A quinazolin-based Schiff-base chemosensor for colorimetric detection of Ni2+ and Zn2+ ions and ‘turn-on’ fluorometric detection of Zn2+ ion

Herein, we have reported a novel quinazolin-based Schiff base chemosensor (E)-2-benzamido-N′-(1-(pyridin-2-yl)ethylidene)benzohydrazide (L). L has been designed, synthesised and characterised by 1H-NMR, IR spectroscopy, ESI-MS spectrometry and theoretical studies. The receptor showed appreciable colorimetric λmax shift for both Ni2+ and Zn2+ ions and fluorometric “turn on” response in presence of only Zn2+ ion. The Jobs plot analysis revealed that receptor forms 2 : 1 complex with both the ions Ni2+ and Zn2+, further confirmed by ESI-MS analysis. The single crystal structure of L–Ni2+ complex (1) has also been determined. The colorimetric detection limits were calculated to 7.9 nM and 7.5 nM respectively for Ni2+ and Zn2+ in methanol–Tris–HCl buffer medium (10 mM, pH 7.2, 1 : 1 v/v). The chemosensor L can be applied for the recovery of contaminated water samples.


Introduction
Metal ion detection continues to be an important area of research both in the environment and in biological systems. 1 Nickel has long been thought of as a potentially toxic metal. Decomposition or degradation of rocks and soil, biological cycles, particularly industrial processes, and water disposal are the main sources of nickel in aquatic systems. 2 The tolerable concentration of Ni(II) ions in drinking water is 2.5 mg mL −1 . 3 Among the transition metal series, Ni 2+ plays a signicant part in biological activities like metabolism, respiration, and biosynthesis. It is a necessary component of various metalloenzymes such hydrogenases, carbon monoxide dehydrogenases, and acireductone dioxygenases. [4][5][6] In the chemical industry, it is widely used for the manufacture of practical electronics devices including Ni-Cd batteries, electroplating, and electroforming. 7,8 One of the nickel alloys utilised in the manufacture of tools, machinery, and weapons is stainless steel. 9 The adverse effects of excessive Ni 2+ accumulation over the recommended limits on human physical health include respiratory issues, lung cancer, pneumonitis, allergies, and central nervous system disorders. 10,11 Thus, one of the difficult issues in current research is the specic detection of Ni 2+ ion at the biological and environmental level.
Zinc ion is the second-most prevalent heavy metal ion and it is well known that zinc plays a signicant role in cellular processes. 12 Because of its structural characteristics, the zinc, a biologically signicant metal ion, is essential for many pathophysiologic processes, including gene expression, cell apoptosis, enzymatic regulation, and neurotransmission. [13][14][15] The breakdown of Zn 2+ ion homeostasis, which is toxic in excess or absence may in the pathology of intellectual development as well as other neurological issues including Alzheimer's and Parkinson's diseases. 16,17 Due to this, the detection of Zn 2+ has been studied intensively. Many selective colorimetric and/or uorescent probes targeting Zn 2+ have been reported, that are mostly based on quinoline, uorescein, benzazole, anthracene, coumarin, BODIPY, Schiff bases, indole, thiazole, terpyridin, naphthalimide, nanoparticles and so on.  From the above reports most of them are uorescent based and few are colorimetric uorescent chemosensors. Xu et al. (2009) has reported an NBD-based colorimetric and uorescent chemosensor for Zn 2+ and its use for detection of intracellular zinc ions. 41 S. Lee et al. (2016) has reported a Zn 2+ colorimetric sensor using label-free silver nanoparticles. 42 On the other hand, a uorescent chemosensor that is "switching on" and selective to Zn 2+ was described by Li et al. 43 and applied in MCF-7 cells. Two new ratiometric chemosensors were also created and tested by Lee et al. 44 for the detection of potentially harmful free Zn 2+ ions in aqueous media. A novel uorescent chemosensor was recently created by Chen and Li et al. 45 using a cyclen moiety as the ionophore and a helical imide as the uorophore. Moreover, Ding et al. 46 developed selective and responsive "turn-on" uorescentric Zn 2+ sensors based on di-and tri-pyridines with easily modulatable emission wavelengths. Inspired by the above research and our search on uorescent colorimetric chemosensor, 47,48 in this work, we have reported an optical sensor for colorimetric detection of Ni 2+ and Zn 2+ ions and selective uorometric detection of Zn 2+ ion. A quinazolinone derived Schiff base chemosensor (E)-2-benzamido-N ′ -(1-(pyridin-2-yl)ethylidene)benzohydrazide (L) has been designed, synthesised and characterised by 1 H-NMR, IR spectroscopy, ESI-MS spectrometry and single crystal XRD analysis. The receptor showed colorimetric l max shi for Ni 2+ and Zn 2+ (detection limit was 7.9 nM and 7.5 nM respectively) and uorometric "turn-on" response for Zn 2+ ion with detection limit 1.7 nM in methanol-Tris-HCl buffer medium (10 mM, pH 7.2, 1 : 1 v/v). The Jobs plot analysis revealed that receptor forms 2 : 1 complex with both the ions Ni 2+ and Zn 2+ , further conrmed by ESI-MS analysis. The single crystal structure of L-Ni 2+ complex, 3 has also been isolated. The chemosensor L can be applied to the recovery of contaminated water samples.

Materials and general information
The necessary components were purchased from Sigma-Aldrich and employed for synthesis without further purication. For the majority of the experiments, analytical-grade solvents were utilised, and double-deionized water was used for dilution and the preparation of Tris buffer (pH = 7.2) solution. The metal ion solutions were made from their nitrate salts. 1 H-NMR and 13 C-NMR spectra were recorded on a Bruker DRX spectrometer operating at 400 MHz in CDCl 3 and chemical shis were recorded in ppm relative to TMS. On a Shimadzu UV 1800 spectrophotometer, absorption spectra were captured using a quartz cuvette with a 10 mm path length and a wavelength of between 200 and 800 nm. Using a Waters mass spectrometer with mixed solvent HPLC methanol and triple-distilled water, high resolution mass (HRMs) spectra were captured. The pH measurements were done using a digital pH meter (Merck) by adjusting dilute hydrochloric acid and sodium hydroxide in buffer solution. Solutions of the receptor L (1 × 10 −5 M) and metal salts (1 × 10 −4 M) were prepared in CH 3 OH-H 2 O (1 : 1, v/v) and H 2 O respectively.
X-ray data collection and structural determination X-ray single crystal data were collected using MoKa (l = 0.7107 A) radiation on a BRUKER APEX II diffractometer equipped with CCD area detector. Data collection, data reduction, structure solution/renement were carried out using the soware package of SMART APEX. 49 The structures were solved by direct methods (SHELXS-97) and standard Fourier techniques, and rened on F 2 using full matrix least squares procedures (SHELXL-97) using the SHELX-97 package 50 incorporated in WinGX. 51 In most of the cases, non-hydrogen atoms were treated anisotropically. Hydrogen atoms were xed geometrically at their calculated positions following riding atom model. Structural information of 3 have been deposited at the Cambridge Crystallographic Data Center (CCDC number 2205292 †).
Synthesis of 2-phenyl-4(3H)-3,1-benzoxazinone (1) The 2-phenyl-4H-benzoxalin-4-one (1) has been synthesized by following the reported procedure 52 with some modication. To a stirred solution of anthranilic acid (2.74 g, 0.020 mol) in pyridine (12 mL), benzoyl chloride (2.81 g, 0.020 mol) was added dropwise, maintaining the temperature about 0-5°C for 1 h. The reaction mixture was stirred for another 3 h at room temperature until a solid product was formed. The reaction mixture was neutralized with saturated sodium bicarbonate solution and the pale yellow solid which separated was ltered, washed with water and re-crystallized from ethanol. Yield, 3.8 g, 85%; mp 112-115°C. 13

Photo physical measurements
Chemosensor L (3.58 mg) was dissolved separately in 10 mL of homogeneous solvent CH 3 OH-H 2 O (1 : 1, v/v) to make a solution of 1 × 10 −3 M and 30 mL of each of this solution were diluted with 2.97 mL of CH 3 OH-Tris buffer (1 : 1 v/v) mixture to make a nal concentration of 10 mM. The analyte solutions have been prepared using nitrate salt of metals in the order of 10 −4 M in double-distilled water. Aer mixing L with each of the metal ions for a few seconds, absorption and uorescence spectra were obtained at room temperature.

Job's plot measurements
A methanol-Tris buffer (1 : 1) solutions containing L (10 mM) and aqueous solution of Ni(NO 3 ) 2 and Zn(NO 3 ) 2 (10 mM each) were prepared separately. Then changing the mole ratio of the L from 0.1 to 0.9 in such a manner that the sum of the total metal ion and L volume remained constant (2 mL). All the solutions were diluted to 3 mL. Aer shaking them for a minute, UV-vis spectra were obtained at room temperature.

Computational details
The program package GAUSSIAN-09 Revision C.01 was employed for all calculations. 54 The gas phase geometries of the compound were fully optimized symmetry restrictions in singlet ground state with the gradient-corrected DFT level coupled with B3LYP. 55 Basis set LanL2DZ was used for the whole molecules L and NiL 2 (3). The HOMOs and LUMOs of molecular ions were calculated with the same basis set and functional.

Result and discussion
Synthesis and structure of L The coupling reaction between aldehyde and amine generate the imine functionalised ligand L (Scheme 1) with high yield (over 85%) and it was characterised by elemental analysis, FTIR, 1 H NMR and ESI-mass spectra.

UV-vis spectroscopic studies of L towards different metal ions
The receptor contains imine linker as well as several amide functional moieties thus interaction with cations and anions were expected. Thus absorption studies of L in presence of common cations and anions were performed at room temperature in methanol-Tris-HCl buffer (1 : 1 v/v, 10 mM, pH 7.2) solution. The receptor L shows main absorption band at 280 nm due to p-p* transition. This absorption band of L signicantly red shied to around 375 nm only in presence of 2 equiv. of Ni 2+ and Zn 2+ ions with the change of colour of the solution from colourless to yellow. Addition of same amount of several metal like Cu 2+ , Co 2+ , Cd 2+ , Cr 3+ , Mn 7+ , Sm 3+ , Al 3+ to the solution of receptor L, also absorption band shi observed, but the color and absorbance intensity produced by these ions is not very prominent when compare with Ni 2+ and Zn 2+ ions. The remaining cations and anions e.g. Fe 3+ , Hg 2+ , Pb 2+ , Cr 3+ , Ag + , Mn 2+ , Fe 2+ etc. cannot produced any signicant change of receptor solution at that condition (Fig. S11 †).
Here successive amount addition of analyte ions to the ligand L, absorption band gradually enhanced at ∼375 nm for Ni 2+ whereas 333 nm and 375 nm for Zn 2+ ion. As a results the absorption band at 280 nm goes on decreasing simultaneously and well-dened isosbestic point developed at 332 nm and 331 nm for Ni 2+ and Zn 2+ ions respectively. This change in absorption behaviour of L with addition of Ni 2+ and Zn 2+ ions remained consistent up to 0.5 equiv. for both ions and then remained well saturated (Fig. 1).

Scheme 1 Synthesis procedure of ligand L.
From the titration experiments the calibration curves were plotted where the absorbance intensity around 375 nm for both the metal ions varies almost linearly with the concentration. Thus detection limit was calculated to 7.9 × 10 −9 M for Ni 2+ and 7.9 × 10 −9 M for Zn 2+ using convenient formula DL = 3s/m, here 's' is standard deviation of the blank measurement and 'm' is the slope of the linear calibration curve (Fig. 2). This low level DL values are far below the guideline provided by WHO in drinking water and several reported Ni 2+ and Zn 2+ sensors. As per WHO, the permissible limits of Ni 2+ and Zn 2+ ions in drinking waters are 0.0008 and 5.0 mg L −1 respectively. 56 The precise detection of analyte like Zn 2+ and Ni 2+ ion in real water sample can be hampered in presence of other interfering ions due to their cross responses. Thus analyte signalling of receptor L was performed in presence of common competiting ions as a background. The presence of same equivalent of other interfering cations with analyte ion, the absorption spectra of host-guest complex cannot be disturbed (Fig. 3). Even 3 fold excessive addition of competitive metal ions than analyte, the   The Job's plot indicates that here 2 : 1 binding stoichiometric ratio obtained between receptor L and both the analyte metal ions Ni 2+ and Zn 2+ (Fig. S12 †), which was further conrmed by ESI-MS analysis ( Fig. S7 and S9 †) and single crystal structure of the L-Ni 2+ (3) was isolated complex (vide infra). In the ESI-MS spectra peaks at m/z 772.2 (Fig. S7 †) (Fig. 4). The values obtained as 2.37 × 10 3 M −1/2 and 2.31 × 10 3 M −1/2 for Ni 2+ and Zn 2+ ion respectively, indicates very strong affinity between receptor and analytes.
As we are interested on analyte sensing in aqueous or semi aqueous medium, thus pH of this medium plays a very crucial role as receptor contain pH sensitive amide (-CONH-) and imine (>C]N) moieties. For that purpose, receptor solutions with variable pH range 2 to 12 were adjusted by dilute HCl and NaOH and then analyte ions were added independently. The sequential UV-vis studies of each set of solution indicates that absorbance intensity at 375 nm for L and its metal complexes are mostly increases with pH. At pH = 2-4, there is less absorbance intensity difference observed between L and L + M 2+ (M = Zn and Ni), thus not very suitable to sense at this pH range. Thus in a wide range of pH = 5-12 the proposed chemosensor can successfully sense both Ni 2+ and Zn 2+ ion including physiological environment (Fig. 5).
Generally, metal ions like Ni 2+ and Zn 2+ ion form stable chelate complex with multidentate ligand Na 2 EDTA. Thus reversibility was checked by absorption studies L + M 2+ (M = Zn and Ni) solution with Na 2 EDTA in working medium. The characteristic absorption bands of host-guest complexes around 375 nm, instantly disappeared when L + M 2+ (M = Zn and Ni) complex reacted with 1 equiv. Na 2 EDTA (Fig. 6). Thus absorbance intensity at 280 nm restored with solution turns yellow to colourless due to generation of L. Further addition of analytes, the absorbance intensity at 375 nm was almost recovered. The alternative addition of chelator and analytes, absorption switching between L and L + M 2+ were almost consistent up to 4 cycles with negligible loss of absorbance intensity which indicate that sensing process is reversible than any irreversible metal catalysed reaction.

Fluorescence studies
The emission properties of the receptor L in presence of selective metal ions was also examined in methanol-Tris-HCl buffer (10 mM, pH 7.2) mixture (1 : 1 v/v). Among the selected metal ions like Fe 3+ , Cu 2+ , Co 2+ , Ni 2+ , Zn 2+ , Cd 2+ , Hg 2+ , Pb 2+ , Cr 3+ , Ag + , Al 3+ , Mn 2+ and Fe 2+ only addition of 2 equiv. Zn 2+ ion to the ligand solution emission intensity at 463 nm signicantly enhanced whereas the same amount of other metal ions including Ni 2+ induced very signicant emission change of receptor L (Fig. 7). To  get the details aspect of the emission spectra, titration experiment was conducted. When excited at 290 nm free receptor L showed weak emission at 455 nm. Upon increasing the Zn 2+ ion concentration in the receptor solution, emission intensity gradually enhanced with slightly red shied to 463 nm that got saturated up to 5 equiv. of Zn 2+ ion (Fig. 8a). The metal ion coordination with receptor L via imine side probably restricted both the non-radiative PET and >C]N isomerisation process, thus emission intensity increase due to chelation enhancement uorescence effect (CHEF). At the same time, there is no change in emission intensity of L in case of Ni 2+ ion indicate that in L-Ni 2+ complex both the opposing process i.e. paramagnetic uorescence quenching and chelation enhancement uorescence effect mutually compensate to each other. The emission enhancement of L in presence of Zn 2+ ion, cannot be disturbed in addition of 2-fold higher concentration of other metal ions. The uorometric detection limit was found to 1.7 × 10 −9 M calculated using the same formula 3s/m (Fig. 8b).

DFT calculation
The geometry optimizations staring from the Gauss-view structure of L lead to a global minimum as a stationary level. The optimized structures of the L and its Ni 2+ complex (3) and schematic representation of the energy of MOs and contours of selected HOMO and LUMO orbitals of L and 3 are presented in Fig. 9. The HOMO to LUMO energy gap for L is 3.25 eV, whereas for 3, the gap is 2.92 eV.
X-ray single crystal structure of NiL 2 (3) The ligand L reacts with Ni(NO 3 ) 2 (2 : 1 molar ratio) in methanol at room temperature to form the complex 3. It crystallizes in the monoclinic space group C2/c ( Table 1). The asymmetric unit consists of one L ligand and one Ni 2+ ion ( Fig. 10a) with occupancy half. The overall complex contains one Ni 2+ ion and two L ligands (Fig. 10b). The metal ion shows distorted octahedral coordination with N 4 O 2 donor sets from  two pyridyl N, two amide O and two hydrazide N (Fig. 10c). The Ni-N and Ni-O bond distances and bond angles (Tables S1 and S2 †) were found to be within normal ranges. The charges on Ni(II) ion were internally compensated by two deprotonated hydrazide NH originating from two L. Carefully looking into the structure of 1 revealed that the complex is associated through intermolecular p-p stacking and CH-p interactions (Fig. 10d).
Comparison of the performance of L with other reported fluorescent and colorimetric chemosensors Colorimetric detection of Ni 2+ and Zn 2+ ions and 'turn-on' uorometric detection of Zn 2+ ion using a single probe is not very common in the literature. Compared to the other systems, our system has several attractive analytical features, such as high sensitivity, wide linear range, high selectivity, lower detection limit, simple operation technology, good solubility, sensitive visualization and good practical applicability (Table 2). Moreover, the synthesis of our proposed chemosensor L requires only two steps and less hazardous reagents, and no hazardous by-product is formed.

Application
To explore the Ni 2+ and Zn 2+ sensing ability of the present chemosensor L in real water samples, we have tested the probe L in waste water and we got some result. Articial M 2+ (M = Ni, Zn) polluted tap water samples have been made by adding known amounts of standard M 2+ solutions in order to test its sensing in various samples. The prepared samples were analyzed by the same UV-vis spectrometer and the recovery amounts were calculated using the calibration curve (intensity vs. conc.) with the help of Lambert-Beer law. As shown in Table 3, the M 2+ concentration recovered for each sample are close in agreement with spiked amount with good precision. Thus, the receptor L can be suitable to detect M 2+ ion quantitatively in real water samples, indicating that it could be applied in real environments (Table 3).

Conclusion
In summary, we have successfully designed and synthesized a quinazolinone derived Schiff base chemosensor L for colorimetric nanomolar detection of Ni 2+ and colorimetric uorescent detection of Zn 2+ with high selectivity and sensitivity over other competitive ions in aqueous medium. A 2 : 1 stoichiometry between L and Ni 2+ were supported by Job-plot, ESI-mass spectral analysis and DFT studies. The single crystal of the L + Ni 2+ was also isolated. The colorimetric detection limits reached up to 7.9 nM for Ni 2+ and 7.5 nM for Zn 2+ respectively. Fluorescent measurement of Zn 2+ were obtained with the detection limit of 1.70 nM, which were far lower than those recommended by the WHO guidelines for drinking water. The reversible response to Ni 2+ and Zn 2+ ion was established by the Na 2 EDTA experiments. Moreover, L could operate in a wide range of pH and can be successfully applied for detection and quantication of Ni 2+ in environmental samples.

Conflicts of interest
Authors declare no conicts of interest.