2,7-Naphthyridine based colorimetric and uorescent “Turn Off” chemosensors for selective detection of Ni(II) in aqueous media

A highly selective and sensitive 2,7-naphthyridine based colorimetric, reversible, pH independent and uorescence “Turn Off” chemosensors (L1-L4) for detection of Ni 2+ are being reported in aqueous media. The synthesized sensors are highly ecient in detecting Ni 2+ even in the presence of other metal ions that commonly co-exist with nickel. The receptors (L1-L4) showed a distinct color change from yellow to red by addition of Ni 2+ with spectral changes in bands at 535–550 nm. The detection limit of Ni 2+ for (L1-L4) are in the range of 0.2–0.5 µM which is 2–5 times lower than the permissible value of Ni 2+ (1.2 µM) in drinking water dened by EPA. The binding mode of interactions of L1-L4 for Ni 2+ were found to be 2:1 through job’s plot and ESI-MS analysis. Moreover the receptors can be used to quantify Ni 2+ in real water samples and formation of test strips by dip-stick method increases the practical applicability of Ni 2+ test for “in-the-eld” measurement of Ni 2+ . Importantly the sensing potential of these derivatives have been tuned by the nature of substituents i.e. electron donating (CH 3 ) and electron attracting (F, OCF 3 ), which showed that L4 is highly ecient in sensing of Ni 2+ even at minute level.


Introduction
The development and synthesis of chemosensors for selective and sensitive detection of heavy and transition metal ions is an active area of present day research due to their substantial effects on environment and biological systems [1][2][3] . The sensing of metal ions in aqueous media is provocative task due to presence of competitive interactions between the solvent and guest for receptor binding sites 4,5 . Among the various transition metals, nickel is an important element due to its wide spread use in industry (Ni-Cd batteries), in ceramics and magnetic types of computers, metallurgical processes (electroplating), rods for arc welding, surgical and dental prostheses, pigments for paints [6][7][8] . Nickle has signi cant role in various enzymatic activities such as acireductone dioxygenases, carbon monoxide dehydrogenases, and catalyst for hydrogenation. It is also used as essential trace element in biological systems which has signi cance in biosynthesis and metabolism of some plants and microorganisms. However it is toxic metal in bio-medicine point of view as it can be easily absorbed in our different organs like spleen liver, kidney etc that might cause lungs cancer and nasopharyngeal carcinoma, asthma, disorder of respiratory as well as central nervous system in humans, pneumonitis. The de ciency and excessive use of nickle also affects the life of many prokaryotic and eukaryotic organisms [9][10][11][12][13] .
Considering all these facts, the selective monitoring of nickel is very important in environmental, biological and industrial samples. Although various analytical methods such as ame atomic absorption spectrometry-electro thermal atomization (FAS-ETA), atomic absorption spectrometry (AAS), inductively coupled plasma atomic emission spectrometry (ICP-AES) are widely used for detection of metal ions [14][15][16][17][18][19] . However, most of the methods need trained operators, sophisticated equipment and tedious sample preparation procedures therefore there is still a need of simple, e cient and cost-effective methods for the micro level detection of heavy metals ions. Now a days, colorimetric, ratiometric, potentiometric and uorescence sensors have gained the attention for selective detection of metal ions (Ni 2+ ) in biological and environmental samples. Colorimetric chemosensors showed a distinct visible color change without the use of expensive equipment. Therefore colorimetric sensors seem to be more promising due to low cost, rapid detection and simple-to-use than classical techniques and uorescent sensors as well. Only a few number of colorimetric chemosensors for the detection of Ni 2+ at parts per million level are reported to date 20 .
Thus in perpetuation of our research work for the development in the eld of molecular recognition, we herein report benzo[c]pyrazolo [2,7] naphthyridine-5,6-diamine based e cient colorimetric and/or uorescent off sensors that can detect Ni 2+ with sensitivity and selectivity in aqueous solutions.
To the best of our knowledge, this is the rst report in which benzo[c]pyrazolo [2,7]naphthyridines have been explored as chromophore and two amino groups as binding sites for the selective detection of Ni 2+ in aqueous solutions. Moreover the novel synthesized chemosensors L1-L4 could be used as practical sensor for quantitative determination of nickel at ppm level in real water samples. Importantly the sensing potential of these derivatives can be tuned by the nature of substituents i.e. electron donating (CH 3 ) and electron attracting (F, OCF 3 ), which affect the metal ions sensing property.
The Ni 2+ complex of receptor L4 was synthesized by mixing Ni 2+ salt with L4 using 1:2 ratio in DMSO-H 2 O solvent mixture. The yellow solution of the ligand immediately turned to red colored solution. The solution was further re uxed to get the solid product (Scheme 2).
The binding interaction of L1-L4 with different metal ions was further monitored by investigating UVvisible absorption spectral changes and shown in Table 1. The UV-visible spectra of model receptor L4 showed a remarkable bathochromic shift in absorption spectrum at 537 nm which is in good agreement with color change, may possibly be ascribed to fast metal-ligand binding kinetics and high thermodynamic a nity of Ni 2+ for N-donor ligands 32 . The other examined metal ions did not exhibit any distinct spectral changes in UV-visible spectrum at 537 nm under identical conditions. Similar pattern of absorption spectral changes were observed for L1-L3 ( Figure S1-S3).
The coordination between receptor L4 and Ni 2+ was further rati ed by UV-visible absorption spectral titrations involving sequential addition of Ni 2+ (0-12 µM) to L4 (20 µM). It was observed that intensity of absorption bands at 537 nm and 438 nm increased while the absorption bands at 396 nm and 376 nm began to decrease until it reached its limiting value. Moreover emergence of isosbestic points at 365 nm and 410 nm during spectral titrations indicate the formation of stable complex with some stoichiometric ratios between L4 and Ni 2+ 33 ( Figure 3). The similar coordination behavior was observed for L1-L3 ( Figure S4-S6). These results suggest that receptors L1-L4 could be employed as colorimetric and ratiometric sensor for Ni 2+ and discriminating among different transition metal ions (Fe 3+ , Cu 2+ , Co 2+ , Pb 2+ , Hg 2+ ) which are normally di cult to differentiate.
The binding stoichiometry of the complexes were further explored by Job's continuous variation method 34 by plotting mole fraction versus changes in absorption intensity at 535 nm for L1, 538 nm for L2, 550 nm for L3 and 537 nm for L4, respectively. The Job's plot ( Figure S7) indicate maximum value at 0.7 corresponding to the formation of complex with 2:1 stoichiometry between L1-L4 and metal ions The association constant K a of receptors L1-L4 with Ni 2+ were determined by Benesi-Hildebrand equation 35 ( Figure S8) and are listed in  The detection limit of L1-L4 for Ni 2+ as colorimetric sensors were determined both by naked eye and absorption spectral changes. The results are shown in Table 2. For naked eye detection, the receptor L4 showed a distinct color change at minimum concentration of 1 x 10 -6 M for Ni 2+ (Figure 4/S9). Moreover, the detection limit determined by absorption spectral changes on the basis of 3S B /S 37 for L4 and Ni 2+ was found to be 2.43 x 10 -7 M. This value is ve times lower than EPA drinking water guidelines 1.2 x 10 -6 M for Ni 2+ 38 and revealed that L4 is highly e cient in sensing Ni 2+ even at minute level.
The possible binding mode of receptor L4 and Ni 2+ in the complex showed that the nitrogen atoms of naphthyridines coordinates Ni 2+ in 2:1 ratio and showed bathochromic shift in absorption spectra that can be rationalized by ICT 32 . The coordination of Ni 2+ to the nitrogen of naphthyridine moiety increases its electron withdrawing character which showed stronger ICT from electron donating methyl group to metal complex moiety.

ESI-MS and IR titrations
The  Figure S10).
FT-IR titrations were performed by using Bruker Alpha FT-IR and Figure 5 showed comparison of IR spectra of L4 before and after the addition of Ni 2+ . The sharp peaks present at 3420, 3294 and 3109 cm -1 due to NH stretching frequencies in free receptor L4 were broadened by adding Ni 2+ , suggesting the involvement of NH 2 group in coordination with Ni 2+ to form complex. 39 Metal ion selectivity An important feature of receptor L4 is to examine its selectivity towards analyte by competitive titration experiments ( Figure 6). The intensity of absorption band at 537 nm due to complex formation of L4- The UV-visible absorption spectra of L4-Ni 2+ complex with various anions was recorded to check the stability of complex, no change in absorption band at 537 nm was observed (Figure 7). This clearly depict that the stability of complex is unaffected in the presence of various anions.

pH effect study
In order to investigate the effect of pH on absorption response of receptor L4 to Ni 2+ , a series of solution with pH value ranging from (2.0 -12.0) were prepared ( Figure S11).
At pH 2.0-3.0, the receptor L4 has no substantial response to Ni 2+ in absorption spectroscopy. The absorption at 537 nm is maximum and constant in pH range 7.0 -8.0 and above pH 8.0, absorbance decreased gradually. The results warranted its biological and environmental applications at physiological pH. The color of L4-Ni 2+ complex remained red between pH 4-11, which indicate that Ni 2+ could be clearly detected over a wide range of pH 4-11.

Reversibility of receptor L4
The reversibility of receptor L4 towards Ni 2+ was examined by adding ethylenediaminetetracetic acid (EDTA, 1 equiv.) to the complexed solution of L4 and Ni 2+ (Figure 8). The solution color changed from red to light yellow (original color of L4). Upon addition of Ni 2+ again the absorbance at 537 nm was recorded. The absorption changes in spectral bands were reversible even after several cycles with alternative sequential addition of Ni 2+ and EDTA. These results indicate that receptor L4 could be recyclable through reagent EDTA. Such regeneration and reversibility could be valuable for the fabrication of sensors to sense Ni 2+ .

Fluorescence study
The uorescence study of L4 via uorescence titrations were examined at room temperature and exhibited emission maximum at 470 nm (λ ex = 390nm). The sequential addition of Nli 2+ (0 -12 µM) to the receptor L4 caused a reasonable decrease of emission intensity in emission maxima at 470 nm and gave bounteous information regarding "turn-off" behavior of receptor ( Figure 9). The quenching of uorescence (CHEQ) may be due to coordination of Ni 2+ with NH 2 group of receptor L4 as amine group loses its donating ability to uorophore and emission potential is quenched 40 .

Chemistry synthesized of L4-Ni 2+ complex
The synthesized complex of L4-Ni 2+ was characterized in terms of molar conductance, SEM and ESMIS analysis. The 10 -3 M solution of L4-Ni 2+ complex exhibited molar conductance value of 0.4 S cm 2 M -1 which suggest its non-electrolyte behavior in DMSO solution. Furthermore, SEM analysis was carried out to get better understanding of morphological difference before and after the addition of Ni 2+ to L4 receptor ( Figure 10). SEM images of receptor L4 has dense sprinkled elliptical shape like structure which has been transformed into rough stone like structure after complexation with Ni 2+ . The ESI-MS of synthesized L4-Ni 2+ complex ( Figure S12) showed the molecular ion peak at m/z 685.58 which resembled very well with experimental molar mass of [2L4+Ni+Cl 2 ] complex.

Practical application
In order to investigate the potential use of newly synthesized receptor L4 in real water samples, a calibration curve was drawn, which showed good linear relationship (R2 = 0.9996, n = 3) between the absorbance of L4-Ni 2+ complex and Ni 2+ concentration (0-5 µM) at 537 nm ( Figure 14/S11).
The receptor L4 was used for estimation of Ni 2+ in drinking water, tap water and industrial waste water samples (Table 3). All water samples were analyzed in triplicate with good recoveries and RSD values.
The results indicate that receptor L4 is highly speci c and sensitive for Ni 2+ estimation in environmental samples. To explore another application of receptor L4, test kits were prepared by immersing lter paper in receptor L4 (1x10 -3 M, HEPES buffer, pH = 7.4) and then air dried to check the suitability of "dip-stick" method for detection of Ni 2+ . When the prepared test strips were immersed into water solution of Ni 2+ with different concentrations, clear color change from yellow to red was observed ( Figure 11). The results showed that discernible concentration of Ni 2+ can be as low as 1x10 -5 M. The development of "dip-stick" method did not require any additional equipment for detection of Ni 2+ and showed extreme attraction "in-the-eld" measurements.

Conclusion
In summary, we have successfully characterized the photophysical properties of benzo[c]pyrazolo [2,7]naphthyridines (L1-L4) for the rst time which were prepared by green synthetic route. The receptor (L1-L4) promotes selective and sensitive sensing of Ni 2+ in aqueous media over a wide range of pH (4)(5)(6)(7)(8)(9)(10)(11) even in the presence of competitive ions i.e Fe 3+ , Cu 2+ , Co 2+ . A unique colorimetric response to Ni 2+ is observer (yellow to red) through coordination of receptor and Ni 2+ complex could be recyclable through treatment with EDTA. The detection limit of Ni 2+ were found to be in range of 0.2-0.5 µM for (L1-L4) which is 2-5 times lower than than the permissible value of Ni 2+ (1.2 µM) in drinking water de ned by American Environmental Protection Agency (EPA). The binding mode of interactions of (L1-L4) for Ni 2+ were found to be 2:1 through job's plot and ESI-MS analysis. The uorescence properties of receptor were evaluated with uorescence quenching by coordination with Ni 2+ . As a practical application, the most e cient receptor L4 could be used to quantify and detect Ni 2+ in real water samples and also applied for fabrication to test kit by using "dip-stick" method. The result indicate that to the best of our knowledge, receptor (L1-L4) are the rst reported multifunctional, naked eye chemosensors for sensing of Ni 2+ in aqueous solutions.

Materials and equipment
All the solvents and reagents used for synthesis were of analytical grade and used as received. Infrared (IR) spectra were recorded by Bruker Alpha FT-IR spectrophotometer. Mass spectra were recorded by Thermo Scienti c LTQ-XL system tted with electrospray ionization (ESI) source, Jeol 600 MS Route, and Jeol Hx110 mass spectrometer (EI-HR). Pre-coated aluminum sheets of silica jel 60 GF254 (Merck) were used as TLC plates to check the purity of compounds. Quantitative determination of nickel was carried out by Inductively Coupled Plasma-Optical Emission Spectrometer (iCAP6500 ICP-OES , Thermo Scienti c, Cambridge, United Kingdom). The pH was measured by using Metrohm, 78l pH/ion meter.
Synthesis of receptors (L1-L4) The synthesis of receptors (L1-L4) was carried out in two steps following our previously reported added to 4 mL of the solution of receptor L4 to give 10 equivalent of metal ions. Then Ni 2+ solution was added into mixed solution of each metal ion to make 1 equivalent. After few minutes of mixing them, the UV-visible spectra was recorded at room temperature.

Water sample collection and Ni 2+ determination
The drinking water samples, tap water and industrial waste water samples were collected, preserved and stored in plastic containers for Ni 2+ analysis. Industrial waste water samples were ltered prior to analysis. Each sample was analyzed in triplicate using receptor L4 and ICP-OES as standard method (Table 3). Spiking and recovery method was used in order to validate chemosensing performance of our newly developed sensor L4. UV-visible spectral measurement of water samples containing Ni 2+ was carried out by adding 0.5 mL of receptor L4 to 2.5 mL of sample solutions and pH of solution was maintained at 7.4 using HEPES buffer. The solutions were allowed to stand for 10 min at room temperature and absorption measurements were taken at 537 nm. Filtered water samples were directly used for ICP-OES analysis. Figure 1 Some structural motifs of amino group containing sensors and present study.   Naked eye detection limit for receptor L4.

Supplementary Files
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