Introduction

Copper ion is one of essential heavy metal ions in human body and plays an important role in various physiologic processes [1, 2]. But disruption of copper homeostasis can result in a variety of diseases such as Menkes [3], Wilson’s diseases [4], familial amyotropic lateral sclerosis [5], Alzheimer’s disease [6], and prion diseases [7]. Moreover, long-term exposure to high levels of Cu2+ can induce liver and kidney damage [8]. Therefore, a sensitive method for detecting copper in biological samples is necessary and indispensable. A number of methods have been developed for the detection of copper, including atomic absorption spectrometry [9, 10], inductively coupled plasma atomic emission spectrometry [11, 12], voltammetry [13], electrochemical method [14, 15] and fluorescent chemosensors [16, 17]. However, most of these methods need relatively high cost apparatus and cannot be used in biological applications because they entail the destruction of the sample. Consequently, fluorescent chemosensors have attracted researchers’ attention due to their high sensitivity, selectivity and easy operational use [18, 19].

Up to now, a lot of fluorescent sensors for monitoring Cu2+ based on calcein [20], rhodamine derivatives [21], Schiff base [22, 23], quinoline [24], fluorescein [25], coumarin [26, 27], indole [28] and naphthalendiimide [29] have been reported. However, major shortcomings of them are inferior selectivity [23], reversibility [22–25] or water solubility [26, 27]. Therefore, the development of water soluble and reversible fluorescent probes for monitoring Cu2+ in living cells is more appealing.

In recent years, pyrazoline derivatives have gained much attention due to their outstanding properties, such as high fluorescence quantum yields and excellent stability [30–34]. As an continuation of our work on the development of fluorescent probe for monitoring metal ions [33–37], herein we report a new pyrazoline-based fluorescent probe 3 (Scheme 1) for Cu2+ recognition. This probe with high sensitivity and selectivity for monitoring Cu2+ in aqueous solution is suitable for imaging Cu2+ in living cells.

Scheme 1
scheme 1

Synthesis of probe 3

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Experimental Details

Apparatus

Thin-layer chromatography (TLC) was conducted on silica gel 60 F254 plates (Merck KGaA). 1H NMR and 13C NMR spectra were recorded on a Bruker Avance 300 (300 MHz and 75 MHz) spectrometer, using DMSO as solvent, and tetramethylsilane (TMS) as internal standard. Melting points were determined on an XD-4 digital micro melting point apparatus. IR spectra were recorded with an IR spectrophotometer VERTEX 70 FT-IR (Bruker Optics). HRMS spectra were recorded on a QTOF6510 spectrograph (Agilent). UV–vis spectra were recorded on a U-4100 (Hitachi). Fluorescent measurements were recorded on a Perkin–Elmer LS-55 luminescence spectrophotometer. All pH measurements were made with a Model PHS-3C pH meter (Shanghai, China) and operated at room temperature about 298 K.

Reagents

Deionized water was used throughout the experiment. All the reagents were purchased from commercial suppliers and used without further purification. The salts used in stock aqueous solutions of metal ions were NaNO3, Fe(NO3)3 9H2O, AgNO3, KNO3, Co(NO3)2 6H2O, Mg(NO3)2 6H2O, Ca(NO3)2 4H2O, Al(NO3)3 9H2O, Ba(NO3)2, Cr(NO3)3 9H2O, Ni(NO3)2 6H2O, Cd(NO3)2 4H2O, Pb(NO3)2, Cu(NO3)2 3H2O, Zn(NO3)2 6H2O and HgCl2.

Synthesis of 2-(5-Phenyl-3-Thiophen-2-yl-4,5-Dihydro-Pyrazol-1-yl)-Pyridine (3)

The synthetic route of proposed compound 3 is shown in Scheme 1. Starting materials chalcone (1) and 2-hydrazinylpyridine (2) were prepared according to literature [38, 39]. A mixture of chalcone (1) (0.419 g, 2 mmol), 2-hydrazinylpyridine (2) (0.258 g, 2.4 mmol), NaOH (0.250 g, 6 mmol) and ethanol (25 ml) was stirred at reflux for 1 h. After cooling, water (100 mL) was added to the mixture. The mixture was filtered and the crude product was crystallized from ethanol to give compound 3 as white crystals, Yield: 45.2 %; mp: 164–165 °C; IR (KBr, cm−1): 3084.8, 2917.6, 1588.6, 1472.6, 1430.1, 1127.9, 766.0, 698.8; 1H NMR (300 MHz, DMSO): δ 3.14 (dd, 1H, J = 5.1, 17.4 Hz, 4-Htrans), 3.91 (dd, 1H, J = 12.3, 17.4 Hz, 4-Hcis), 5.76 (dd, 1H, J = 5.1, 12.3 Hz, 5-H of pyrazoline), 6.67 (t, 1H, J = 6 Hz, pyridine-H), 7.11 (dd, 1H, J = 3.6, 5.1 Hz, pyridine-H), 7.17–7.22 (m, 3H, Ar-H), 7.27 (s, 1H, Ar-H), 7.29–7.33 (m, 3H, Hz, thiophene-H), 7.96 (d, 1H, J = 3.7 Hz, pyridine-H); 13C NMR (75 MHz, DMSO): 155.33, 147.97, 146.63, 143.70, 137.86, 135.76, 129.03, 128.81, 127.34, 126.03, 114.86, 108.85, 60.87, 43.40. HRMS: calcd for [M + H]+ C18H16N3S: 306.1065; found: 306.1076.

A single crystal of 3 was obtained from ethanol solution and was characterized using X-ray crystallography (Fig.1).

Fig. 1
figure 1

Crystal structure of probe 3

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Cell Culture and Imaging

Hela cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) containing 10 % calf bovine serum (HyClone) at 37 °C in humidified air and 5 % CO2. For fluorescence imaging, the cells (5 × 104 mL-1) were seeded into 24-well plates, and experiments to assay Cu2+ uptake were performed in the same media supplemented with 1 μM of CuCl2 for 0.5 h. The cells were washed twice with PBS buffer before the staining experiments, and incubated with 1 μM of probe 3 for 1 h in the incubator. After washing twice with PBS, the cells were imaged under a Phase Contrast Microscope (Nikon, Japan).

Results and Discussion

Absorption Properties

The absorption spectra of probe 3 in aqueous solution (EtOH:HEPES = 1:1, v/v, 0.02 M, pH = 7.2) were investigated. As shown in Fig. 2, in the absence of Cu2+ ion, probe 3 showed an absorption maximum at 356 nm with ε 2.1 × 104 M-1 cm−1 (Fig. S1, Fig. S2). Upon the addition of Cu2+, the absorption peak at 356 nm decreased with a red shift of 10 nm. Because of the binding of the probe and Cu2+, conjugate electrons of the complex delocalized more than free probe, resulting in a red shift of maximum absorption. It is obvious that none of other cations produced such a decrease in their absorption spectra.

Fig. 2
figure 2

Absorption spectra of probe 3 (10 μM) with addition of Al3+, Fe3+, Co2+, Ni2+, Ba2+, Ca2+, Cd2+, Cr3+, K+, Mg2+, Na+, Ag+, Hg2+, Zn2+ and Cu2+ in aqueous solution (EtOH:HEPES = 1:1, v/v, 0.02 M, pH = 7.2) with an excitation at 356 nm

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Selectivity Studies

The fluorescence spectra of probe 3 with various metal ions in aqueous solution (EtOH:HEPES = 1:1, v/v, 0.02 M, pH = 7.2) were conducted to examine the selectivity. As shown in Fig. 3, the fluorescence spectra of probe 3 showed a strong fluorescence emission at 452 nm and the addition of 5 equiv. of Cu2+ induced a significant decrease in fluorescence intensity. However, the addition of other metal ions, including Al3+, Fe3+, Co2+, Ni2+, Ba2+, Ca2+, Cd2+, Cr3+, K+, Mg2+, Na+, Ag+, Hg2+, Zn2+ did not induce obvious fluorescence change of the probe, which indicates the selectivity of the probe to Cu2+. The fact was also confirmed by color change from stronger blue fluorescence in absence of Cu2+ to almost no-fluorescence in presence of Cu2+ under the irradiation at 365 nm. For other metal ions, no changes were observed under the same condition. Therefore, it also proved the selectivity of the probe to Cu2+ (Fig. 3 inset).

Fig. 3
figure 3

Fluorescence spectra of probe 3 (10 μM) with addition of Al3+, Fe3+, Co2+, Ni2+, Ba2+, Ca2+, Cd2+, Cr3+, K+, Mg2+, Na+, Ag+, Hg2+, Zn2+, Cu2+ in aqueous solution (EtOH:HEPES 1:1, v/v, 0.02 M, pH = 7.2) with an excitation at 356 nm and emission at 452 nm. Inset: the color change of probe 3 (10 μM) in aqueous solution (EtOH:HEPES 1:1, v/v, 0.02 M, pH = 7.2) solution with 5 equiv. Cu2+

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Anion effect on the selectivity of the probe for Cu2+ was also carried out. The results showed that the fluorescence intensity of 3-Cu2+ did not change in the cases of CuSO4, CuCl2, Cu(NO3)2 and Cu(OAc)2 (Fig. S3).

Tolerance Over Other Metal Ions

In order to test the practical application of the probe for Cu2+ ion, the interference of other common foreign ions on the fluorescence intensity of probe 3 was also studied. As shown in Fig. 4, only Cr3+ and Hg2+ slightly disturbed the fluorescence intensity of 3-Cu2+ and the initial fluorescence intensity of 3-Cu2+ did not change significantly with other metal ions such as Al3+, Fe3+, Co2+, Ni2+, Ba2+, Ca2+, Cd2+, K+, Mg2+, Na+, Ag+ and Zn2+. The results indicated that probe 3 had a high selectivity for Cu2+ in the presence of other related species.

Fig. 4
figure 4

Fluorescence spectra of probe 3 (10 μM) with addition of Al3+, Fe3+, Co2+, Ni2+, Ag+, Hg2+, Zn2+, Ba2+, Cd2+, Cr3+ (5 equiv.) and Mg2+, Ca2+ (100 equiv.) and K+, Na+ (200 equiv.) in aqueous solution (EtOH:HEPES = 1:1, v/v, 0.02 M, pH = 7.2) containing 5 equiv. of Cu2+ with an excitation at 356 nm and emission at 452 nm

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Cu2+-Titration

As shown in Fig. 5, fluorescence titration experiments clearly showed fluorescence “turn-off”. At the beginning, with the addition of Cu2+ at a concentration lower than 1.0 equiv. of probe 3, a significant decrease in the fluorescence intensity was observed and the fluorescence of probe 3 was almost completely quenched with only 2 equiv. of Cu2+ ions. As a further increase in Cu2+ concentration, the fluorescence intensity did not change (Fig. 5, inset). As shown in Fig. S5, the detection limit of probe 3 for the determination of Cu2+ was estimated to be 1.919× 10–7 M (R = 0.995). Therefore, the data demonstrate that compound 3 can be used as an excellent “turn-off” probe for detection of Cu2+ ion.

Fig. 5
figure 5

Fluorescence spectra of probe 3 (10 μM) with addition of various concentrations of Cu2+ in aqueous solution (EtOH:HEPES = 1:1, v/v, 0.02 M, pH = 7.2) with an excitation at 356 nm

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Binding of Probe 3 with Cu2+

To investigate the coordination information between probe 3 and Cu2+, the stoichiometry between probe 3 and Cu2+ in the complex system was determined by the changes in the absorption response of probe 3 with varying concentrations of Cu2+. In Fig. 6, there is an inflection point when the molar fraction was 0.5, indicating a 1:1 stoichiometry of the Cu2+ to probe 3 in the complex. The association constant (Ka) of Cu2+ with probe 3 in HEPES buffered solution at pH 7.20 was found to be 6.796× 104 M−1 (Fig. S4). In addition, the Cu2+-titration was also supposed a 1:1 Cu2+ complex formation. A proposed complex binding was presented in Scheme 2. Cu2+ was bound to one nitrogen atom from pyridine, one nitrogen atom from pyrazoline and one sulfur atom from thiophene. The model was confirmed by a theory calculation. Based on first-principles Density Functional Theory (DFT), the calculations were performed using the CASTEP code [40]. The exchange and correlation interactions were modeled using the generalized gradient approximation (GGA) with the PW91 functional [41]. In the geometrical optimization, all forces on atoms were converged to 0.05 eV∕Å, the maximum ionic displacement was within 0.002 Å and the total stress tensor was reduced to the order of 0.1 GPa (Fig. 7).

Fig. 6
figure 6

Job’s plot evaluated from the absorption spectra of probe 3 and Cu2+ in aqueous solution (EtOH:HEPES = 1:1, v/v, 0.02 M, pH = 7.2) with absorption at 356 nm (the total concentration of probe 3 and Cu2+ is 4.0 × 10−5 M)

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Scheme 2
scheme 2

The proposed binding mode

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Fig. 7
figure 7

Optimized structure of 3-Cu2+ by calculation

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Reversibility and Effect of pH

The response of probe 3 to Cu2+ was confirmed to be reversible by the EDTA. As shown in Fig. 8, upon addition of 1 equiv. EDTA to the mixture of probe 3 (10 μM) and Cu2+ (10 μM) in aqueous solution (EtOH:HEPES = 1:1, v/v, 0.02 M, pH = 7.2), the fluorescent intensity was almost completely recovered, indicating that the EDTA replaced the receptor 3 to chelate Cu2+. Thus, the experimental observations suggested that probe 3 should be a reversible for Cu2+. On the other hand, the effect of pH on the fluorescence response of probe 3 to Cu2+ was investigated. As shown in Fig. S5, in absence of Cu2+, nearly no substantial change in fluorescence intensity of probe 3 was observed in the pH range from 5.5 to 9.0, but the fluorescence intensity of 3-Cu2+ has different responsive behaviors in different pH ranges. With increasing pH value from 5.5 to 7, the fluorescence intensity of 3-Cu2+ decreases because the deprotonation of probe 3 increases the conjugation between probe 3 and Cu2+. The significant increase of the fluorescence intensity with the increasing pH value from 7.5 to 9.0 should be attributed to the displacing of Cu2+ from the complex, leading to the recovery of the fluorescence of probe 3. Thus, the coordination between probe 3 and Cu2+ is stable in the pH range of 7.0–7.5, indicating that the probe is promising for biological applications.

Fig. 8
figure 8

Effect of EDTA (1 equiv.) on the fluorescence spectra of probe 3 (10 μM) with Cu2+ (1 equiv.) in aqueous solution (EtOH:HEPES = 1:1, v/v, 0.02 M, pH = 7.2) with an excitation at 356 nm and emission at 452 nm

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Imaging of Intracellular Cu2+

The ability of probes to sensitively and selectively detect analyte in living cells is significant for biological application. Considering that higher level of Cu2+ in tumors takes a possible key role in promoting angiogenesis, we carried out assay in Hela cells.

From Fig. 9, we can clearly observe significant confocal imaging changes of probe 3 (1 μM) in the medium upon addition of Cu2+ (1 equiv.) for 1 h at 37 °C. Hela cells incubated with probe 3 initially display a strong fluorescent image, but the fluorescence image completely quenched in the presence of Cu2+. When EDTA (0.5 mM) was added to the medium containing probe 3 (1 μM) and Cu2+ (1 μM), fluorescent image was returned because EDTA chelated strongly Cu2+ to lead to free probe 3.

Fig. 9
figure 9

Fluorescence microscope images of living Hela cells. a: Fluorescence image of probe 3 (1 μM); d: Fluorescence image of probe 3 (1 μM) in the presence of Cu2+ (1 μM); g: Fluorescence image of probe 3 (1 μM) in the presence of Cu2+ (1 μM) and EDTA (0.5 mM); b, e and h: Bright-field; c, f and i: Overlay image

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Conclusions

In summary, a new pyrazoline-based probe 3 was developed. The probe can monitor Cu2+ with high sensitivity and selectivity over other competitive metal ions in aqueous solution (EtOH:HEPES = 1:1, v/v, 0.02 M, pH = 7.2). The binding ratio of probe 3 and Cu2+ was determined to be 1:1, which was confirmed by the Job’s plot and the Cu2+-titration results. The binding constant (Ka) for 3-Cu (II) was calculated to be 6.796× 104 M−1 and the detection limit of probe 3 for Cu2+ was 1.919× 10−7 M. Additionally, the probe could serve as a reversible fluorescent probe to image Cu2+ in living cells.