A colorimetric and fluorometric investigation of Cu(II) ion in aqueous medium with a fluorescein-based chemosensor

Abstract

As a selective colorimetric and fluorometric chemosensor, a novel fluorescein-based Schiff base (FNSB), 1,4-bis(1-fluorescein)-2,3-diaza-1,3-butadiene (L, 3) exhibits efficient binding for Cu²⁺ ion in water and allows naked-eye detection as a result of the formation of a 1 : 1 copper–ligand complex. The complexations of Cu²⁺ ion due to molecular interaction with 3 resulted in a rapid change in color from colorless to deep yellow with a characteristic change in the UV-vis absorption to a longer wavelength by 130 nm in acetonitrile and significant quenching of the fluorescence intensity (∼2.5 fold for Cu²⁺ ion) with maxima at 519 nm in 1% DMSO–Tris buffer solution at physiological pH (7.4). The detection limit of Cu²⁺ ion in acetonitrile is found to be 20 μM by UV spectroscopy, whereas in aqueous solution, the limit is reduced to 1.25 μM by fluorescence spectroscopy.

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In recent times, the toxicity of environmental pollutants containing metal ions is a problem that has received global attention due to its critical effects on physiology, ecology, evolution, nutrition, and the environment.¹ In this respect, there is an immense need for detection and identification of metal ions in aqueous solution for environmental monitoring and biomedical science, ensuring the regular functioning of ecosystems, health and humanity. It has been found that the involvement of specific concentrations of metal ions is essential in a number of biological processes.² In particular, copper plays a pivotal role in different biological processes, such as iron absorption, haemopoiesis, and various enzyme-catalyzed and redox reactions.³ A neural copper concentration on the order of 0.1 mM is required in the body and brain for biological processes; however, higher concentrations cause vomiting, lethargy, increased blood pressure and respiratory rates, acute hemolytic anemia, liver damage, neurotoxicity, and neurodegenerative diseases.^4,5 Furthermore, copper ions can disrupt natural ecosystems due to their adverse effects on microorganisms.⁶ Thus, there is an upsurge of interest in the development of selective and reliable detection methods for the qualitative and quantitative analysis of metal ions. In this perspective, chemosensors are of great importance to chemistry, biology, and medicine because they allow rapid detection of different compounds in living organisms and the environment.²

Colorimetric and fluorometric chemosensors are of particular interest; they have distinct advantages in terms of sensitivity, selectivity, response time, real time analysis and easy operation. To date, several molecular design strategies have been exploited to develop chemosensors for the selective recognition of different species on the basis of different host–guest interactions, such as hydrogen-bonding, electrostatic forces, metal-receptor coordination, hydrophobic interactions and van der Waals forces;⁷ however, effective sensors with good compound selectivity, solvent specificity and detection limits are rare.

Recently, several fluorophores based on boron–dipyrromethene (BODIPY), naphthalene, pyrene, anthraquinone, coumarin, xanthane, rhodamine and fluorescein, which are effective either in the UV or visible/near IR regions, have been studied.⁸ Among these, fluorescent chemosensors for Cu(II)-selective detection were reported and have been used with some success in biological applications.⁹ However, poor water solubility and selectivity makes these sensors unsuitable for environmental and cellular applications. To address these limitations, water soluble fluorescein-based chemosensors are significant due to their intrinsic advantages of high quantum yield and absorption and emission maxima in the visible region compared to other organic dyes.^10,11

In addition to the signaling unit of a fluorophore, a built-in Lewis base receptor, i.e., a Schiff base receptor with N-donor atoms in the imine group and the O-donor of the phenol as a recognition center, can coordinate to a cation as a Lewis acid, affording stable complexes with various transition metals.^12a–e Thus, the presence of amines/imines in the receptor plays a key role in establishing electronic communication between fluorophores and analytes.^12f–g

Many methods have been used to detect metal ions, including fluorescence spectroscopy, UV-vis absorption, atomic absorption, ICP emission spectroscopy, and voltammetry. Amongst these, UV-vis absorption and fluorescence emission spectroscopy are attractive approaches because of their high sensitivity, facile operation, and widespread availability of equipment for analysis. As a result, fluorescent chemosensor molecules have been designed and synthesized for metal ion detection, in some cases yielding enhanced (turn-on) fluorescence signals and quenched (turn-off) signals^13,14 at low concentrations for use as fluorescence turn-on/off sensors.

In this contribution, we report a new assay for the detection of Cu²⁺ ion using a fluorescein-based Schiff base (FNSB), 1,4-bis(1-fluorescein)-2,3-diaza-1,3-butadiene (3), as a highly selective colorimetric and fluorometric chemosensor in organic solvent as well as in aqueous solution at physiological pH (7.4). Indeed, the inherent ability¹⁵ of Cu²⁺ ion to quench fluorescence over other quenching metal ions (Hg²⁺, Ni²⁺, Pb²⁺, and Ag⁺) has been exploited to develop a selective fluorescence quenching sensor.

Though numerous ligands have been reported in the literature for selective detection of Cu²⁺ in organic solvents,⁹ and a few can be used in aqueous medium,^9v ligands are rarely found to be active both in organic and aqueous media.^9w Fortunately, our synthesized imine-functionalized fluorescein-based ligand 3, which is obtained in a two-step reaction from naturally occurring, inexpensive fluorescein with moderate to good yield, selectively binds Cu²⁺ in both organic and aqueous media without affecting its specificity or selectivity over other cations.

Several factors, such as high quantum yield, the absorption and emission maxima of fluorescein, and the participation of the Schiff base as a recognition unit for coordination to a metal cation as a Lewis acid, were considered in the design of the fluorescein-derived Schiff base ligand. In addition to the above parameters, pH, as an exogenous factor, affected the chromogenic behavior (i.e. the selectivity and sensitivity) of the receptor. Generally, fluorescein exists in a fluorescent ring opened carboxylic acid form and in a non-fluorescent ring closed lactone form, and this equilibrium is very sensitive to the pH of the solution (Fig. 1).¹⁶ Thus, the pH-tunable sensitivity of fluorescein can be used to sense changes in a local environment by monitoring its ring opening and closing equilibrium, in addition to other spectroscopic effects, to detect metal ions in industrial and commercial pollutants.¹⁷ In this regard, the development of a water-soluble fluorescein-derived Schiff base fluorophore for the tunable detection of Cu²⁺ ion in aqueous solution at physiological pH is an attractive and challenging approach.

Fig. 1 UV-vis absorbance of ligand 3 in various solvents at 2.5 × 10⁻⁵ M concentration.

Experimental section

General information and methods

All materials were obtained from commercial suppliers and were used without further purification. All solvents were dried according to standard methods prior to use. All solvents used in the optical spectroscopic studies were either HPLC or spectroscopic grade, and all reactions were monitored by TLC analysis on aluminium plates pre-coated with silica gel 60 F₂₅₄. Column chromatographic purifications were carried out on silica gel (200–400 mesh size). NMR spectra were measured on a Bruker Avance instrument. The ¹H NMR (500 MHz) chemical shifts are given in ppm relative to the internal reference TMS. The ¹³C NMR (125 MHz) chemical shifts are given using CDCl₃, MeOH-d₄ and DMSO-d₆ as the internal standards. ESI-MS data were recorded on an Agilent Q-TOF MicroTM LC-MS mass spectrometer. Fluorescence excitation and emission spectra were obtained using an Edinburgh Instruments Xenon-900 spectrofluorophotometer. UV-vis absorption spectra were recorded on a Spectro 2060+ UV-visible spectrophotometer.

Synthesis of 1,4-bis(1-fluorescein)-2,3-diaza-1,3-butadiene (3)

Scheme 1 illustrates the two step synthesis of the fluorescein-derived Schiff base (FNSB), 1,4-bis(1-fluorescein)-2,3-diaza-1,3-butadiene (3), as a cationic ligand (L). The Reimer–Tiemann reaction of fluorescein (1) with chloroform in alkaline aqueous methanolic solution at 55 °C produced fluorescein monoaldehyde (2)¹⁸ in 24 to 28% yield. The appearance of a peak at 10.63 ppm in the ¹H NMR spectrum (Fig. SI 1†), the C–H stretching of –CHO due to Fermi coupling at 2928 and 2853 cm⁻¹, and the C=O stretching at 1727 cm⁻¹ in the FTIR spectra (Fig. SI 6†) confirmed the functionalization of fluorescein with an aldehyde group by the Reimer–Tiemann reaction. The Schiff base formation was performed by a reaction between fluorescein monoaldehyde and a slight excess of hydrazine hydrate in ethanol at 90 °C, affording the final 1,4-bis(fluorescein)-2,3-diaza-1,3-butadiene (3) in 35% yield. The appearance of a mass peak at m/z = 717.1558 for [M + H]⁺ in the mass spectra (Fig. SI 5†), the disappearance of the aldehyde peak at 10.63 ppm in the ¹H NMR spectra, C–H stretching at 2928 and 2853 cm⁻¹ and C=O stretching at 1727 cm⁻¹ in the FTIR spectra (Fig. SI 6†) indicate the formation of Schiff base 3 from aldehyde 2 with hydrazine hydrate (Scheme 1).

Scheme 1 Synthesis of 1,4-bis(1-fluorescein)-2,3-diaza-1,3-butadiene (3) as a cationic ligand.

Spectral studies

The selectivity, binding and stoichiometry of the complex system were also determined by NMR and mass spectroscopy. The NMR and mass spectroscopy data support 1 : 1 ligand-to-metal complex formation (Fig. SI 7 and 8†) with a change of the binding constant in the fluorogenic response of ligand 3 in the presence of varying concentrations of Cu²⁺, indicating the formation of a 1 : 1 ligand-to-metal complex (by Job plot (Fig. 13), NMR, and MS) with an association constant of 2.23 × 10⁴ M⁻¹ (Fig. 12). The conversion of L to L–Cu²⁺ (i.e. 1 : 1 L : Cu²⁺) (Scheme 2) was confirmed by ESI-TOF-MS and ¹H NMR spectra. The peak at m/z 779.20 in positive ESI mode and 778.32 in negative ESI mode corresponding to the mass of [L–Cu²⁺] (calcd m/z 779.07) was found after 1 equiv. of CuClO₄ was added to the solution of 1 equiv. ligand (Fig. SI 8†). The cation binding by ligand 3 was also detected by changes in the ¹H NMR spectrum. In the ¹H NMR spectrum, upon addition of variable amounts of Cu²⁺, the shifting of the ligand protons and the change of splitting of the ligand protons showed the formation of ligand-to-Cu²⁺ complex. Moreover, after the cation concentration has reached the ligand concentration, further addition of Cu²⁺ is ineffective. Overall, TLC (thin layer chromatography) also clearly shows that the ligand–Cu²⁺complex is formed by the R_f value of 0.0 in comparison to the R_f = 0.50 of free ligand in 1 : 20 MeOH/DCM (Fig. SI 9†). Thus, the ESI-TOF-MS, NMR and TLC results firmly support the inter-conversion of L to L–Cu²⁺ complex. The melting point of the ligand was also determined and was found to be 350 °C.

Scheme 2 Proposed mechanism of the sensoring system.

The details of the procedure and the spectral data of the compounds can be found in the ESI.†

Results and discussion

Solubility studies

Investigation of the selectivity of the synthesized cationic chemosensor 3 towards different metal ions necessitates choices of organic solvent, aqueous buffer and overall solvent composition. The solubility study of the cationic chemosensor (3) was performed with UV-vis spectroscopy at a concentration of 2.5 × 10⁻⁵ M in different organic solvents and aqueous media. The outcome revealed that the solubility in organic solvents with polarity indices ranging from 5.1 to 7.2 (DMF, DMSO, pyridine, acetone, acetonitrile) is noticeable, with the highest intensity broad peak at 350 nm. The synthesized ligand was non-fluorescent in aqueous solution, and its solubility (pH < ∼7.0) is not noteworthy. However, compound 3 is highly soluble in 1% DMSO in aqueous Tris–buffer at pH = 7.4, appearing fluorescent with an absorption peak near 491 nm. The solubility of the sensor in different organic solvents resulted in a color change visible to the naked eye, and this phenomenon is also supported by the UV-vis spectroscopy studies. In this regard, it should be mentioned that although the ligand was highly soluble in DMF and DMSO, with an absorption peak near 350 nm, the intrinsic coordination, oxidizing properties, and high boiling points of these solvents limit their use as experimental media. Thus, the comparably good solubility of ligand 3 in acetonitrile at 2.5 × 10⁻⁵ M concentration with an absorbance peak at 350 nm favors the detection of metal ions with the naked eye and the measurement of peak shifting by spectroscopic studies (Fig. 1).

pH studies

To inspect the pH effect and the solubility of 3 in aqueous solution, the absorbance of the chemosensor at a concentration of 2.5 × 10⁻⁵ M was investigated in the pH range of 2 to 10 in 1% DMSO–Tris buffer solution. It was found that ligand 3 shows no solubility in aqueous media at acidic and even at neutral pH. The absorbance of 3 in basic media ranging from 7.4 to 10.0 pH in Tris base at concentrations of 1 mmol and 10 mmol exhibits similar fluorescence activity, with the same absorbance maxima at 491 nm but with different intensities (Fig. SI 11†). The result in acidic or neutral pH is not significant, presumably due to the stability of the ring closure form of fluorescein, which is non-fluorescent. The changes in pH value from 7 to 7.4 and the addition of 1% DMSO in an aqueous Tris buffer solution, which is compatible with biological systems, show very good absorbance properties of the synthetic ligand 3 at a higher wavelength of ca. 141 nm in comparison to organic solvents due to the extended conjugation of the phenolate anion at pH 7.4 (Fig. 2).

Fig. 2 pK_a-Dependent structure of fluorescein.

UV-vis spectral studies of the metal–ligand complex

The metal-binding properties of ligand 3 in organic as well as in aqueous solution were examined. Colorimetric studies of metal–ligand interaction were performed in acetonitrile to determine the selectivity and sensitivity of the following metal cations (Fig. 3): Li⁺, Na⁺, K⁺, Mg²⁺, Ca²⁺, Ni²⁺, Cu²⁺, Zn²⁺, Ag⁺, Cd²⁺, Hg²⁺, Pb²⁺, Pr³⁺, Sm³⁺, and Gd³⁺. The study commenced with nitrate salts of lanthanide metals and perchlorate salts of the remaining metal cations with ligand 3 in a 10 : 1 stoichiometric ratio. In this scenario, all metal ions in acetonitrile exhibit an instant color change which can be detected by UV spectroscopy. When the ratio of metal and ligand 3 was changed to 2 : 1 (Fig. 3a), the selective detection of Cu²⁺ and Hg²⁺ cations was witnessed, with the formation of new bands at 480 nm and 440 nm, respectively. This result demonstrates that Cu²⁺ ion only gives a maximum quantifiable change with a red shift of 130 nm along with a change in colour from light pink to yellow. However, it is interesting to note that the absorbance intensity of Cu²⁺ and Hg²⁺ cation decreases, whereas the intensity of Ni²⁺ cation increases with time, and the solution becomes pink (Fig. 3b).

Fig. 3 (a) UV-vis spectra of a fresh solution of ligand 3 at 2.5 × 10⁻⁵ M in acetonitrile in the presence of metal ions at 5 × 10⁻⁵ M. (b) UV-vis spectra of the same solution of ligand 3 and Ni²⁺ after 6 h.

Studies of sensitivity of the ligand

To determine the limit of detection (LOD) for the Cu²⁺ cation in acetonitrile, different concentrations of Cu²⁺ ion (10 to 100 ppm) were titrated against ligand 3 at a concentration of 2.5 × 10⁻⁵ M and the solution was monitored by UV-vis spectroscopy. The LOD for the Cu²⁺ ion from the absorbance spectra was found to be 20 μM (Fig. 4). Surprisingly, after 1 h, the absorbances of the metal–ligand solutions began to change, which indicated that the complex formed during metal–ligand binding studies in acetonitrile is not stable in solution for a long time.

Fig. 4 Titration curve of Cu²⁺ against ligand 3 at a concentration of 2.5 × 10⁻⁵ M in acetonitrile.

After colorimetric study of ligand 3 in organic solvent, the lower detection limit of the synthesized ligand with Cu²⁺ ion as a fluorescent probe at physiological pH 7.4 was also determined through colorimetric methods. To execute this experiment, spectroscopic measurements of ligand 3 were investigated in 1% DMSO–Tris buffer at physiological pH (10 mM Tris buffer, pH 7.4) under ambient conditions. Addition of biologically and environmentally relevant alkali, alkaline earth, transition, lanthanide or heavy metal ions did not produce any discernible change in the absorption spectral profile of the receptor at lower concentrations of metal ions (∼10⁻⁵ M). This observation suggests that there is no interaction between the synthesized chemosensor and the added metal ions. However, in the presence of Cu²⁺ ion, prominent changes in the UV-vis spectra are observed. The metal-free receptor gives strong fluorescence emission (I = 672 420) upon excitation at 491 nm due to high affinity with chemosensor 3 through efficient photoinduced electron transfer (PET) from the fluorophore to the receptor at physiological pH (7.4). This emission profile does not change to any noticeable extent in terms of both intensity and peak positions in the presence of alkali, alkaline earth, transition, or lanthanide metal ions. Meanwhile, the addition of the perchlorate salt of Cu²⁺ elicits a significant diminution of the fluorescence intensity (∼2.5 fold for Cu²⁺ ion) with a wavelength of maximum absorption at 519 nm for Cu²⁺ (Fig. 5a). The turn-off fluorescence response was found to be reversible, as the addition of an aqueous solution of ligand to the L–Cu²⁺ solution changes the fluorescence output to the level of the metal-free receptor; this observation was also supported by the Job plot.

Fig. 5 (a) Fluorescence spectra of chemosensor 3 at 1.5 × 10⁻⁵ M in 1% DMSO–Tris buffer (10 mmol) at pH 7.4 in the presence of metal ions (1.5 × 10⁻⁴ M) at λ_ex = 491 nm and slit width 2 nm. (b) UV-vis absorption spectra of 3 at 1.5 × 10⁻⁵ M in the presence of metal ions (1.5 × 10⁻⁴ M) in 1% DMSO–Tris buffer at pH 7.4.

When the ligand concentration is 15 μM and the metal ion concentration is 10 times greater, a change is observed in the absorbance peak in the UV spectra from 490 to 496 nm with an intense absorbance peak, whereas ligand 3 shows an absorption peak at 496 nm (Fig. SI 12†). This small change in absorbance makes it difficult to determine the selectivity of the metal cations (Fig. 5b). The association of the receptor with a complementary analyte can suppress either an electron or an energy transfer process and switch on the fluorescence of the adjacent fluorophore.

When the experiment was performed using a fluorescence spectrophotometer, and the metal ion concentration was 10 times that of the ligand (3), excellent selectivity was observed towards Cu²⁺ ion; the fluorescence was quenched compared to all other metal cations and a change in quenching intensity was observed from 6.7 × 10⁵ to 2.9 × 10⁵. However, a significant fluorescence change was not observed by fluorescence spectroscopy for any of the other metal cations. This phenomenon unambiguously indicates that the binding interaction between ligand 3 and Cu²⁺ is stronger than the interaction between ligand 3 and other quenching metal ions (Fig. 6).

Fig. 6 Fluorescence intensity of ligand 3 at 1.5 × 10⁻⁵ M in 1% DMSO–Tris buffer (pH 7.4) in the presence of various metal ions (1.5 × 10⁻⁴ M) at λ_max = 519 nm.

Studies of fluorometric titration of Cu²⁺ against ligand

For the limit of detection (LOD) at pH 7.4, the titration curve of Cu²⁺ was determined by considering concentrations of metal ions from 0.2 to 100 equivalents with respect to ligand 3 in 1% DMSO–Tris buffer (Fig. 7). Surprisingly, the detection limit was found to be 1.25 μM in aqueous solution (Fig. 8) by fluorescence spectroscopy, while according to UV spectroscopy, the detection limit of Cu²⁺ cation was found to be 20.0 μM in acetonitrile (organic solvent). Therefore, the detection limit via fluorescence spectroscopy provides sixteen-fold increase in an aqueous medium compared to UV spectroscopy in organic solvent.

Fig. 7 Fluorometric titration of Cu²⁺ against ligand concentration at 5.0 μM in 1% DMSO–Tris base at pH 7.4.

Fig. 8 LOD curve for Cu²⁺ against ligand concentration at 5.0 μM in 1% DMSO–Tris base at pH 7.4.

Sensing mechanism studies

The addition of acid maintains the lactone form of the chemosensor, activating an energy transfer pathway and effectively quenching the fluorescence of the receptor.¹⁹ The addition of base converts the chemosensor into the carboxylic acid form (Fig. 9), preventing the electron transfer process and switching on the fluorescence of the receptor.

Fig. 9 Molecular interactions.

The mechanism of Cu²⁺-mediated fluorescence quenching in chemosensors

Non-radiative deactivation of photoexcited fluorophore 3 by the interaction of Cu²⁺ ion can be achieved either through “energy transfer” or “electron transfer”.²⁰ This is because transition metal ions such as Cu(II) possess empty or half-filled orbitals which can be involved in a Dexter type energy-transfer mechanism, as per the presentation in Fig. 10A. On the other hand, the coordination of the N-atom of Schiff base 3 favors access to the Cu(III) state²¹ in the Dexter type electron-transfer mechanism. Thus, an electron can be transferred from the complexed Cu(II) centre to the photoexcited fluorophore moiety F* of 3, as depicted in Fig. 10B.

Fig. 10 Quenching of the excited fluorescein-based Schiff base in the presence of Cu(II) ion: (A) Dexter type energy transfer mechanism; (B) Dexter type electron transfer mechanism.

Determination of Stern–Volmer and association constants

This unambiguous result is also corroborated by the changes in the binding constant of the fluorogenic response of ligand 3 in the presence of varying concentrations of Cu²⁺, with a Stern–Volmer constant of K_sv = 1.69 × 10⁴ M⁻¹ (Fig. 11) and an association constant of K_ass = 2.23 × 10⁴ M⁻¹ (Fig. 12); this indicates the affinity of Cu²⁺ with the ligand via a static quenching pathway.

Fig. 11 Stern–Volmer plot for ligand 3 as a function of the concentration of Cu²⁺ cation.

Fig. 12 Determination of binding constant (association constant) to find the binding affinity of ligand 3 for Cu²⁺ in 1% DMSO–Tris buffer at pH 7.4. I₀ is the fluorescence intensity of L, I is the fluorescence intensity of L + [Cu²⁺] and I_α is the minimum fluorescence intensity of L + [Cu²⁺].

Studies of Job plots

The observed peak abscissa values of 0.50 in Fig. 13 indicate a 1 : 1 stoichiometry of binding (ligand 3/Cu²⁺) for ligand 3 (theoretical value is 0.50). Hence, from the above study, the proposed mechanism for the detection of Cu²⁺ is revealed, as shown in Scheme 2.

Fig. 13 Job plot of Cu²⁺ complex formation with 3 at 5 μM concentrations of each in 1% DMSO–Tris buffer at pH 7.4. X = [3]/[3] + [Cu²⁺] is the mole fraction of 3, I₀ is the fluorescence intensity when X = 1 and I is the fluorescence intensity at the respective values of X. The samples contained 5 μM concentrations of 3 and Cu²⁺ at 519 nm.

Conclusion

In summary, we have synthesized a novel fluorescein-based chemosensor (3) for selective binding to Cu²⁺ metal cation in acetonitrile as the organic solvent and in water (1% DMSO–Tris buffer solution) at pH 7.4. According to UV spectroscopy, the detection limit of Cu²⁺ was found to be 20 μM in acetonitrile, whereas the detection limit in 1% DMSO–Tris buffer at pH 7.4 was reduced to 1.25 μM in fluorescence spectroscopy. In this buffer solution at pH 7.4, the receptor shows fluorescence quenching. This sensing ability at physiological pH in aqueous media may be useful for the detection of Cu²⁺ in vivo, as copper is a widely used industrial metal that is toxic at high concentrations and is involved in brain diseases such as Alzheimer's and Parkinson's disease. Even though some colorimetric/fluorescent chemosensors have been developed for the detection of Cu²⁺ to date, sensing methods for fast detection of Cu²⁺ in aqueous solution, especially using colorimetric sensors without resorting to instruments, are relatively rare. This fluorescent dye works in aqueous medium at physiological pH and, hence, it can be used in live cell imaging studies with a detection limit in the ppm region for Cu²⁺ ion in cases of poisoning. The resulting Cu(II) complex is further being explored as a probe for other substances, and this investigation will be published in due course.

Conflict of interest

The authors declare no competing financial interest.

Acknowledgements

The authors are greatly thankful to UGC for financial support and Central University of Gujarat, Gandhinagar for instrumental and infrastructural facilities. Reena Rathod also acknowledged Dr Parimal Paul, Chief Scientist and Madhuri Bhatt, Research Scholar, CSIR-Central Salt and Marine Chemicals Research Institute, India for fluorescent spectroscopic data.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: Experimental procedures, spectroscopic data and different calculated data and graph (PDF). See DOI: 10.1039/c6ra03021a

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