N’-(4-(diethylamino)-2-hydroxybenzylidene) isonicotinohydrazide based chemosensor for nanomolar detection of Ni(II) ion

ABSTRACT A novel Schiff base, N’-(4-(diethylamino)-2-hydroxybenzylidene) isonicotinohydrazide (S1) was synthesised by the condensation reaction between isonicotinohydrazide and 4-(diethylamino)-2-hydroxybenzaldehyde. The chemo-sensing behaviour of S1 towards Ni2+ was investigated and confirmed that the S1 exhibited fair selectivity and sensitivity. Furthermore, it was noted that there was no significant effect of co-existing cations and anions on the detection of Ni2+. The complexation ratio of S1 and Ni2+ was confirmed as 1:1 with Jobs continuous variation method and two isosbestic points on absorption titration further supported by DFT calculations. The binding constant (Kb) for S1+Ni2+ was calculated as 22,769 M−1 and 22,134 M−1. Moreover, the obtained limit of detection (LOD) for Ni2+ was found to be 375 nM. Finally, chemosensing applications of S1 as a paper test strip kit in the environmental monitoring assessment laboratory for the recovery of Ni2+ from real water samples were successfully designed and evaluated.


Introduction
Recently, the responsive and selective detection of metal ions has been attracted researchers due to several applications including environment to health and industrial sectors [1,2].In the past years, many chemosensors for Fe 3+ , Hg 2+ , Cu 2+ , Al 3+ and Pb 2+ ions were reported in the literature, but, limited chromogenic and fluorescent sensors for the detection of Ni 2+ ions [3,4].Numerous analytical techniques have been employed including electrochemical, voltammetry, and ion chromatography for the detection of metal ions.However, these techniques have several disadvantages such as expensiveness, timeconsuming, and skill analyst requirement, but colorimetric and fluorescent-based chemosensors are cost-effective and handy for real-time detection [5][6][7][8][9][10][11].
Nickel is one of the significant micronutrients for the human body that catalyses various physiological metabolisms [12,13].On the other hand, it plays a vital role in the physiological activities of animals and plants and has equal industrial importance.It is used in making many alloys and also acts as an important catalyst in several organic reactions.Moreover, the toxicity of nickel is of greatest concern to humans due to its adverse effects such as lung tumour, dermatitis, and intense pneumonitis [14][15][16].According to the World Health Organization, the tolerance limit of nickel in drinking water is ~0.07 mg/litre.The literature reports confirmed that the leaching of nickel from pipes is the primary source of water pollution.However, vehicle emissions, metal mining, burning of fossil fuels, and smelting are also responsible for the release of nickel into the environment [17,18,[19][20][21].
Generally, Schiff bases have hetero-atoms and form highly stable complexes with metal ions in a short period, leading to the development of chemosensors.The Schiff bases and their complexes are vastly used in the research and development divisions in most industries.Interestingly, it was observed that these complexes have fair medical applications and are being used as anticancer, anti-inflammatory, analgesic, anticonvulsant, antimicrobial, antitubercular drugs, etc [22][23][24].
For the past few years, our team is involved in the synthesis of materials that have been used for the removal of contaminants from the various drinking waters [25][26][27][28][29][30][31][32][33][34][35].In recent decades, the literature review suggests that few chemosensors have been reported for Ni 2+ , compared to other analytes such as Fe 3+ , Hg 2+ , Cu 2+ , Al 3+ , and Pb 2+ .The earlier reports on chemosensors have demonstrated some drawbacks including complicated synthesis procedures, use of expensive chemicals, low detection limit, less practical ability, and no regeneration ability.To overcome the above-mentioned shortcomings, this study was aimed at synthesising the N'-(4-(diethylamino)-2-hydroxybenzylidene) isonicotinohydrazide (S1) and investigated its recognition properties for the Ni 2+ ions.The Job's plot and DFT studies have confirmed the complexation ratio as 1:1 for S1 and Ni 2+ ions.The limit of detection (LOD) and the binding constant (Kb) have been evaluated using the isotherm equations.Finally, S1 is explored as the paper test kit for the real analysis of various water samples.

Apparatus
The UV-visible absorption and FTIR specta were obtained from UV-1800 UV/Vis spectrometer and Shimadzuand Brukar IR spectrometer, respectively.An NMR spectrum was obtained from Bruker DTX-400 spectrometer in CDCl 3 , with internal standard as TMS.A mass spectrum was obtained from micro TOF-Q II mass spectrometer.

Synthesis of S1
In 50 ml of CH 3 OH, isoniazide (0.411 g, 3 mmol) and 4-(diethylamino)-2-hydroxybenzaldehyde (0.579 g, 3 mmol) were added with a constant stirring for 2 h at room temperature (RT) [36].The obtained yellow-coloured product (S1) was filtered, dried at room temperature, and recrystallised with 85% C 2 H 5 OH.The synthesis procedure is illustrated in Scheme 1.The purity of the molecule S1 is evaluated with FTIR, 1 H NMR and mass spectroscopic techniques.

Synthesis of S1+Ni 2+ complex
The S1 (0.312 g, 1 mmol) was added to 25 ml of CH 3 OH with a constant stirring followed by the addition of 1 mmol of NiCl 2 .Then the mixture was refluxed while stirring for 2 h.The obtained darkish brown solid (S1) was filtered, air-dried, and further purified by recrystallisation in ethanol.

Theoretical calculations
The DFT calculation was performed by the Gaussian 09 programme with DFT-B3LYP methodology, using a 6-31G** and LANL2DZ basis set with full optimisation of ligand (S1) and complex (S1+Ni 2+ ).

Binding constant
The K b was calculated on the basis of Benesi-Hildebrand and Scatchard plots.The high value of binding constant shows the stability of the complex that was further supported by the DFT calculation.

Job's plot measurements and LOD calculation
The continuous variation method is used to evaluate stoichiometry between S1 and Ni 2+ .The working solution of S1 and Ni 2+ was prepared from the stock solution (solution preparation is given in the supplement file).The solutions of S1 and Ni 2+ were prepared at ratios of 1:9 to 9:1 and recorded on a UV-visible spectrometer.
The limit of detection (LOD) is calculated with below expression: δ = Standard deviation for five blank measurements, S = Slope of the fit lines

Spectroscopic characterisation of S1
The purity of the molecule S1 was performed using FTIR, 1 H-NMR and Mass techniques.

Absorption studies of S1
It is important to optimise the solvent ratio of the solution in which S1 was dissolved and experiments were performed.The S1 was highly soluble in methanol.Therefore, it was selected for the further steps.The spectrum of S1 was recorded in methanol and started adding the deionised distilled water into methanol to optimise the ratio.It was observed that 40% of the water in the solution does not affect the spectrum.
The spectrum was reproducible and uniform, however, beyond 40% of deionised distilled water, the spectrum of S1 diminished and the solution becomes turbid.The interaction of S1 was investigated with various metal ions in 60:40, v/v of CH 3 OH/H 2 O. Figure 1   stating that there was no significant interference effect after addition of various metal ions.Thus, the proposed sensor S1 is suitable for the sensing of Ni 2+ with a fair sensitivity [41].
The fluorescence properties of S1 were analysed by adding different concentrations (c = 1 × 10 −4 M) of cations in 60:40, v/v of CH 3 OH/H 2 O.The fluorescence emission of S1 was observed at 475 nm upon excitation at 415 nm.Among the measured cations, a selective enhancement in the fluorescence of S1 was observed as depicted in Figure 4(a).In contrast to the other metal ions, the addition of Ni 2+ displayed a significant and discriminating fluorescence enhancement at an emission band of 475 nm.The enhanced fluorescence of S1 with Ni 2+ is confirmed based on the reticence of isomerisation of C = N due to complex formation.Figure 4(b) illustrates the fluorescence spectra of titration experiments with standard addition of Ni 2+ ion solution ranging from 0 to 200 µL to the S1 solution.The linear regression shows that by displaying linearity of 0.985, the receptor binds linearly to the nickel ion (Figure 5).The obtained data on the non-linear fit on the stoichiometry of S1 and Ni 2+ was found to be 1:1.In the FTIR spectrum of pure ligand, broad bands appeared nearly at 3200 cm −1 and 3400 cm −1 corresponding to -NH and -OH stretching frequencies, respectively.The sharp bands that appeared at 1654 cm −1 and 1628 cm −1 are characteristic bands for carbonyl and imine stretching frequencies, respectively.Interestingly, the characteristic bands correspond to -OH and -NH are disappeared in the FTIR spectra of the corresponding Ni 2+ complex and also the band for the carbonyl stretching was disappeared while the band for imine is shifted to lower wavelength (i.e.below 1600 cm −1 ) in the complex.The above information reflects that the amide functionality of the ligand must have undergone amidoimidol tautomerism to become imidol structure as the prominent one during complexation.Further, the protons of phenolic -OH and imidol -OH must have been deprotonated while complex formation.Besides, the shifting of the band for the imine functional group to a lower wavelength is due to the coordination of nitrogen of the imine with the metal.Since the imino nitrogen plays a role as an electron donor during the complex formation, the imine stretching frequency is lowered (Figure S4 supplimentry information).The geometry of the complex probably be a distorted square planar wherein phenolic

DFT study
The energies and dipole moments of the optimised structures of S1 and S1+Ni 2+ are found as −1028.9830a.u., −2524.1808a.u., 10.5456 debye, and 8.1474 debye, respectively.The obtained results demonstrated that the sharp decrease in the HOMO-LUMO energy gap, conformed the development of a stable complex between S1 and Ni 2+ .The HOMO-LUMO gap of S1 was 2.1639 eV and 0.0751 eV for S1+Ni 2+ -complex (Figure 6 and Table 1) resulted in the change of the photophysical properties [42,43].The small value of the HOMO-LUMO gap in complex (pyramidal geometry) revealed the formation of the stable complex with self-occurring spontaneous reaction.The obtained DFT results were in good agreement with the experimental results.

Limit of detection, binding constant and Job's plot
The LOD is based on the IUPAC definition (CDL = 3Sb/m) and was found to be 375 nM from 10 blank solutions that were less than the literature reports (500 nM).It is also much lower than the allowable concentrations of Ni 2+ in drinking water specified by the World Health Organization.The binding constant (Kb) was calculated and found to be 22,769 M −1 and 22,134 M −1 using Benesi-Hildebrand [44] and Scatchard [45] plot, respectively, indicating a strong and stable complexation between Ni 2+ ion and S1 (Figure 7(a,b)).The binding stoichiometry of 1:1was confirmed between S1 and Ni 2+ ions by (Figure 8) Job's continuous variation method [46].

Reversibility of S1
The sample's reversibility is a core character in which the sample comes under the model sample class.A variety of anions were used to analyse the reversible binding of Ni 2+ ions using the absorption spectrum.The wine red colour of the sample S1+Ni 2+ is missing with the inclusion of DMG 2-alone.It would appear that the addition of DMG 2-will split the S1 +Ni 2+ sample complex.The DMG 2-is the reversible reactant that de-metals the complex and reappears S1 into its original form.The S1 demonstrated superior 6-cycle reversibility (Figure 9(a,b)).The reversibility of the sensor indicates regeneration ability several times to transform it into a low-cost sensor.

Application as a paper test strip kit
To shows the practical applicability and for commercialisation, the current study uses S1 as a paper test strip for the Ni 2+ sensing.The discriminating colour transforms of S1 with the addition of Ni 2+ solution; encourage us to show S1 as a test kit.In the current study, the solution of S1 and Ni 2+ solution is prepared separately.The two paper strips cut into a rectangular form having the capability to absorb the solution has been taken and deep into the solution of the S1 and dry it for a few minutes by exposing it to the air [47].The solution of Ni 2+ is sprayed onto the second strip and dries for a few minutes by exposing it to the air.The second test strip instantaneously transforms from yellow to wine red colour (Figure 10). Figure 11 shows the physical images of the S1, NiCl 2 , and S1+Ni 2+ complex.The colour of the test strips shifted from yellow to deep wine red as the Ni 2+ ion concentration was increased, and it was easily distinguishable by naked eyes.The test paper can clearly detect Ni 2+ in aqueous solution at a low concentration.Other cations in the study had no discernible effects.In fact, monitoring of Ni 2+ in natural aquatic environments without use of spectroscopic instrumentation has proven to be quite effective in reducing Ni 2+ toxicity in under developed areas.As a result, the simple test kit may be used to detect and estimate Ni 2+ ion concentrations in a rough and quantitative manner.

Ni 2+ recovery from real water sample
The high selectivity of S1 towards Ni 2+ ions makes S1 potentially useful for analysing low levels of Ni 2+ ion in different water samples.The two water samples, which included mineral and tap water samples were taken into consideration.Both water samples were purified before being spiked with a Ni 2+ solution of varying concentrations.All samples were analysed for Ni 2+ ions using S1 via absorption experiments.The outcome of this study demonstrated the high-quality recovery of Ni 2+ from both samples (Table 2) [48,49].The absorption spectra of each sample were tested three times (n = 3) with a relative standard deviation (RSD) of <1.
Rapidity, ease of synthesis, low costs, and less time-consuming are all advantages of this probe, which is a difficult feat for a sensor to achieve.Furthermore, unlike a quenched fluorescence event, the probe is reversible.In aqueous media, particularly, the probe is good for very little competitive fluorescence switch on sensors.In contrast to the previous sensor, the LOD is fair enough.However, in some instances, the level of identification was not less as per WHO guidelines.The summary of LOD's of previously reported and current sensors were tabulated in Table 3. From Table 3, it was found that LOD is low compared to that of previously reported sensors and in some reports, it was comparable.On the other hand, the synthesis procedure of S1 was cost-effective compared to the other probes.

Conclusions
In the present study, a colorimetric Schiff base was synthesised and investigated for the detection of Ni 2+ ions in various water samples.The absorption studies showed the discriminating spectrum of S1+Ni 2+ when compared to S1 and supported with DFT.The HOMO-LUMO gap of S1 was 2.1639 eV and 0.0751 eV for S1+Ni 2+ -complex resulted in the change of the photophysical properties.The small value of the HOMO-LUMO gap revealed the stability of the complex as well as showed the reaction as a self-occurring
(a) depicts the absorption spectra of S1 (c = 2.5 × 10 −5 M) showing a maximum absorbance due to π→π* (245 nm, ε = 8.16 × 10 4 ) and n→π* (380 nm, ε = 4.03 × 10 4 ).Interestingly, the obtained results revealed that the addition of various metal ions solutions, except Ni 2+ (c = 2.5 × 10 −4 M), showed no significant change in the absorption maximum.With the addition of Ni 2+ ion solution, the spectrum of S1 has been changed and a new band appeared at 450 nm (ε = 1.93 × 10 4 ), and concurrently the band at 380 nm disappeared (Figure1(a)).This behaviour was attributed due to the interaction of Ni 2+ ions with the functional groups such -OH, -C = N of S1, and changes in the photophysical properties via internal charge transfer (ICT)[37][38][39].In the Inset Figure1(a), left side S1 solution (Yellow) and right side S1+Ni 2+ solution (Wine red colour) were observed with a naked eye.An in-depth study on titration experiments has been carried out between S1 and Ni 2+ ion solution by varying the volume of Ni 2+ ion (0 to 200 µL) and the change in the absorption was recorded and illustrated in Figure1(b)[40].The consecutive addition of Ni 2+ generates a ratiometric response with two isosbestic points at 340 nm and 415 nm as shown in Figure1(b).The two isosbestic points in the absorbance titration showed a complexation ratio of 1:1 between the S1 and Ni 2+ ion which was further confirmed by the fluorescence titration and DFT calculations.The linear plot obtained from the titration study and linear regression displaying an R 2 = 0.988, which indicates that the S1 binds linearly to the Ni 2+ ions (Figure2).

Figure 3 (
a) shows the ratiometric graph of S1 with the various metal ions as obtained from UV-Visible selectivity experiment.The selectivity studies were performed by interacting S1 with Ni 2+ in presence of other metal ions and absorption maximum was recorded.Figure 3(b) demonstrates the results of interference study,

Figure 1 .
Figure 1.(a) Recognition behaviour of S1 with the addition of different metal ions solutions.Inset, left side S1 solution (Yellow) and right side S1+Ni 2+ solution (Wine red colour), and (b) Absorption profile of S1 upon regular addition of Ni 2+ ion solution (0 to 200 µL).

Figure 2 .
Figure 2. Normalised absorbance signal with respect to the change in Ni 2+ concentrations.

Figure 3 .
Figure 3. (a) Effect of various metal ions on the absorbance intensity of S1 and (b) Interference studies of S1 towards Ni 2+ in presence other metal ions.

Figure 4 .Figure 5 .
Figure 4. (a) Emission spectrum (λ ex = 415 nm) of S1 in the presence of the metal ions, and (b) Fluorescence titration spectrum of S1 after continues addition of small amount of Ni 2+ .

Figure 7 .
Figure 7. (a) The plot of Benesi-Hildebrand for receptor S1 where R 2 = 0.982 and the K a value at 22,769 M −1 , and (b) The plot of Scatchard plot for receptor S1 where R 2 = 0.981 and the K a value at 22,134 M −1 .

Figure 8 .
Figure 8.The plot showing the stoichiometric ratio of 1:1 between S1 and Ni 2+ using Job's method.

Figure 9 .
Figure 9. (a) Reversible cycle of S1 with Ni 2+ and DMG 2-, and (b) graph of the reversible study.

Table 2 .
The summary of recovery data of Ni 2+ from mineral and tap water samples using S1.The LOD of S1 for Ni 2+ is found to be at 375 nM.The visual colour appearance and low-level detection made S1 application in the paper test kit and real water sample analysis.

Table 3 .
A comparison of sensors reported in the literature with present work.