illustrated Fig. 8a, the cumulated Sensor 1 in the living SGC-7901 cells induced obvious intracellular fluorescence, this demonstrated that Sensor 1 had successfully immersed in the cells, then added 2×10^–4 mol L^–1 Cu²⁺ solution into the Sensor 1-immersed SGC-7901 cells, as shown in Fig. 8b, the intracellular fluorescence quenched obviously, a tentative conclusion was drawn that the intracellular Cu²⁺ induce the change of the fluorescence. In addition, Fig. 8c depicted that the experimental cell profiles were clear and integrated, the introduction of Sensor 1 and Cu²⁺ did not damage the cell activity. Generally speaking, Fig. 8 demonstrated the fluorescence signal transformation of Sensor 1-supplemented SGC-7901 cells before and after treated with Cu²⁺, the above cell-permeable experiments had proven that Sensor 1 had potential values in biological application.

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Fig.8

Fluorescence microscope imaging of SGC-7901 cells incubated with 1×10^–4 mol L^–1 Sensor 1 before and after treating with 2×10^–4 mol L^–1 of Cu²⁺ (a) SGC-7901 cells incubated with Sensor 1, (b) SGC-7901 cells incubated with Sensor 1 and Cu²⁺, (c) bright field image of SGC-7901 cells incubated with Sensor 1 and Cu²⁺.