A highly selective ratiometric fluorescent probe for the cascade detection of Zn²⁺ and H₂PO₄⁻ and its application in living cell imaging

Abstract

A simple ratiometric sensor (L₁) for the cascade detection of Zn²⁺ and H₂PO₄⁻ with high selectivity was reported based on the intermolecular charge transfer (ICT) mechanism. This new sensor could distinguish Zn²⁺ from Cd²⁺, and features high fluorescence quantum yield with L₁ (0.45) and L₁–Zn²⁺ (0.27). There is a good linear relationship between the fluorescence ratio I_{525 nm}/I_{430 nm} upon addition of Zn²⁺ and the limit of detection (LOD) was evaluated to be 41.0 nM. Furthermore, the in situ prepared L₁–Zn²⁺ complex also displayed good selectivity and ratiometric response for H₂PO₄⁻ and the detection limit of L₁–Zn²⁺ was found to be 49.0 nM. L₁ exhibits permeability and can been applied to the detection of intracellular Zn²⁺ and H₂PO₄⁻ in HepG-2 cells.

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Introduction

Zinc plays important roles in gene transcription, cellular metabolism, immune function, and so on.¹ It is also involved in neurological disorders such as Alzheimer's disease, ischemic stroke, cerebral ischemia and infantile diarrhea.² Therefore, unravelling the processes in facilitating essential biological functions of mobile zinc ions in living cells is of significant interest.³ On the other hand, a simple and rapid method for determination of dihydrogen phosphate (H₂PO₄⁻), which is the predominant equilibrium species of inorganic phosphate at physiological pH, is still in high demand.⁴

Fluorescent chemosensors have played an important role in the recognition of biologically important species, such as metal ions and anions, due to the simplicity and high sensitivity of fluorescence assays.⁵ The receptor, which is referred to as the central processing unit (CPU) of a chemosensor, plays a significant role in the designing of new fluorescent chemosensors, especially for the quinoline-based receptor.⁶ In recent years, numerous chemosensors with good recognition sites for a single species have been developed. However, fluorogenic receptors for the fluorescent chemosensors that allow the cascaded detection of multiple analytes with high selectivity are still desirable.⁷ Although, several fluorescent chemosensors with good recognition site has been designed for both zinc and dihydrogen phosphate,⁸ the ratiometric fluorescent probe with excellent selectivity for the cascade detection of the Zn²⁺ and H₂PO₄⁻ is still a big challenge.⁹

Our research group involves the design and synthesis of novel 8-aminoquinoline derivatives and its application in the biological fields.¹⁰ In continue our work, herein; we have reported a novel receptor for the cascade detection of Zn²⁺ and H₂PO₄⁻ with high selectivity through introducing of the tetrahydrofuran on to the carboxamidoquinoline. Furthermore, based on the intermolecular charge transfer (ICT) mechanism,¹¹ a new fluorescent chemosensors L₁ was designed with excellent ratiometric fluorescence response for the detection of Zn²⁺ ions and H₂PO₄⁻. And this new sensor has displayed a good application prospect in cell image.

Results and discussion

Synthesis

The synthesis of the L₁ was described in Scheme 1. L₁ was synthesized in two steps. Starting from compound 1, compound 2 was achieved in the presence of NaBr, PhI(OAc)₂ and PivOH with Cu(OAc)₂ as the catalyst. Then compound 2, phenylacetylene, PdCl₂(PPh₃)₂ and Et₃N were added in DMF and reacted at 60 °C for 24 h, the target product L₁ was obtained.

Scheme 1 The facile synthesis of L₁. Reagents, conditions and yields: (a) Cu(OAc)₂, PhI(OAc)₂, PivOH, NaBr, 1,4-dioxane, air, 50 °C, 15 min, 87%; (b) phenylacetylen, PdCl₂(PPh₃)₂, Et₃N, DMF, 60 °C, 24 h, 72%.

Fluorescence response for Zn²⁺

We implemented the spectroscopic properties of L₁ for Zn²⁺ using the UV-Vis and fluorescence spectroscopy studies in DMSO/H₂O (8 : 2, v/v). The absorption spectrum of L₁ exhibited two absorption bands at 280 nm and 360 nm corresponding to phenylene-ethynylene.¹² Upon addition of Zn²⁺ ions (0–8 equiv.) to the solution of L₁, resulted in the red shift of the absorption band center at 360 nm to 420 nm, along with the formation of a clear isosbestic point at 385 nm, indicating formation of L₁–Zn²⁺ complex (Fig. S1†).

In order to evaluate the selectivity of L₁ for Zn²⁺, the fluorescence changes of L₁ to various metal ions (Mg²⁺, Na⁺, Cu²⁺, Hg²⁺, K⁺, Al³⁺, Fe³⁺, Ca²⁺, Ag⁺, Ba²⁺, Co²⁺, Pb²⁺, Mn²⁺, Ni²⁺, Cr³⁺, Zn²⁺ and Cd²⁺) were investigated in DMSO/H₂O (8 : 2, v/v). Sensor L₁, upon excitation at 390 nm, displayed a strong emission band centered at 430 nm which contribute to the introducing of the phenylene-ethynylene. Upon addition of 12 equiv. Zn²⁺ ion, the emission band at 430 nm decreased dramatically, meanwhile, a new emission band centered at 525 nm with a remarkable red-shift of 95 nm were observed (Fig. 1a). It can be attributed to an ICT process in the excited state of L₁–Zn²⁺.¹³ In this situation, electron transfer from amide nitrogen adjacent to the quinoline which was substituted with phenylene-ethynylene at C-5 position to Zn²⁺ enhanced the ICT process of L₁, and an obviously red-shift of 95 nm was achieved. Simultaneously, an apparently yellow emission of the solution can be visualized through the naked eye under a UV lamp (365 nm, Fig. 1b). While addition of other metal cations, no significant spectral changes were observed except Cu²⁺ and Hg²⁺, which shown fluorescence quenching effects. These results indicated that L₁ can readily recognize Zn²⁺ from other ions through the obviously red-shift fluorescence. Furthermore, a competition experiment was conducted through adding Zn²⁺ (12 equiv.) to L₁ solution in the presence of other metal ions (12 equiv.), which demonstrated that no obvious interference was observed except for Cu²⁺ and Hg²⁺ (Fig. 2).

Fig. 1 (a) Fluorescence (λ_ex = 390 nm) responses of L₁ (5 μM) upon addition of various metal ions (60 μM) in DMSO/water (v/v = 8/2); (b) visual changes in fluorescence of L₁ in the presence of metal ions.

Fig. 2 The ratio fluorescence responses (I_{525 nm}/I_{430 nm}) of L₁ (5 μM) containing 60 μM Zn²⁺ to the selected metal ions: 12 equiv. of Mg²⁺, Na⁺, Cu²⁺, Hg²⁺, K⁺, Al³⁺, Fe³⁺, Ca²⁺, Ag⁺, Ba²⁺, Co²⁺, Pb²⁺, Mn²⁺, Ni²⁺, Cr³⁺, Cd²⁺ in DMSO/water (v/v = 8/2).

In an attempt to evaluate the sensing property of sensor L₁ to Zn²⁺, fluorescence titration experiments were conducted. As shown in Fig. 3, the emission intensity at 430 nm was gradually decreased, while a new emission band appeared at 525 nm with gradual increasing upon addition of Zn²⁺ ions. And the well-defined isoemissive point was observed at 475 nm. The fluorescence quantum yield of the L₁–Zn²⁺ complex was calculated to be 0.27 (at 525 nm) as compared to that of free L₁ (0.45 at 430 nm). This ratiometric fluorescence behavior of L₁ with Zn²⁺ ions is attributed to internal charge transfer (ICT) mechanisms. From the titration profile, the binding constant of L₁ and Zn²⁺ was evaluated to be 1.94 × 10⁴ M⁻¹ (Fig. S2†).^7a,d The detection limit of L₁ for Zn²⁺ ions was determined to be 41.0 nM which is sufficiently low for the detection of Zn²⁺ ions in intracellular and in vivo^11d (Fig. S3†).

Fig. 3 Fluorescence spectra of L₁ (5 μM) in the presence of different concentration of Zn²⁺ in DMSO/H₂O (v/v = 8 : 2), λ_ex = 390 nm.

For better understanding the interaction of L₁ with Zn²⁺, ¹H NMR spectra of L₁ in the absence and presence of Zn²⁺ (12.0 equiv.) were conducted (Fig. 4). On complexation of L₁ with Zn²⁺, amide proton was diminished, ascribed to the interaction between metal and carboxamido group.¹⁴ And all protons of the quinoline or tetrahydrofuran moieties changed accordingly, demonstrated that the direct interaction between the ligating groups of the probe and Zn²⁺. Moreover, the release of L₁ when added H₂PO₄⁻ to the L₁–Zn²⁺ solution was displayed through the ESI-Mass spectra, in which two peaks at m/z 343.1444 assigned to [L₁ + H⁺] and at m/z 406.2726 assigned to [L₁ + Zn²⁺ + H⁺] were observed (Fig. S4†). The Job's plot analysis indicated that 1 : 1 binding stoichiometry between L₁ and Zn²⁺ (Fig. 5). And the proposed mechanism of L₁ for the cascade detection of Zn²⁺ and H₂PO₄⁻ was displayed in Fig. S5.†

Fig. 4 ¹H NMR spectrum of L₁ (DMSO-d₆) in the absence and presence of Zn²⁺.

Fig. 5 Job's plot for L₁–Zn²⁺ complex in DMSO/H₂O (v/v = 8 : 2). The total concentration of Zn²⁺ and L₁ is 10 μM. λ_ex = 390 nm.

Subsequently effects of pH on the fluorescence of L₁ with Zn²⁺ also were investigated. As shown in Fig. 6, L₁ displayed weak fluorescence response to Zn²⁺ in an acidic environment (pH < 5) contribute to the protonation of the amino group in L₁, resulted in weak coordination ability to Zn²⁺. However, the Zn²⁺ detection abilities of L₁ can be achieved when the pH increased from 6 to 11. At pH 7.5, the fluorescence intensity reached its optimum point at 525 nm, indicating that L₁ possess the highest sensing ability under the physiological pH window. Therefore, all the fluorescence detection processes were performed at pH 7.5.

Fig. 6 Effect of pH on the fluorescence intensity of L₁ (5 μM) with Zn²⁺ (12 equiv.) in DMSO/water (v/v, 8/2) (λ_ex = 390 nm, λ_em = 525 nm).

Fluorescence sensing of L₁–Zn²⁺ complex for H₂PO₄⁻

Then L₁–Zn²⁺ complex were further conducted as a secondary sensor system towards H₂PO₄⁻ recognition via the relay recognition approach. To evaluate the selective detection of H₂PO₄⁻ with various anions, F⁻, Cl⁻, Br⁻, AcO⁻, PO₄³⁻, PPi, CO₃²⁻, HSO₄⁻, CN⁻ and SO₄²⁻ (14 equiv.) were added to the solution of the L₁–Zn²⁺ complex. As shown in Fig. 7, the addition of H₂PO₄⁻ (14 equiv.) to the solution of L₁–Zn²⁺ complex resulted in the appearance of fluorescence emission at 430 nm along with simultaneous decease at 525 nm. And the solution turned to the original blue color from yellow (Fig. 7). The revival of probe L₁ fluorescence at 430 nm is contributed to the coordination of H₂PO₄⁻ with Zn²⁺. However, the addition of other inorganic anions such F⁻, Cl⁻, Br⁻, AcO⁻, PO₄³⁻, PPi, CO₃²⁻, HSO₄⁻, CN⁻ and SO₄²⁻ (14 equiv.) did not show any obvious changes in fluorescence response (Fig. S6†).

Fig. 7 Fluorescence spectra of L₁ (5 μM) upon addition of various anions to the L₁–Zn²⁺ complex in DMSO/H₂O (v/v = 8 : 2); the inset shows visual changes in fluorescence of L₁–Zn²⁺ in the presence of anions.

The fluorescence titration experiments also were conducted to estimate the sensing property of sensor L₁–Zn²⁺ to H₂PO₄⁻ (Fig. 8). The emission intensity at 430 nm was gradually increased, while emission intensity at 525 nm with gradual decreasing upon addition of H₂PO₄⁻ ions. Addition of 14 equiv. H₂PO₄⁻ (70 μM) to the solution resulted in absolutely quenching of the fluorescence emission of the L₁–Zn²⁺ complex. Furthermore, the L₁–Zn²⁺ complex provided an excellent ratiometric system for the detection of H₂PO₄⁻ in DMSO/H₂O (8 : 2, v/v). And the detection limit of L₁–Zn²⁺ for H₂PO₄⁻ ions was determined to be 49.0 nM [Fig. S7†].

Fig. 8 Fluorescence spectra of L₁–Zn²⁺ (5 μM) in the presence of different concentration of H₂PO₄⁻ in DMSO/H₂O (v/v = 8 : 2), λ_ex = 390 nm.

Application of fluorescence detection of Zn²⁺ and H₂PO₄⁻ ions in living cells

To further assess fluorescence detection of Zn²⁺ and H₂PO₄⁻ ions in biological samples, fluorescent imaging inside HepG-2 cells were performed by the bio-imaging process. Firstly, the cytotoxicity which plays the important factor in live cell imaging of the L₁ was assessed by MTS assays in HepG-2 cells (Fig. S8†). The result indicating that low micromolar concentrations of L₁ were almost free of toxicity to the HepG-2 cells. Although treatment with 50 μM of L₁ at 24 h, the cell survival decreased, no significant loss of cells were noticed, suggesting that this new sensor can be applied in the cell imaging.

Incubation of HepG-2 cells with 5 μM of L₁ in TBS buffer before imaging for 30 min at 37 °C showed in the blue channel which indicated the emissive nature of L₁ in the intracellular system (Fig. 9a–c). While, the addition of Zn²⁺ (40 μM) to cells with receptor L₁ (5 μM) showed fluorescence emission in the yellow channel (Fig. 9d–f). These results suggest that L₁ is cell permeable and can be further designed as a biomaterial for the probing of bioactive analytes in the cellular environment with the change in fluorescence emission from blue to yellow. Moreover, the successive interaction of H₂PO₄⁻ (60 μM) towards the L₁–Zn²⁺ complex was conducted. The yellow fluorescence turned off upon the addition of NaH₂PO₄, along with the fluorescence revival of probe L₁ (Fig. 9g–i). Hence, L₁ has potential applicability for detecting Zn²⁺ and H₂PO₄⁻ in biological samples via the relay recognition approach.

Fig. 9 Fluorescence and bright field images of HepG-2 cell lines. Image (b) is the fluorescence image of cells in the blue channel treated with probe L₁ (5 μM) for 30 min at 37 °C; (a) is the bright field image of (b); image (c) is the overlay of (b) and (a). Image (e) is the fluorescence image of cells in the yellow channel upon treatment with L₁ (5 μM) and then Zn²⁺ (40 μM) for 30 min at 37 °C; (d) is the bright field image of (e); image (f) is the overlay of (d) and (e). Image (h) is the fluorescence image of cells in the blue channel upon treatment with L₁ (5 μM), Zn²⁺ (40 μM) and then H₂PO₄⁻ (60 μM) for 30 min at 37 °C; (g) is the bright field image of (h); image (i) is the overlay of (g) and (h); λ_ex = 400 nm.

Conclusions

In summary, we have developed a novel fluorescent L₁ with tetrahydrofuran-2-carboxamidoquinoline as the new receptor. Sensor L₁ displayed highly selective and ratiometric fluorescence responses to Zn²⁺ at physiological pH condition. Moreover, the in situ formed L₁–Zn²⁺ complex demonstrated reversible, selective and good ratiometric response for the H₂PO₄⁻. The detection limit of L₁ to Zn²⁺ and L₁–Zn²⁺ complex to H₂PO₄⁻ was found to be 41.0 nM and 49.0 nM respectively. Furthermore, the intracellular detection of Zn²⁺ ions by L₁ and H₂PO₄⁻ ions by the L₁–Zn²⁺ complex was also conducted in the HepG-2 cell lines. These results suggest that L₁ and the in situ formed L₁–Zn²⁺ complex are cell permeable and can be further designed as a biomaterial for the probing of bioactive analytes in the cellular environment with the change in fluorescence emission between blue to yellow.

Experimental section

Materials and general methods

The reagents and solvents for the present study were purchased from commercial suppliers and used as received. Absorption measurements were carried out using a JASCO-V630 spectrophotometer. Fluorescence spectra were recorded on SpectraMax M5. ¹H NMR and ¹³C NMR spectra were recorded using a Bruker DRX-500 spectrometer. All of the chemical shifts were reported in ppm and coupling constants (J) in hertz. High-resolution mass spectroscopy (HRMS) was measured on a Bruker micrOTOF-Q mass spectrometer (BrukerDaltonik, Bremen, Germany). The pH measurements were made using a Mettler Toledo FE20 pH meter. Fluorescence imaging experiments were performed using a Zeiss Axiovert 200M inverted epifluorescence microscope with a Hamamatsu EM-CCD digital camera C9100 and a MS200 XY Piezo Z stage (Applied Scientific Instruments, Inc.). The cytotoxicity of L₁ was evaluated by the MTS assay (BB-4204-250T).

Cell imaging experiments

The HepG-2 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM, GIBCO), supplemented with 10% heat-deactivated fetal bovine serum (FBS) and 1% penicillin/streptomycin, at 37 °C in a humidified atmosphere with 5% CO₂.¹⁵ In order to get the cell image, the HepG-2 cells were seeded in 6-well flat-bottomed plates one day before. 5 μm of L₁ was added to the HepG-2 cells and incubated for 0.5 h at 37 °C under 5% CO₂ and the fluorescence imaging of intracellular Zn²⁺ was observed under inverted fluorescence microscope, the image last for 5 min. And then the cells were washed with TBS for three times, subsequently incubating with 40 μm Zn²⁺ for another 0.5 h, then the image was achieved. After rinsed with TBS for another three times of the cells, NaH₂PO₄ solutions (60 μm) were added in the cells. The imaging was started immediately upon NaH₂PO₄ solution addition.

Synthesis

The synthesis of the L₁ was described in Scheme 1. Compound 1 and 2 were achieved according to our previous work.^10b The mixture of compound 2 (0.64 g, 2.0 mmol), phenylacetylene (0.25 g, 2.5 mmol), PdCl₂(PPh₃)₂ (15.8 mg, 0.2 mmol), Et₃N (0.40 g, 4.0 mmol), and DMF (15 mL) was heated at 60 °C for 24 h. After completion of the reaction, the mixture was filtered to remove salts and the precipitate was washed with 100 mL of ethyl acetate, and then the organic phase was added into 100 mL of water. 100 mL of ethyl acetate was used to extract the crude product from the mixture. The organic phase was removed under reduced pressure to obtain the crude product, which was purified by silica gel column with a mixture solvent of petroleum ether/ethyl acetate as the eluent.

L₁. Yield: (615.6 mg, 90%). Mp: 119–120 °C. ¹H NMR (500 MHz, CDCl₃) δ 10.99 (s, 1H), 8.90 (dd, J = 4.2, 1.6 Hz, 1H), 8.79 (d, J = 8.1 Hz, 1H), 8.71 (dd, J = 8.4, 1.6 Hz, 1H), 7.80 (d, J = 8.1 Hz, 1H), 7.66–7.58 (m, 2H), 7.55 (dd, J = 8.4, 4.2 Hz, 1H), 7.42–7.33 (m, 3H), 4.62 (dd, J = 8.4, 5.6 Hz, 1H), 4.28–4.19 (m, 1H), 4.07 (dd, J = 15.2, 7.0 Hz, 1H), 2.45–2.38 (m, 1H), 2.27 (ddd, J = 13.0, 7.5, 6.0 Hz, 1H), 2.04–1.95 (m, 2H); ¹³C NMR (125 MHz, CDCl₃) δ 171.23, 147.94, 137.56, 133.88, 133.45, 130.61, 130.56, 127.45, 127.27, 122.11, 121.16, 114.99, 114.13, 93.04, 85.35, 78.17, 68.78, 29.42, 24.55. HRMS (ESI⁺): found [M + H]⁺ 343.1437 C₂₂H₁₈ClN₅O₂ requires [M + H]⁺ 343.1444.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

Financial support from the Natural Science Foundation of Zhejiang Province (No. LZ13B020001), the National Natural Science Foundation of China (No. 21376213, No. 21376058).

Notes and references

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c7ra08020d

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