A high selective “turn-on” fluorescent chemosensor for detection of Zn2+ in aqueous media

A simple Schiff base fluorescent probe (SW) composed of oxime and salicylaldehyde units was designed and synthesized. The probe has high selectivity and sensitivity for Zn2+ ion in mixed solvent (DMSO/H2O, V/V = 9:1). The fluorescence has obvious redshift phenomenon and visible color change. The stoichiometric ratio of the probe to Zn2+ ion was confirmed to be 1:2 (mole) by 1H NMR, MS analysis and job curves. And the detection limit of fluorescence response of the SW to Zn2+ is down to 2.53 × 10–8 mol/L. At the same time, the probe SW can be applied to the test paper detection of Zn2+ ions under 365 nm UV light and the detection of Zn2+ ions in actual water samples.


Introduction
Zinc is the second most abundant transition metal ion in the human body after iron. It plays an important role in various physiological and pathological processes, including DNA synthesis, gene expression, enzyme regulation structure and neuron signal transmission (Wang et al. 2020a, b, c, d;Zhang et al. 2021). The lack of Zn 2+ in adults can lead to neurological disorders, Alzheimer's disease and diabetes. Lack of Zn 2+ in children can lead to decreased immune function, diarrhea and even death (Vetriarasu et al. 2019;Liu et al. 2020a, b). In recent years, zinc ion is widely used in electroplating industry, which causes more and more serious environmental pollution. Therefore, it is very important for human health and environment to design a high selective zinc ion detection probe Yu et al. 2017b, a). Although many chemical sensors for zinc detection have been studied before, new fluorescent probes for selective detection of zinc ions in physiological pH conditions and environmental systems are still in great demand. Due to its d 10 configuration, Zn 2+ sensor is usually interfered by other d 10 metal ions (such as Cd II and Hg II ) Bian et al. 2021b, a). As we all know, Schiff base fluorescent probe is a kind of metal ion probe which is relatively simple to synthesize and widely used. It has the advantages of convenient operation, simple detection method, fast detection and high sensitivity, and has attracted great attention.
Because of the C = N group in its structure, its rigid structure and fluorescence are enhanced after chelating with metal ions (Patil et al. 2018;Xu et al. 2021a, b).
In this paper, we designed and synthesized a fluorescent probe SW with high selectivity and sensitivity for the detection of Zn 2+ ions, which can be used for visual detection. The probe has the advantages of simple preparation and low cost Pannipara et al. 2018). It has a strong practical value for the test paper detection of Zn 2+ ions under 365 nm UV light and the detection of Zn 2+ ions in actual water samples.

Materials and methods
The O-benzylhydroxylamine (99%), 4-aminoacetophenone (99%), and salicylic aldehyde (98%) used in the experiment were purchased from Alfa Aesar. The remaining reagents and solvents are all analytical reagents and can be used without further purification. The water used in the experiment is distilled water. The X-4 microscopic melting point 1 3 instrument produced by Beijing Tyco Instrument Limited company was used for melting point measurement, and no calibration was performed before use. German Vario EL V3.00 automatic element analyzer was used for the analysis of C, H, and N elements. 1 H NMR spectra were recorded in DMSO-d6 solution using Bruker AV series DRX-500 MHz nuclear magnetic resonance instrument. Fluorescence spectra were recorded using Hitachi (Japan) F-7000 fluorescence spectrophotometer. Ultraviolet-visible absorption spectrum is measured by Hitachi UV-3900 spectrometer. The B3LYP/6-31G function is used as the basis of geometric optimization, and the Gaussian 09 software program is used for DFT calculation.

Fluorescence recognition of Zn 2+ by probe SW
The response of probe SW to different metal cations was studied by the fluorescence method at room temperature (Li et al. 2021a, b, c). As shown in Fig. 1a, fifteen cations (Na + , Al 3+ , Ba 2+ , Ca 2+ , Cd 2+ , Co 2+ , Cr 3+ , Fe 3+ , Mg 2+ , Mn 2+ , Zn 2+ , Ni 2+ , Pb 2+ , Hg 2+ and Cu 2+ ) (1.0 × 10 -2 mol/L) were added to the solution of probe SW (2.0 × 10 -4 mol/L) (DMSO/H 2 O, V/V = 9:1), respectively. When the excitation wavelength is 368 nm, the probe SW has a weak fluorescence emission peak at 437 nm. After the addition of other metal ions, the intensity of emission peak has no obvious enhancement except Zn 2+ ions. Furthermore, the emission peak was redshifted from 437 to 502 nm and the intensity increased by 16 times upon addition of Zn 2+ ions (Dong et al. 2017;Wang et al. 2020a). Under the UV lamp, the solution of 15 kinds of metal ions to be measured was added to DMSO/H 2 O solution (V/V = 9:1) of SW in turn, a strong bright green fluorescence is produced only added Zn 2+ ions, and other metal cations have basically no obvious effect (Anand et al. 2017;Pan et al. 2020a, b), the fluorescence remains unchanged or quenched (Fig. 1b). It can prove that probe SW could selectively identify Zn 2+ ion among other metal ions to be measured. It is helpful to study the high sensitivity of probe SW to Zn 2+ ion by anti-interference experiment (Ozdemir 2016;Sun et al. 2019). In Fig. 2, after adding Zn 2+ ion to Scheme 1 Synthetic routes of SW probe SW solutions containing different metal ions, other metal cations had no markedly effect on the fluorescence recognition to Zn 2+ ions except for the slight quenching of Cu 2+ ion and Co 2+ ion had a slight effect (Liu et al. 2020a). The results show that SW has good selectivity for Zn 2+ ions.
In order to quantitatively evaluate the fluorescence sensing behavior of SW, we carried out fluorescence titration experiment. As shown in Fig. 3, the Zn 2+ solution (1.0 × 10 −3 mol/L) was gradually added to the probe SW solution (2.0 × 10 −4 mol/L) (Upadhyay et al. 2018;Wang et al. 2021a, b), the free SW exhibited a weak emission peak at 437 nm, a new peak emission present at 507 nm and its intensity gradually increased when the concentration of Zn 2+ ion increases continuously (Wang et al. 2017;Chang et al. 2020). When the Zn 2+ content reaches 0.5 equivalent, the fluorescence emission intensity reaches the maximum, indicating that the optimal binding ratio of Zn 2+ to probe SW is 1:2. Bringing the results of titration experiments into the Benesi-Hildebrand equation (Sudipa et al. 2019;Xu et al. 2021a), and the binding constant is calculated as K a = 7.7 × 10 4 M −1 (Fig. S2a). Where, F 0 , F and F max are the fluorescence intensity without Zn 2+ , the fluorescence intensity at any given Zn 2+ concentration and the fluorescence intensity after titration saturation, respectively. The limit of detection LOD for Zn 2+ toward SW was calculated using LOD = 3σ/slope and was found to be 2.53 × 10 −8 mol/L ( Fig. S2b) (Wang et al. 2020b;Purkait et al. 2019). Among them, the standard deviation σ was calculated by measuring five consecutive fluorescence intensities of probe SW, and the slope was obtained by plotting the relationship between the emission intensity of SW and the concentration of Zn 2+ Bian et al. 2021b; Fan et al.  1 3 2020). The calculated results are lower than the acceptable limit (7.0 × 10 −6 mol/L) of WHO drinking water Kang et al. 2019b). Compared with other Zn 2+ sensors reported previously, the detection limit is lower and the sensitivity is higher (Table 1).
In order to test and verify the rapid detection performance of probe SW for Zn 2+ , the Zn 2+ response time experiment was carried out ). As shown in Fig. S3, after adding Zn 2+ , the response time of probe SW was 3 min (Pan et al. 2020a;Li et al. 2021a). The fluorescence reversibility studied by adding Zn 2+ and ethylenediaminetetraacetic acid (EDTA, c = 1.0 × 10 -3 mol/L) to SW solution. In Fig S4, when EDTA was introduced to SW and Zn 2+ mixed solution, the fluorescence intensity at 502 nm was diminished. Then, Zn 2+ solution was added again, and the fluorescence intensity was close to the initial fluorescence value, and the above experimental steps were repeated, and the fluorescence intensity continued to decrease and increase (Wang et al. 2021a;Mu et al. 2020). Thus, the probe SW can monitor Zn 2+ reversibly.

UV-Vis spectroscopic studies of Zn 2+ by probe SW
In the UV-Vis spectrum, as shown in Fig S5a, the free ligand SW exhibited a weak absorb peak at 425 nm. When Zn 2+ ions were added to probe SW solution, a new absorption peak appeared at 458 nm, and the solution changed from colorless to yellow (Fig. S5b). At the same time, the addition of Co 2+ , Fe 3+ and Cu 2+ metal ions also slightly changed the color. Therefore, UV-Vis spectrum can be used as an assistant method to detect the Zn 2+ by this probe SW. As shown in Fig. S6, the Zn 2+ solution (1.0 × 10 −3 mol/L) was gradually added to the probe SW solution (2.0 × 10 −4 mol/L) for UV-visible titration experiments (Wang et al. 2020c;). With the continuous increase of Zn 2+ concentration, a new absorption peak appears at 458 nm, while the absorption peak at 352 nm gradually decreases. At the same time, an isoabsorptive point appeared at 285 nm and until the content of Zn 2+ solution reached 0.5 equivalents, the peaks at 458 nm and 352 nm remained unchanged (Fig. S7b). The UV-Vis spectral characteristics support the molar ratio of metal to ligand of 1:2, and the association constant K a = 5.6 × 10 4 M −1 (Fig. S7a).

The detection mechanism of the probe SW for Zn 2+
protons H1 (δ = 12.87 ppm) gradually disappeared until completely disappeared, indicating the deprotonation process of hydroxyl induced by Zn 2+ . With the addition of Zn 2+ , azomethine H2 (HC = N) shifts from 9.02 ppm to 8.65 ppm, which may be due to the coordination between imine-N and Zn 2+ . In the complex SW-Zn 2+ , the 1 H NMR titration data supports 1:2 metal-ligand ratio Yu et al. 2017a). Job's plot further analyzes the coordination ratio between SW and Zn 2+ (Fig. S8), when the mole fraction of Zn 2+ ion is 0.345, the fluorescence intensity reaches the maximum value, showing that the binding ratio of Zn 2+ ion to SW is 1:2 (Liu et al. 2020b;Xue et al. 2019).
As shown in Scheme 2, the fluorescence enhancement recognition mechanism of Zn 2+ by probe SW may be mainly due to the presence of Zn 2+ hindering the PET (light-induced electron transfer) effect of SW. Owing to the rotation of imine group (-CH = N) in free SW, there is a very weak fluorescence in SW solution. The addition of Zn 2+ ions can coordinate with the probe SW, which inhibits the free rotation of imine group of SW molecules and produces CHEF effect Zhang et al. 2018), resulting enhanced fluorescence. The mass spectrum peak of the complex appeared at 751.22 (Fig. S9), which further confirmed that the complex was consistent with the conjecture.

DFT computation
In order to further study the geometry and interaction of SW and Zn 2+ , density functional theory (DFT) calculation was carried out. The geometry structures of SW and SW-Zn 2+ were optimized by 6-31G/LanL2DZ basic setting program using Gausans-09 software Rout et al. 2019). As shown in Fig. 5, one the Zn 2+ ion coordinated with two ligand SW molecules. Among, the coordination atoms are hydroxyl O atoms and the imine N atoms on C = N groups of two SW molecules, respectively. The LUMO and HOMO energies of SW were −1.551 eV and −5.659 eV, respectively. The energy gap (∆E = E LUMO -E HOMO ) was 4.108 eV, and the SW molecule was delocalized on the entire conjugated skeleton except for the oxime phenyl group. After SW coordinating with Zn 2+ , LUMO and HOMO energies were −1.931 eV and −5.386 eV, respectively. Correspondingly, the energy gap was 3.455 eV, and the electron density was mainly delocalized on the two Schiff groups and mainly on entire salicylaldehyde and aminoacetophenone conjugated skeleton except for the oxime phenyl group. The electron density of HOMO-LUMO transition showed the fluorophore-metal charge transfer, indicating that with the PET effect, the excited electrons were readily averted to metal ions (Cui et al. 2019;Li et al. 2021b). The decrease of

Practical application of probe SW
In practical application, the probe SW is designed as a Zn 2+ responsive strip sensor with good selectivity, and it can quickly and simply detect Zn 2+ ions by changing the wavelength, which has great practical value (Kang et al. 2019a). The filter strips were immersed in DMSO/H 2 O solution of probe SW for 1 h, and dried at low temperature, then immersed in different concentrations of Zn 2+ ions and other metal ions for 30 min, and the changes were observed under 365 nm UV lamps (Diao et al. 2018). As shown in Fig. 6a, with the increase of Zn 2+ concentration, the color of the test paper changed from colorless to green. Whereas the other metal ions did not cause obvious changes in the color of test paper except Zn 2+ ions (Fig. 6b), indicating that SW probe had high selectivity for Zn 2+ .
In order to test the practicability of Zn 2+ ions in real water samples, drinking water, tap water and Yellow River Water were collected for sensing experiments (Sarkar et al. 2020). All samples were filtered through 0.2 mm filter membrane and tested for three times. The calibration curve was obtained by measuring the Zn 2+ ions concentration  (Ke et al. 2020). As shown in Table 2, the detection results show that the probe SW can effectively detection Zn 2+ ions and has high recovery (98%-103%), good analytical precision (RSD < 3%), which meets the detection requirements. Therefore, the probe SW can be effectively used for the detection of Zn 2+ ion concentration in actual water samples and has practical value in environmental analysis.

Conclusions
In summary, a simple Schiff base ligand SW has been designed and synthesized, and it has higher sensitivity and selectivity to Zn 2+ in DMSO/H 2 O (v/v = 9:1) solution. And the detection limit of the SW to Zn 2+ is down to 2.53 × 10 −8 mol/L, which was lower than the limited value defined by WHO. By means of 1 H NMR, MS analysis and theoretical calculation, we obtained the binding mode of probe SW to Zn 2+ is 2:1. In addition, the probe SW can be used for the detection of Zn 2+ ions in the test paper under the UV light at 365 nm and the detection of Zn 2+ ions in actual water samples, which has a potential application prospect.