Dual-response detection of Ni2+ and Cu2+ ions by a pyrazolopyrimidine-based fluorescent sensor and the application of this sensor in bioimaging

Herein, a dual-response fluorescent sensor, L, based on pyrazolopyrimidine was designed and developed for the simultaneous detection of Ni2+ and Cu2+ ions in the presence of other metal ions; the structural characterization of L was carried out by FTIR spectroscopy, NMR spectroscopy, HRMS and X-ray diffraction analysis. The sensor L effectively displayed fluorescence quenching towards the Ni2+ and Cu2+ ions with high sensitivity without interference from other metal ions. The results reveal that L binds to Ni2+ and Cu2+ in a 2 : 1 pattern, which matches well with the result of the Job's plot. The association constants of L with Ni2+ and Cu2+ were 3.2 × 104 M−1 and 7.57 × 104 M−1, respectively. The detection limits (DLs) are down to 8.9 nM for Ni2+ and 8.7 nM for Cu2+. The fluorescence imaging of L in T-24 cells was investigated because of the low cytotoxicity of L, indicating that L could be used to detect Ni2+ and Cu2+ in living cells.


Introduction
Heavy metal ions have gained extensive attention due to their importance in many biological and environmental processes. However, the ingestion of these metals, including arsenic, mercury, copper, nickel, and lead, 1 even at very low concentrations can lead to numerous health issues or toxic effects. 2 Copper is associated with various biological processes 3 as it is utilized in important cofactors of several proteins and enzymes. [4][5][6][7] The imbalance of Cu 2+ level causes various diseases; moreover, nickel at high concentrations can cause severe harmful effects such as pneumonitis, lung cancer, respiratory problems, and kidney damage through its bioaccumulation. [8][9][10] If the accumulation of excess amounts of copper and nickel ions or their misregulation in the body, it is very dangerous to man and animals. 11,12 Hence, the sensing of copper and nickel ions in aqueous media is necessary because of the vital roles of these ions in biological systems.
Nowadays, spectroscopic techniques, such as atomic absorption spectroscopy (AAS), ion-chromatography ultraviolet-visible spectroscopy (IC-UV-vis), inductively coupled plasma mass spectroscopy (ICP-MS), and chromatography, are being used to detect the Cu 2+ and Ni 2+ metal ions. [13][14][15][16][17] These traditional methods have high sensitivity; 18 however, they require expensive instruments, trained professionals, difficult sample preparation processes and high time consumption; 19 therefore, continuous efforts have been focused on the development of methods for the rapid and convenient sensing of particular heavy metal cations. 20,21 Although many uorescent sensors have been reported for the Cu 2+ and Ni 2+ ions, there is still an intense demand for new, efficient Cu 2+ and Ni 2+ ion uorescent probes that can work in aqueous media with high selectivity and sensitivity at low concentrations.
Herein, the luminescent pyrazolopyrimidine 2-(3,5-diphenylpyrazol-1-yl)-4-methyl-6,8-diphenyl-1,5,8a,9-tetraaza-uorene (L) was studied. It possesses both pyrimidine and pyrazole moieties as backbones with a phenyl substituent, which can be modied to enhance the uorescence intensity. 22 Moreover, due to their good binding characteristics and structures that enable easy modication, 23-28 pyrazolopyrimidine and its derivatives are vital chelators and sensors for metal ions. The ingenious sensor L is an "N-C-N"-type ligand containing two coordination sites, which can provide a special coordination environment that increases the binding efficiency of L with metal ions. [29][30][31][32] In addition, the introduction of a typical electron-withdrawing substituent, phenyl, can effectively improve the selectivity of L towards metal ions. 33 The most important thing is that the sensor L shows low toxicity in living cells and may have potential therapeutic value in the diagnosis and treatment eld. Therefore, herein, a dual function and low cytotoxic sensor for the label-free detection of Cu 2+ and Ni 2+ cations with very low detection limit of nanomolar level in the presence of other cations was synthesized, and then, its potential applications in human cancer cell imaging were investigated.

Materials
All the materials and solvents were applied as received without further purication. The stock solutions of the sensor L (2 Â 10 À3 M) were prepared by dissolving L in ethanol and then diluted to desired concentration with ethanol when needed. The stock solutions of metal ions (2 Â 10 À3 M) were prepared from the salts LiCl, NaCl, KCl, 3 and Pb(NO 3 ) 4 using pure water.
Cell culture and uorescence imaging for Cu 2+ and Ni 2+ in living cells The living cells were obtained from the Shanghai Cell Bank of the Chinese Academy of Science, which were grown in the Dulbecco ' s modied Eagle medium (DMEM, Gibco), containing 10% fetal bovine serum (FBS, Gibco) (V/V), under an atmosphere of 5% CO 2 and 95% air at 37 C. These cells were incubated with the compound L (5 mM) under the same abovementioned experimental conditions for 4 h. The cells were washed with PBS buffer and simultaneously imaged using a confocal laser scanning microscope. Then, the cells were incubated with the CuCl 2 and NiCl 2 solutions (30 mM) for another 6 h, washed with PBS buffer and again imaged using a confocal laser scanning microscope.

Synthesis and characterization
As illustrated in Scheme 1, the target chemosensor L was easily synthesized by the condensation reaction of pyridyl hydrazine A with 1,3-diphenylpropane-1,3-dione. Subsequently, its structure was characterized by 1 H NMR spectroscopy, FTIR spectroscopy, HRMS and single-crystal X-ray crystallography; all the spectroscopic data were in agreement with the formula of L. Both pyrazole and pyrimidine units from the same ligand coordinated to the same Ni/Cu atom forming a conjugated plan motif. As shown in Fig. 1, multiple binding sites were provided by the electron-donating N atom; a chelating model was suggested based on the crystal structure when L was treated with the metal ions.
intense emission band at 494 nm (l ex ¼ 330 nm) with a high quantum yield determined using quinine sulfate as a reference. 35 The addition of the Cu 2+ and Ni 2+ ions to the ethanol solution of probe L caused a remarkable uorescence quenching ((I 0 À I)/I 0 Â 100% ¼ 81.9% for the Ni 2+ ions and 84.8% for Cu 2+ ) at 494 nm, and slightly quenched (addition of Fe 3+ , Co 2+ or Ag + ) or no uorescence spectral changes were observed upon the addition of other metal ions under the same conditions (Fig. 2a). This result clearly suggests that L may be used to detect the Ni 2+ and Cu 2+ ions due to their higher binding affinity with the chelator of L that leads to the formation of stable complexes between L and the Ni 2+ or Cu 2+ ions. [38][39][40] However, other metal ions caused a slight change in the uorescence intensity due to the unsuitable coordination geometry conformation of the chemosensor L and inappropriate ionic radius and insufficient binding energy of these metal ions. 41 As shown in Fig. 2b, the uorescence clearly changed from bright green to colourless under UV light (365 nm) aer the addition of the Ni 2+ and Cu 2+ ions, whereas no change was observed aer the addition of other metal ions tested herein under UV light. These ndings revealed that the sensor L could be used to easily detect the Ni 2+ and Cu 2+ ions by the naked eye under 365 nm UV light.

Competitive studies
To conrm the selectivity of L, competitive experiments towards Cu 2+ or Ni 2+ (2.5 equiv.) over other competitive coexisting metal ions (2.5 equiv.) were carried out. The changes in the uorescence intensity are displayed in Fig. 3a and b. As shown in Fig. 3, except for Ni 2+ and Cu 2+ , other competitive metal ions had no obvious effect on the uorescence of L. This performance clearly indicated that the binding interaction between L and Ni 2+ or Cu 2+ was stronger than that between L and other metal ions. Therefore, this shows that the recognition of Ni 2+ and Cu 2+ by L is barely interfered by other coexisting metal ions; this indicates that L is a dual-response uorescent sensor for the detection of Ni 2+ and Cu 2+ ions in an ethanol solution.

Titration analysis
To obtain a better insight into the sensitivity of the probe L, we increased the concentration of Cu 2+ (0-1.2 equiv.) or Ni 2+ (0-1.5 equiv.) ions to detect the uorescence intensity of L (20.0 mM). The strong uorescence intensity of the sensor L at 494 nm (quantum yield, F F ¼ 0.293) was attributed to the uorescence enhancement effect of the N dopant atoms on the surface of L in the excited state. 34 The changes in the uorescence intensity as   a function of the concentration of Cu 2+ and Ni 2+ ions are shown in Fig. 4a and c, respectively. The uorescence intensity gradually decreased and then reached minimum with an increase in the concentration of the Cu 2+ and Ni 2+ ions ( Fig. 4b and d), respectively. Based on these concentration-dependent experiments, the corresponding association constants of L with Ni 2+ and Cu 2+ were calculated to be 3.2 Â 10 4 M À1 and 7.57 Â 10 4 M À1 by the Benesi-Hildebrand plot, with the correlation coefficient R ¼ 88.617 and 97.475, respectively (Fig. 5). A linear regression curve was tted to the titration data of the uorescence intensity and the concentration of Ni 2+ and Cu 2+ , and then, the detection limit (DL) values were calculated to be 8.9 nM and 8.7 nM (Fig. S5 †), respectively, which were determined reasonably by the equation DL ¼ K Â s/S (eqn (S1) †), where K ¼ 3, s is the standard deviation of the blank solution (10 times) and S is the slope of the calibration curve; 42 moreover, these detection limits were signicantly less than the maximum contaminant levels of Ni 2+ (17 mM) and Cu 2+ (30 mM) in potable water according to the EPA guidelines; 43 this indicated that the as-prepared L could be deemed as an excellent uorescent sensor and employed for the recognition and detection of the Ni 2+ and Cu 2+ ions instantly in biological and environmental systems.

Binding mode studies
To obtain further information about the binding mechanism, the binding process was studied by Job's plot and HRMS mass spectral analysis. The Job's plot for L with the metal ions was obtained for achieving the binding stoichiometry between L and Ni 2+ and Cu 2+ (Fig. 6). It exhibited a maximum point at a mole ratio fraction of 1/3, indicating a 2 : 1 stoichiometry of the binding mode of L with Ni 2+ or Cu 2+ . 44 The HRMS spectra illustrated that the binding ratio of L to Cu 2+ was 2 : 1 (Fig. S4 †). Therefore, the results obtained from the mass spectral analysis correlate well with those obtained by the Job's method.

Fluorescence imaging in T-24 cells
Cytotoxicity tests were carried out to ascertain the potential biological applications of L in vitro. The cytotoxicity of L against seven human tumor cell lines (T-24, HeLa, Hep-G2, MGC803, A549, NCI-H460, and SK-OV-3) and a normal liver cell line (HL-7702) was investigated by the MTT method. We treated different cell lines with different concentrations of L for 48 h, and the IC 50 values ranged from 30 to 40 mM (Table S2 †     Inspired by the low cytotoxicity and the strong uorescence intensity of L, uorescence imaging to recognize the Ni 2+ and Cu 2+ ions in living cells was conducted (Fig. 7). The uorescence lter of the blue channel with an excitation of 330 nm was chosen as the signal output. The T-24 cells were rst treated with 5 mM of L for 4 h at 37 C and then washed with PBS buffer, which displayed a blue uorescence (Fig. 7b and c). Consequently, it was observed that the uorescence was quenched aer the incubation of cells with 30 mM of a CuCl 2 $6H 2 O or NiCl 2 $6H 2 O solution for another 6 h ( Fig. 7e and h). These imaging experiments prove good permeability of L into the membrane of cells, and thus, L can be used to recognize these two metal ions in living cells.

Conclusions
In conclusion, herein, an easy-to-prepare and efficient dualresponse uorescent sensor, L, for the detection of Ni 2+ and Cu 2+ in an ethanol solution was designed and synthesized, which was investigated in detail by employing uorescence measurements. The presented sensor L exhibited good sensitivity and biocompatibility for the detection of Ni 2+ and Cu 2+ ions over several metal ions with the detection limit of as low as 8.9 nM for Ni 2+ and 8.7 nM for Cu 2+ . The 2 : 1 binding mode between L and the Ni 2+ or Cu 2+ ions was suggested via the Job's method and HRMS spectra. The probe L could be used as a staining reagent in cells due to its good biocompatibility, which provided a sensitive response to the uorescence of Ni 2+ and Cu 2+ in living cells. Therefore, it can be a good candidate to recognize Ni 2+ and Cu 2+ in a biological environment.

Conflicts of interest
There are no conicts to declare.