Dual Sensing Performance of 1,2-Squaraine for the Colorimetric Detection of Fe3+ and Hg2+ Ions

A simple 1,2-squaraine based chemosensor material (SQ) has been reported to show dual sensing performance for colorimetric detection of Fe3+ and Hg2+ ions. Compared to common instrumental analysis, this method could provide fast and direct detection though colorimetric changes by the naked eye. The sensor has shown excellent selectivity over the other metal ions by tuning different solvent environments. The detection limit for Fe3+ could reach to 0.538 μM, which was lower than that in the environmental agency guideline (U.S. Environmental Protection Agency, U.S. EPA) in drinking water. And for Hg2+ detection, the limit was calculated as 1.689 μM in our case. A 1:1 binding mode between SQ–Fe3+ and SQ–Hg2+ ion were evidenced by Job’s plot measurement and IR analysis. The proposed different binding mechanisms were also supported by Density Function Theory (DFT) calculation. All these findings provide a unique material and a simple, facile, and low cost colorimetric method for dual metal ions analysis and have shown preliminary analytical applications in industrial water sample analysis.


Introduction
Development of selective and sensitive chemosensor materials for metal cation ions has attracted considerable attention owing to their key role and potential applications in chemistry, materials, and the environment [1][2][3][4][5][6][7][8][9][10][11]. Among heavy metals, iron is one of the essential elements playing an important function in a wide range of biological process, such as cellular metabolism, oxygen carrying, and regulation of enzyme reactions [12][13][14][15][16]. Meanwhile, mercury is considered a toxic element that has harmful effects on the human health and the environment. Accumulation of mercury in the body will cause severe health problem, especially damage of the central nervous system [17][18][19][20]. Therefore, a convenient and rapid method for the analysis of Fe 3+ and Hg 2+ is highly in demand. Current analytical techniques such as atomic absorption spectroscopy, inductively coupled plasma mass spectroscopy, and electrochemical analysis require sophisticated instruments and complex sample preparation procedures, which limit their wide applications [21][22][23][24][25]. It is imperative to develop simple material and effective methods for the detection of these ions. The use of a chemosensor material provides unique advantages in a view of sensitivity and response time. One strategy for the design of such molecules involves introducing an appropriate binding ligand to the chromophore in which The synthesis of sensor SQ was followed as reported literature [48] and the characterization for the compound was summarized in Figures S1-S3. The product was distinct from common squaraine as two carbonyl groups (C=O) on squaric core were adjacent to form 1,2-regioisomer instead of 1,3-regioisomer. It has two cross-conjugated-electron systems from hetero aromatic donor to the squaric core as donor acceptor. There are potential binding sites including two carbonyl groups (C=O) on squaric core and sulfur atom on the substituted 3-butyl-2-methylbenzo[d]thiazol-3-ium iodide which could provide extra electron pairs to the metal ions. There are many literatures that reported 1,3-regioisomer can be applied in the recognition of several metal ions [49][50][51][52]. We believe that besides 1,3-regioisomer, 1,2-regioisomer may also function as a metal ion detector in some circumstances.
An important characteristic of squaraines is their tendency to form different patterns of aggregations, resulting in a dramatic color modulation [53,54]. We carried out the dilution experiments in some selected solvents (pure acetic acid and 4 mM SDS solution) by UV-Vis measurements to see if there were aggregations for SQ and the results were shown in supporting information ( Figure S4a,b). It has shown that the absorption intensities decreased both in pure acetic acid and 4 mM SDS solution with the decrement of the SQ concentrations (10 −4 -10 −6 M). The linear range of concentration of SQ in two different solvents was 1 µM to 10 µM by using the Beer-Lambert law. No absorption wavelength of SQ was shifted which suggest SQ was sensitive to the solvent environment but no aggregation can be concluded from the current data. The molar absorption coefficient of SQ in the linear range was not changed. A series of other solvents was applied in the UV-Vis measurements. Two microliters was extracted from 10 mM SQ stock solution and diluted with 198 µL corresponding solvent to get the total volume of 200 µL. The final concentration of SQ was 100 µM (100 times dilution). The absorption data for SQ in different solutions were summarized in Figure 1. SQ exhibited a maximum absorption wavelength at 475 nm in pure distilled water. While in boric acid buffer (10 mM, pH = 6.8) and phosphate buffer (10 mM, pH = 6.6), it gave a broad flat band in absorption range of 400 nm to 600 nm. Interestingly, it was found that in pure acetic acid, SQ showed two maximum absorption

Colorimetric Sensing for Fe 3+ and Hg 2+
To evaluate the sensing properties of SQ (final concentration: 100 μM) towards metal ions, the UV-Vis spectral changes were investigated with addition of various metal ions including Na + , K + , Li + , Ag + , Zn 2+ , Hg 2+ , Cd 2+ , Fe 2+ , Co 2+ , Ca 2+ , Fe 3+ (final concentration: 100 μM) in pure acetic acid. As shown in Figure 2, there were no significant spectral changes in the presence of most metal ions, whereas Fe 3+ caused the distinct spectral changes with absorption intensity decreased in 525 nm. And Fe 3+ pronounced color changes from orange to cream yellow by direct visualization. The water effect on detection of Fe 3+ for SQ in AcOH-H2O solutions was further evaluated (Figure 3). There were gradually decreased absorption bands at 525 nm of SQ with increasing portion of water in acetic acid. The pH effect on the selectivity of SQ for Fe 3+ has been observed as well. With increasing amount of AcOH in solution, the pH decreased gradually but the absorption intensity of SQ itself dramatically enhanced. In addition, only a distinct difference in absorption bands was observed in pure acetic acid for SQ with or without addition of Fe 3+ . We have also monitored the absorption changes of SQ in pure acetic acid and after 24 h, there were no significant changes in view of UV-Vis spectral ( Figure S5a). In addition, 1 HMR spectrum of SQ in CD2Cl2 after 24 h ( Figure S5b) shown with no decomposition preliminarily revealed its stability in this condition. All these results indicated that SQ can be a good probe for detecting Fe 3+ .
Another interesting phenomenon was discovered in the detection of Hg 2+ in SDS surfactant solutions. As shown in Figure 4, when various metal ions were added into SQ solutions in presence of 4 mM SDS, the single absorption band of SQ at 455 nm (maximum absorption wavelength without metal ions) was red shift to 535 nm except for Hg 2+ . Upon addition of Hg 2+ , the absorption band at 535 nm decreased gradually, while the absorption in the range of 650 nm to 750 nm increased with one clear isosbestic point, which indicating the binding between SQ and Hg 2+ afforded only one species. This new peak might be ascribed to a metal-to-ligand charge transfer [55,56].
To evaluate the SDS effect on the Hg 2+ detection, wide concentrations (1 μM-4 mM) of SDS solutions were adopted in Figure 5a. It was shown that when SDS concentration reached at 4 mM which was higher than critical micelle concentration (CMC) of SDS, the maximum absorption wavelength of SQ without metal ions exhibited a blue shift to 455 nm. It was found that the relative changes of SQ absorption intensity in 4 mM SDS micellar solution at 535 nm discriminate the most compared to that in other concentrations in the case of Hg 2+ detection (Figure 5b). The solution of SQ in 4 mM SDS displayed an orange color, and the addition of Hg 2+ caused an instant vivid color change

Colorimetric Sensing for Fe 3+ and Hg 2+
To evaluate the sensing properties of SQ (final concentration: 100 µM) towards metal ions, the UV-Vis spectral changes were investigated with addition of various metal ions including Na + , K + , Li + , Ag + , Zn 2+ , Hg 2+ , Cd 2+ , Fe 2+ , Co 2+ , Ca 2+ , Fe 3+ (final concentration: 100 µM) in pure acetic acid. As shown in Figure 2, there were no significant spectral changes in the presence of most metal ions, whereas Fe 3+ caused the distinct spectral changes with absorption intensity decreased in 525 nm. And Fe 3+ pronounced color changes from orange to cream yellow by direct visualization. The water effect on detection of Fe 3+ for SQ in AcOH-H 2 O solutions was further evaluated ( Figure 3). There were gradually decreased absorption bands at 525 nm of SQ with increasing portion of water in acetic acid. The pH effect on the selectivity of SQ for Fe 3+ has been observed as well. With increasing amount of AcOH in solution, the pH decreased gradually but the absorption intensity of SQ itself dramatically enhanced. In addition, only a distinct difference in absorption bands was observed in pure acetic acid for SQ with or without addition of Fe 3+ . We have also monitored the absorption changes of SQ in pure acetic acid and after 24 h, there were no significant changes in view of UV-Vis spectral ( Figure S5a). In addition, 1 HMR spectrum of SQ in CD 2 Cl 2 after 24 h ( Figure S5b) shown with no decomposition preliminarily revealed its stability in this condition. All these results indicated that SQ can be a good probe for detecting Fe 3+ .
Another interesting phenomenon was discovered in the detection of Hg 2+ in SDS surfactant solutions. As shown in Figure 4, when various metal ions were added into SQ solutions in presence of 4 mM SDS, the single absorption band of SQ at 455 nm (maximum absorption wavelength without metal ions) was red shift to 535 nm except for Hg 2+ . Upon addition of Hg 2+ , the absorption band at 535 nm decreased gradually, while the absorption in the range of 650 nm to 750 nm increased with one clear isosbestic point, which indicating the binding between SQ and Hg 2+ afforded only one species. This new peak might be ascribed to a metal-to-ligand charge transfer [55,56]. from orange to grain yellow. The difference in color response allowed SQ to easily distinguish between Fe 3+ and Hg 2+ in aqueous solution.    from orange to grain yellow. The difference in color response allowed SQ to easily distinguish between Fe 3+ and Hg 2+ in aqueous solution.        To evaluate the SDS effect on the Hg 2+ detection, wide concentrations (1 µM-4 mM) of SDS solutions were adopted in Figure 5a. It was shown that when SDS concentration reached at 4 mM which was higher than critical micelle concentration (CMC) of SDS, the maximum absorption wavelength of SQ without metal ions exhibited a blue shift to 455 nm. It was found that the relative changes of SQ absorption intensity in 4 mM SDS micellar solution at 535 nm discriminate the most compared to that in other concentrations in the case of Hg 2+ detection (Figure 5b). The solution of SQ in 4 mM SDS displayed an orange color, and the addition of Hg 2+ caused an instant vivid color change from orange to grain yellow. The difference in color response allowed SQ to easily distinguish between Fe 3+ and Hg 2+ in aqueous solution. To validate the selectivity for Fe 3+ and Hg 2+ , respectively, the competition experiments have also been performed. Other different ions as interferences were added to the mixture of SQ-Fe 3+ or SQ-Hg 2+ solution. The final concentration of other cations (100 μM) was 5 times more than that of Fe 3+ (20 μM) and Hg 2+ (20 μM) in the SQ solution. As shown in Figure 6a,b, there were no significant influences on Fe 3+ and Hg 2+ detections even extra amounts of other ions were added into the SQ-Fe 3+ and SQ-Hg 2+ system. All these findings suggest SQ exhibit high selectivity for Fe 3+ and Hg 2+ compared to other metal ions and it could be used as a colorimetric chemosensor for dual analytes analysis.
For the purpose of exploring the relationship between absorption intensity and response time, a dynamic study of SQ in the detection of Fe 3+ and Hg 2+ was carried out. After direct addition of Fe 3+ , the cream yellow color of SQ-Fe 3+ was faded instantly and to total colorless after 10 h and for SQ-Hg 2+ , it took a longer time to get a total colorless solution ( Figure S6a,b).
The reversibility and regeneration are essential for materials in practical applications. The reversibility of the recognition process of sensor SQ was performed by a reversible binding experiment. Ethylenediaminetetraacetic acid (EDTA) as a strong chelator was added into both SQ-Fe 3+ and SQ-Hg 2+ complex systems. Addition of 1 equivalent EDTA to SQ-Fe 3+ system resulted in an increasing of absorption signal at 525 nm, indicating the regeneration of free SQ ( Figure S7). With the  To validate the selectivity for Fe 3+ and Hg 2+ , respectively, the competition experiments have also been performed. Other different ions as interferences were added to the mixture of SQ-Fe 3+ or SQ-Hg 2+ solution. The final concentration of other cations (100 µM) was 5 times more than that of Fe 3+ (20 µM) and Hg 2+ (20 µM) in the SQ solution. As shown in Figure 6a,b, there were no significant influences on Fe 3+ and Hg 2+ detections even extra amounts of other ions were added into the SQ-Fe 3+ and SQ-Hg 2+ system. All these findings suggest SQ exhibit high selectivity for Fe 3+ and Hg 2+ compared to other metal ions and it could be used as a colorimetric chemosensor for dual analytes analysis.

Binding Constant (Ka) and Limit of Detection (LOD) for Fe 3+ and Hg 2+
To get a further insight into the colorimetric sensing properties of SQ, a quantitative investigation of the binding affinity of sensor SQ with Fe 3+ and Hg 2+ was studied by titration. The UV-Vis absorption spectra of 100 μM SQ in acetic acid and SDS solution were recorded during the titration of various concentrations of Fe 3+ (1 nM-100 μM) and Hg 2+ (1 nM-100 μM), respectively. The binding constant (Ka) was estimated using a Benesi-Hilderbrand plot, which was calculated by absorption changes of consequent titration (A0/A0 − A) against 1/[M]. The magnitude of Ka was calculated from the intercept and slope of the straight line. The estimated value was about 1.24 × 10 6 M −1 for SQ-Fe 3+ complex and 1.1 × 10 6 M −1 for SQ-Hg 2+ (Figures 7a and 8a).
From the absorption titration, the detection limit for Fe 3+ and Hg 2+ on the basis of 3σ/k was therefore calculated and determined to be 0.538 μM (lower than the U. S. Environmental Protection Agency guideline for drinking water, 5.37 μM) and 1.689 μM (Figures 7b and 8b) for Hg 2+ which were superior than many recent reported Fe 3+ and Hg 2+ related sensors ( Table 1). The limit of detection for Hg 2+ can be further improved by modifying the structure through introducing more functional groups to increase the solubility of SQ and hydrogen interaction to the metal ions. For the purpose of exploring the relationship between absorption intensity and response time, a dynamic study of SQ in the detection of Fe 3+ and Hg 2+ was carried out. After direct addition of Fe 3+ , the cream yellow color of SQ-Fe 3+ was faded instantly and to total colorless after 10 h and for SQ-Hg 2+ , it took a longer time to get a total colorless solution ( Figure S6a,b).
The reversibility and regeneration are essential for materials in practical applications. The reversibility of the recognition process of sensor SQ was performed by a reversible binding experiment. Ethylenediaminetetraacetic acid (EDTA) as a strong chelator was added into both SQ-Fe 3+ and SQ-Hg 2+ complex systems. Addition of 1 equivalent EDTA to SQ-Fe 3+ system resulted in an increasing of absorption signal at 525 nm, indicating the regeneration of free SQ ( Figure S7). With the alternate addition of constant concentrations of Fe 3+ to SQ solution, the instant color change was observed again which exhibited good stability with a little signal decay for several cycles ( Figure S8). For Hg 2+ , while after addition of 1 equivalent EDTA to SQ-Hg 2+ system, a distinct absorption band with observed and the alternate addition of constant concentrations of Hg 2+ has shown no dominant absorption changes (Figures S9 and S10). These clear findings of colorimetric ON/OFF behavior of the sensing system suggested that SQ serve as a good reversible sensor for Fe 3+ detection.

Binding Constant (Ka) and Limit of Detection (LOD) for Fe 3+ and Hg 2+
To get a further insight into the colorimetric sensing properties of SQ, a quantitative investigation of the binding affinity of sensor SQ with Fe 3+ and Hg 2+ was studied by titration. The UV-Vis absorption spectra of 100 µM SQ in acetic acid and SDS solution were recorded during the titration of various concentrations of Fe 3+ (1 nM-100 µM) and Hg 2+ (1 nM-100 µM), respectively. The binding constant (Ka) was estimated using a Benesi-Hilderbrand plot, which was calculated by absorption changes of consequent titration (A 0 /A 0 − A) against 1/[M]. The magnitude of Ka was calculated from the intercept and slope of the straight line. The estimated value was about 1.24 × 10 6 M −1 for SQ-Fe 3+ complex and 1.1 × 10 6 M −1 for SQ-Hg 2+ (Figures 7a and 8a). From the absorption titration, the detection limit for Fe 3+ and Hg 2+ on the basis of 3σ/k was therefore calculated and determined to be 0.538 µM (lower than the U. S. Environmental Protection Agency guideline for drinking water, 5.37 µM) and 1.689 µM (Figures 7b and 8b) for Hg 2+ which were superior than many recent reported Fe 3+ and Hg 2+ related sensors ( Table 1). The limit of detection for Hg 2+ can be further improved by modifying the structure through introducing more functional groups to increase the solubility of SQ and hydrogen interaction to the metal ions.

Complexation Mechanism of SQ-Fe 3+ and SQ-Hg 2+
Job's plot measurement was carried out to determine the complexation mode between SQ and Fe 3+ . A maximum value of the absorption intensity at 525 nm was observed when the mole fraction of Fe 3+ reached 0.5. A signature of 1:1 stoichiometry between SQ and Fe 3+ was determined. In addition, a 1:1 stoichiometry between SQ and Hg 2+ was also determined following the same process. (Figure 9a,b).
The Electron Spray Ionization-Mass data of SQ-Fe 3+ and SQ-Hg 2+ complexes were included in Figures S11 and S12. The involvement of binding sites of SQ in complexation was further confirmed through the IR analysis in the presence and absence of metal ions ( Figure 10). The characteristic carbonyl stretching frequencies of SQ appeared at 1727.44 cm −1 and 1677.48 cm −1 . However, in the SQ-Fe 3+ complex, a new peak at 1617.34 cm −1 was observed instead of those at 1766 cm −1 and 1602 cm −1 . At the same time, another peak appeared at 1584.41 cm −1 instead of 1492.07 cm −1 , indicating the SQ binding with Fe 3+ occurs at the two carbonyl groups of squarely moiety in SQ. The IR spectrum for SQ-Hg 2+ complex was different to that of SQ-Fe 3+ complex. The characteristic carbonyl stretching peak in SQ-Hg 2+ complex at 1766 cm −1 and 1602 cm −1 were replaced by a strong new peak at 1613.52 cm −1 . More importantly, two more new peaks at 3585.58 cm −1 and 3525.71 cm −1 shown up ascribed to proton vibration. This indicated that more electron pairs were involved in the binding with presence of Hg 2+ . Conceptually, in accordance with hard soft acid base (HSAB) principle [64], the complexations of SQ-Fe 3+ and SQ-Hg 2+ were undergone two different pathways, which have been proposed in Scheme 1. For SQ-Fe 3+ complex, Fe 3+ was proposed to bind two carbonyl groups in squaric moiety with 1:1 stoichiometry, while for SQ-Hg 2+ complex, it carried out in a different way. The involvement of binding sites of SQ in complexation was further confirmed through the IR analysis in the presence and absence of metal ions ( Figure 10). The characteristic carbonyl stretching frequencies of SQ appeared at 1727.44 cm −1 and 1677.48 cm −1 . However, in the SQ-Fe 3+ complex, a new peak at 1617.34 cm −1 was observed instead of those at 1766 cm −1 and 1602 cm −1 . At the same time, another peak appeared at 1584.41 cm −1 instead of 1492.07 cm −1 , indicating the SQ binding with Fe 3+ occurs at the two carbonyl groups of squarely moiety in SQ. The IR spectrum for SQ-Hg 2+ complex was different to that of SQ-Fe 3+ complex. The characteristic carbonyl stretching peak in SQ-Hg 2+ complex at 1766 cm −1 and 1602 cm −1 were replaced by a strong new peak at 1613.52 cm −1 . More importantly, two more new peaks at 3585.58 cm −1 and 3525.71 cm −1 shown up ascribed to proton vibration. This indicated that more electron pairs were involved in the binding with presence of Hg 2+ . Conceptually, in accordance with hard soft acid base (HSAB) principle [64], the complexations of SQ-Fe 3+ and SQ-Hg 2+ were undergone two different pathways, which have been proposed in Scheme 1. For SQ-Fe 3+ complex, Fe 3+ was proposed to bind two carbonyl groups in squaric moiety with 1:1 stoichiometry, while for SQ-Hg 2+ complex, it carried out in a different way. Theoretical calculations have been explored as well to understand the nature of the binding of SQ-Fe 3+ and SQ-Hg 2+ complexes. All calculations were carried out with the Gaussian 09 package [65]. The density functional theory (DFT) hybrid model with the B3LYP was used for the gas-phase geometry optimization, Lanl2dz basis set with effective core potential (ECP) for Fe and Hg, and the 6-31G(d) basis set was used for all remaining atoms. Based on the calculations, the favorable binding modes between Fe 3+ and its Hg 2+ complexes were depicted in Figure 11a-c. According to the calculations, the geometry optimized structure of SQ-Fe 3+ complex (Figure 11b) was illustrated the same as our proposed. It showed lower energy when Fe 3+ was bind two carbonyl groups in squarely moiety. However, the simulated spectra are in good agreement with the proposed complexation mechanism for only SQ-Fe 3+ complexes. For SQ-Hg 2+ complex, the Hg atom was far away from the SQ. Theoretical calculations have been explored as well to understand the nature of the binding of SQ-Fe 3+ and SQ-Hg 2+ complexes. All calculations were carried out with the Gaussian 09 package [65]. The density functional theory (DFT) hybrid model with the B3LYP was used for the gas-phase geometry optimization, Lanl2dz basis set with effective core potential (ECP) for Fe and Hg, and the 6-31G(d) basis set was used for all remaining atoms. Based on the calculations, the favorable binding modes between Fe 3+ and its Hg 2+ complexes were depicted in Figure 11a-c. According to the calculations, the geometry optimized structure of SQ-Fe 3+ complex (Figure 11b) was illustrated the same as our proposed. It showed lower energy when Fe 3+ was bind two carbonyl groups in squarely moiety. However, the simulated spectra are in good agreement with the proposed complexation mechanism for only SQ-Fe 3+ complexes. For SQ-Hg 2+ complex, the Hg atom was far away from the SQ. Theoretical calculations have been explored as well to understand the nature of the binding of SQ-Fe 3+ and SQ-Hg 2+ complexes. All calculations were carried out with the Gaussian 09 package [65]. The density functional theory (DFT) hybrid model with the B3LYP was used for the gas-phase geometry optimization, Lanl2dz basis set with effective core potential (ECP) for Fe and Hg, and the 6-31G(d) basis set was used for all remaining atoms. Based on the calculations, the favorable binding modes between Fe 3+ and its Hg 2+ complexes were depicted in Figure 11a-c. According to the calculations, the geometry optimized structure of SQ-Fe 3+ complex (Figure 11b) was illustrated the same as our proposed. It showed lower energy when Fe 3+ was bind two carbonyl groups in squarely moiety. However, the simulated spectra are in good agreement with the proposed complexation mechanism for only SQ-Fe 3+ complexes. For SQ-Hg 2+ complex, the Hg atom was far away from the SQ. (c) Figure 11. The geometry-optimized structures of (a) SQ (yellow, red and blue atoms representing as sulfur, oxygen, nitrogen atoms, respectively); (b) SQ-Fe 3+ complex (purple atom representing as Fe 3+ ); (c) SQ-Hg 2+ complex (individual atom at left representing as Hg 2+ atom) at the SMD (H2O)-TD-PBE0/TZVP+LANL2DZ level.

Preliminary Analytical Application
The SQ has been validated for practical applications in the determination of Fe 3+ and Hg 2+ in industry waste water (Table 2). Control experiments showed no significant effect on the colorimetric change of the sensor, the samples were used for the spike and recovery test after treating with acetic acid and 4 mM SDS solution, respectively. It was revealed that SQ has shown a good recovery at different concentrations ( Figure S13a,b). These results preliminarily demonstrated that SQ could be potential to be used in selectively and sensitively determine Fe 3+ and Hg 2+ in real water samples.

Conclusions
In conclusion, a simple 1,2-squaraine SQ has been developed to performance dual colorimetric sensing for Fe 3+ and Hg 2+ ions. An instant color change for selective Fe 3+ detection in pure acetic acid was observed with a detection limit of 0.538 μM, while Hg 2+ can be detected selectively in 4 mM SDS solution by instant colorimetric response with a detection limit of 1.689 μM. The Job's plot supported the 1:1 biding mode for both SQ-Fe 3+ and SQ-Hg 2+ complexes. IR analysis and DFT calculations demonstrated the SQ-Fe 3+ and SQ-Hg 2+ complexes undergo a different complex mechanism. Our Figure 11. The geometry-optimized structures of (a) SQ (yellow, red and blue atoms representing as sulfur, oxygen, nitrogen atoms, respectively); (b) SQ-Fe 3+ complex (purple atom representing as Fe 3+ ); (c) SQ-Hg 2+ complex (individual atom at left representing as Hg 2+ atom) at the SMD (H 2 O)-TD-PBE0/TZVP+LANL2DZ level.

Preliminary Analytical Application
The SQ has been validated for practical applications in the determination of Fe 3+ and Hg 2+ in industry waste water (Table 2). Control experiments showed no significant effect on the colorimetric change of the sensor, the samples were used for the spike and recovery test after treating with acetic acid and 4 mM SDS solution, respectively. It was revealed that SQ has shown a good recovery at different concentrations ( Figure S13a,b). These results preliminarily demonstrated that SQ could be potential to be used in selectively and sensitively determine Fe 3+ and Hg 2+ in real water samples.

Conclusions
In conclusion, a simple 1,2-squaraine SQ has been developed to performance dual colorimetric sensing for Fe 3+ and Hg 2+ ions. An instant color change for selective Fe 3+ detection in pure acetic acid was observed with a detection limit of 0.538 µM, while Hg 2+ can be detected selectively in 4 mM SDS solution by instant colorimetric response with a detection limit of 1.689 µM. The Job's plot supported the 1:1 biding mode for both SQ-Fe 3+ and SQ-Hg 2+ complexes. IR analysis and DFT calculations demonstrated the SQ-Fe 3+ and SQ-Hg 2+ complexes undergo a different complex mechanism. Our findings provide a simple material and a facile, low cost colorimetric method for dual analytes analysis and have shown preliminary analytical applications in industrial water samples.
Supplementary Materials: The following are available online at http://www.mdpi.com/1996-1944/11/10/1998/s1; Figure S1: 1 H NMR spectrum for compound SQ; Figure S2: 13 C NMR spectrum for compound SQ; Figure S3: high resolution mass spectrum for SQ; Figure S4: (a) absorption for different concentrations of SQ in pure acetic acid and (b) absorption for different concentrations of SQ in 4 mM SDS solution; Figure S5: (a) absorption of SQ in pure acetic acid at different time points; (b) 1 HMR spectra of SQ in CD 2 Cl 2 after 24 hours; Figure S6: (a) dynamic study on the absorption change of SQ-Fe 3+ and (b) dynamic study on the absorption change of SQ-Hg 2+ , Figure S7: reversible study of SQ-Fe 3+ toward addition of EDTA; Figure S8: reversible study of SQ-Hg 2+ upon alternate addition of Fe 3+ ; Figure S9: reversible study of SQ-Hg 2+ complex toward addition of EDTA; Figure S10: reversible absorption changes of SQ upon alternate addition of Hg 2+ and EDTA; Figure S11: high resolution mass spectrum for SQ-Fe 3+ , Figure S12: high resolution mass spectrum for SQ-Hg 2+ ; Figure S13: (a) titration curve of SQ for Fe 3+ (1-6 mM) and (b) titration curve of SQ for Hg 2+ (1-6 mM).