A handy and accessible tool for identification of Sn(II) in toothpaste

An easily accessible colorimetric probe, a carbazole–naphthaldehyde conjugate (CNP), was successfully prepared for the selective and sensitive recognition of Sn(II) in different commercially-available toothpaste and mouth wash samples. The binding mechanism of CNP for Sn2+ was confirmed by UV–Vis, 1H, and 13C NMR titrations. The proposed sensing mechanism was supported by quantum chemical calculations. Selective detection of Sn(II) in the nanomolar range (85 nM), among other interfering metal ions, makes it exclusive. Moreover, Sn2+ can be detected with a simple paper strip from toothpaste, which makes this method handy and easily accessible. The potential application of this system for monitoring Sn2+ can be used as an expedient tool for environmental and industrial purposes.


Results and discussions
To explore the interaction pattern of the probe CNP with Sn 2+ , we have performed several experiments such as NMR titration, absorbance titration, pH titration, selectivity test and theoretical calculations.
Crystallographic analysis. Single crystals suitable for X-ray diffraction were obtained by slow evaporation of an acetonitrile solution of CNP at 3 °C. It crystallises in the monoclinic space group P2 1 /n ( Fig. 1, Figure S4 in ESI †). Different than in solution where CNP is unequivocally present as the enol-imine tautomer (Figures S1, S2 in ESI †), the X-ray structure of CNP revealed that the compound is in the keto-enamine form in solid-state.

NMR titration analysis.
Interactive properties of the probe CNP towards Sn 2+ was investigated through 1 H, 13 C NMR titrations in DMSO-d 6 and D 2 O. In 1 H NMR titration, the aromatic -OH proton peak at 16.37 ppm abruptly disappears after addition of one equivalent of Sn 2+ (SnCl 2 dihydrate), followed by downfield shift of imine proton peak at 9.84 ppm. This phenomena indicates the formation of strong coordination between -OH, -N groups of CNP and Sn 2+ (Fig. 2).
Moreover in 13 C NMR titration with the addition of one equivalent Sn 2+ , the carbon attached with -OH group shifts downfield from 169 to 193 ppm and also the imine 'C' peak at 153 ppm shifted towards 159 ppm with decreased intensity (Fig. 3). The above features also confirm the formation of CNP-Sn 2+ complex.
UV-Vis spectral behaviour of CNP with Sn 2+ . The interactions between the probe CNP and Sn 2+ was demonstrated by absorbance titration in acetonitrile:water (1:8, v/v) at neutral pH value (pH 7.0, 10 mM phosphate buffer). In the UV-Vis absorption spectra, we get a characteristic absorbance peak of CNP at 400 nm which decreases upon incremental addition of Sn 2+ , with an enhancement at 454 nm followed by a rapid colour change from pale yellow to deep orange. Furthermore, a notable isosbestic point at 425 nm indicates possible stronger interaction between CNP and Sn 2+ (Fig. 4a). Ratiometric changes in absorbance with increasing concentration of Sn 2+ have been represented in Fig. 4b.
Effect of pH value. pH titration clearly reflects that CNP is slight sensitive towards acidic pH values whereas the CNP-Sn 2+ complex is pH independent ( Figure S8, ESI †). So, we carried out all the experiments in the pH value range of 7.0 using 10 mM phosphate buffer.
Colorimetric responses of CNP toward various metal ions. The colorimetric behaviour of the sensor probe CNP was evaluated upon the addition of various metal ions in the aqueous medium (10 mM phosphate buffer, pH 7.0). As depicted in Figure S9, the pale yellow color of the probe turned to deep orange with the addition of Sn 2+ . The synthesized probe CNP did not show any notable color changes with the addition of other metal ions. The specific color change of CNP with Sn 2+ was attributed to several electron transitions in the CNP-Sn 2+ complex such as π-π*, d-d, ligand-metal charge transfer, and metal-ligand charge-transfer effects. Furthermore, the comparative spectrophotometric response of CNP was also studied with these metal ions which confirms that our probe CNP selectivity sense Sn 2+ over other metal ions ( Figure S10, ESI †). Therefore, these experimental results indicate that our synthesized probe CNP shows remarkable selectivity and sensitivity towards Sn 2+ over other analytes which could be a beneficial tool for practical approach.
Theoretical calculations. Theoretical calculations were executed for CNP and CNP-Sn 2+ complex systems using quantum chemical calculations at the DFT level LANL2DZ/6-31G** method basis set implemented  www.nature.com/scientificreports/ at Gaussian 09 program 32 and CPCM (Conductor like Polarizable Continuum Model) solvent model was used for solvent (water) effect incorporation. In the optimized structure of CNP, the positions of carbazole and naphthaldehyde units were nearly in the same plane. The optimized structure of the CNP-Sn 2+ complex showed the formation of coordination bonds of Sn 2+ with -OH and -N groups of CNP, which enhanced the stability of the complex ( Figure S11, Table S1, ESI †). From TDDFT calculation, we can see that there is a sharp S 0 -S 1 transition in CNP at 410 nm (oscillator strength f = 0.5188) which is very close to that experimentally observed value at 400 nm, responsible for the absorption of the carbazole moiety. Moreover, in CNP-Sn 2+ , the transition at 448 nm (S 0 -S 1 , f = 0.3563) indicates the π-π* electronic transition from the carbazole to naphthaldehyde moiety which is distinctly executed in the absorbance graph at 454 nm (Table S2, ESI †). Next, the energy distributions of HOMO and LUMO for CNP and its Sn 2+ complex were examined (Table S3, ESI †). As shown in Fig. 5, the energy of the HOMO and LUMO orbital levels for the CNP-Sn 2+ complex is lower than that of the probe CNP. Also, the HOMO-LUMO energy gap of CNP and CNP-Sn 2+ complex was calculated with an energy difference of 0.29 eV.
These outcomes indicate that the effective resonance attraction obtained in the CNP-Sn 2+ complex. The density of the orbital coefficient migrates from the carbazole unit to naphthaldehyde units in CNP, whereas in the CNP-Sn 2+ complex, the orbital coefficient of total framework is moving towards Sn 2+ via N-Sn 2+ coordination  www.nature.com/scientificreports/ bond. Hence, the obtained results imply the formation of a stable complex, which is consistent with the proposed binding mechanism. Furthermore, the mass of the CNP-Sn 2+ complex has been checked which truly validated the binding mechanism ( Figure S12, ESI †). The anticipated coordination mechanism of the CNP-Sn 2+ complex is given in Fig. 6. In 1 H NMR spectrum, the resonances assigned to the hydroxyl and imine groups are 16.37 ppm and 9.84 ppm, respectively. The naphthalene CO and imine C resonances in 13 C NMR spectroscopy are observed at 169.46 and 153.90 ppm, respectively.
Plausible mechanism and explanation. The naphthalene C-O bond length (1.282(3) Å) observed in the X-ray structure is characteristic of ketones rather than phenols, and the C-N bond (1.324(3) Å) is elongated relative to that of a typical imine 33,34 . Accordingly, CNP-enol is more susceptible to bind with Sn 2+ through stronger hydrogen bonding which is also corroborated with IR experiments( Figure S13, ESI †).In consequence, the pale yellow color of CNP becomes the deep orange color due to the non-covalent interactions of CNP with Sn 2+ (Fig. 6).
Quantitative analysis. The excellent photophysical properties of the probe CNP toward Sn 2+ , such as high sensitivity and selectivity at physiological pH encouraged us to further evaluate the potential of the probe for realistic approach. The specific and selective recognition of Sn 2+ by the chemosensor CNP was also examined in three different toothpaste samples. In this work, we took commercially available toothpastes from three different brands (T1, T2, T3). The details procedure of the preparation of toothpaste solutions was described in ESI †. Toothpaste solutions were then added to the CNP solution, a rapid orange color change resulted after few minutes (Fig. 7a). Furthermore, the method was also applied to recognize Sn 2+ by a simple paper strip in different toothpaste solutions. Three paper strips soaked in CNP were dipped separately in the different toothpaste solutions (T1, T2, T3), and Fig. 7b shows the respective color changes of CNP-coated paper strip after dipping in the toothpaste solutions T1, T2, T3 respectively. The concentration of Sn 2+ was also quantified from these three different toothpaste samples (T1, T2, T3). For this work, the above-mentioned toothpaste samples were subjected to colorimetric analysis at pH 7.0 (10 mM phosphate buffer) to quantify the amount of Sn 2+ present therein. Sn 2+ was quantified from these given samples by CNP (1 µM) by virtue of its selective and direct recognition properties. All estimations were done in triplicate. Concentrations of Sn 2+ were estimated by comparison with the CNP-Sn 2+ standard absorbance curve. From the standard curve it was found that the concentration of Sn 2+ were 0.73 µM, 0.70 µM and 0.64 µM in 100 µL of T1, T2 and T3 samples, respectively (Fig. 8, Table 1). Concentration of Sn 2+ was further quantified from two different mouth wash samples (M1, M2) using above mentioned procedure and the respective values are 0.25 µM and 0.28 µM in 100 µL sample solution ( Table 2).

Conclusion
In conclusion, a new carbazole-naphthaldehyde based colorimetric probe CNP was successfully synthesized for selective recognition of Sn 2+ in the aqueous medium under physiological pH value. The structure of the synthesized probe CNP was analyzed by single crystal X-ray diffraction which represents the presence of keto (CNP-keto) form in its solid state. The sensing mechanism has been triggered by the strong coordination bonding of CNP-enol with Sn 2+ , which was confirmed by absorbance, 1 H and 13 C NMR spectroscopy as well as mass spectrometry (HRMS). Theoretical calculations were also performed to justify the binding mechanism and optical behavior of the sensor probe. CNP showed high selectivity and sensitivity for Sn 2+ even in the presence of other metal ions. The detection limit of the probe for Sn 2+ was calculated to be 85 nM, which is much lower than  www.nature.com/scientificreports/ the WHO permissible amount of Sn 2+ in drinking water. We further demonstrated that CNP has been utilized as a colorimetric sensor to detect and quantify trace amounts of Sn 2+ in different toothpaste and mouth wash samples. A handy and accessible paper strip method has been proposed for this purpose. Being a potential probe, it can be used as an expedient 'in-field' approach to estimate Sn 2+ for environmental and industrial purposes for sustainable and environment-friendly industrial production.

Experimental section
Materials and methods. All the reagents were purchased from Sigma-Aldrich Pvt. Ltd. (India). Unless otherwise mentioned, materials were obtained from commercial suppliers and were used without further purification. Solvents were dried according to standard procedures. Elix Millipore water was used in all respective experiments. 1 H and 13 C NMR spectra were recorded on a Bruker 400 MHz instrument. For NMR spectra, DMSO-d 6 and for NMR titration DMSO-d 6 and D 2 O were used as solvent using TMS as an internal standard. Chemical shifts are expressed in δ ppm units and 1 H-1 H and 1 H-13 C coupling constants in Hz. The mass spectrum (HRMS) was carried out using a micromass Q-TOF Micro™ instrument by using methanol as a solvent. UV spectra were recorded on a SHIMADZU UV-3101PC spectrophotometer. FT-IR data were recorded on Shimadzu IRAffinity-1S Fourier transform infrared spectrometer (Spectrum Two) by ATR technique. The following abbreviations are used to describe spin multiplicities in 1 H NMR spectra: s = singlet; d = doublet; t = triplet; m = multiplet. Single crystal X-ray data of CNP was measured using a dual-source Rigaku Super Nova diffractometer equipped with an Atlas detector and an Oxford Cryostream cooling system using mirror-monochromated Cu-K α radiation (λ = 1.54184 Å). Data collection and reduction for both compounds were performed using the program CrysAlisPro 35 and Gaussian face-index absorption correction method was applied 35 . The structures were solved with Direct Methods (SHELXS) [36][37][38] and refined by full-matrix least-squares based on F 2 using SHELXL-2015 [36][37][38] . Non-hydrogen atoms were assigned anisotropic displacement parameters unless stated otherwise. The hydrogen atom bonded to nitrogen was located from Fourier difference maps and refined with an N-H distance restraint of approximately 0.96 Å. Otherhydrogen atoms were placed in idealised positions and included as riding. Isotropic displacement parameters for all H atoms were constrained to multiples of the equivalent displacement parameters of their parent atoms with U iso (H) = 1.2 U eq (parent atom). The single crystal X-ray data, experimental details as well as CCDC number are given in the Supporting Information.
Synthetic procedure of CNP. In a 100 mL round bottom flask, 2-hydroxy naphthaldehyde (1.0 g, 5.8 mmol) in 30 ml ethanol was vigorously stirred at ambient temperature for few minutes. Then, 3-amino-9ethyl carbazole (1.46 g, 6.95 mmol) was dissolved in ethanol (10 mL) and added dropwise to the solution. The reaction mixture was refluxed for 24 h at 83 °C. After completion of the reaction (monitored by TLC), the solvent was evaporated completely under reduced vapor pressure, then extracted with chloroform and water. After drying it over anhydrous Na 2 SO 4 , the organic layer was evaporated completely to get the solid product. This product was purified by column chromatography with the eluent CHCl 3 :PET (5:1, v:v) to get the product CNP with 86% yield (Fig. 9). 1