Synthesis and Characterization of Newly Designed and Highly Solvatochromic Double Squaraine Dye for Sensitive and Selective Recognition towards Cu2+

Synthesis and characterization of a novel and zwitterionic double squaraine dye (DSQ) with a unique D-A-A-D structure is being reported. Contrary to the conventional mono and bis-squaraine dyes with D-A-D and D-A-D-A molecular frameworks reported so far, DSQ dye demonstrated strong solvatochromism allowing for the multiple ion sensing using a single probe by judicious selection of the suitable solvent system. The DSQ dye exhibited a large solvatochromic shift of about 200 nm with color changes from the visible to NIR region with metal ion sensitivity. Utilization of a binary solvent consisted of dimethylformamide and acetonitrile (1:99, v/v), highly selective detection of Cu2+ ions with the linearity range from 50 μM to 1 nM and a detection limit of 6.5 × 10−10 M has been successfully demonstrated. Results of the Benesi–Hildebrand and Jobs plot analysis revealed that DSQ and Cu2+ ions interact in the 2:1 molecular stoichiometry with appreciably good association constant of 2.32 × 104 M−1. Considering the allowed limit of Cu2+ ions intake by human body as recommended by WHO to be 30 μM, the proposed dye can be conveniently used for the simple and naked eye colorimetric monitoring of the drinking water quality.


Introduction
With the progress of industrial civilization, the water in the environment and the soil are polluted by numerous heavy metals in many ways. This threatens the existence of other species living in these bodies. Currently, the environmental contamination caused by the presence of the toxic metal ions is rising necessitating a significant need for the metal ion sensing. Furthermore, the human body has a large number of metal ions, and their imbalances result in altered body function, leading to several physiological disorders [1]. The quest for the development of novel functional dyes for targeted applications is a neverending journey of the curious human mind. Especially, the bright tunable colors, synthetic diversity of organic dyes, sensitivity in the near-infrared (NIR) wavelength region, and imparting of the targeted functionality by judicious molecular design has opened the door for applications, particularly in the area of optoelectronics such as optical data storage, imaging, and optical communication [2][3][4][5][6][7]. Amongst functional organic dyes, squaraine dyes, typically bearing a donor(D)-acceptor(A)-donor(D) zwitterionic framework, are one of the most interesting classes of organic dyes owing to their exceptional light absorption [8,9]. The journey of the squaraine dyes started with the report on the synthesis of squaraine dye by condensation of electron-deficient squaric acid with electron-donating pyrrole units by Triebs and Jacob [10]. This geared the momentum of research for the development of such a class of intensely colored dyes utilizing vastly available electron-donating aromatic rings and squaric acid [11][12][13][14]. This quest for the design and development of novel squaraine dyes started about six decades ago, which is expected to be continued in future owing

Synthesis of the Double-Squaraine (DSQ) Dye (1)
The DSQ dye (1) was successfully synthesized in four steps as shown in Scheme 1.
The key precursor (5) was synthesized in three steps from the commercially available starting compound 1,1,2-trimethyl-1H-benz[e]indole and diethyl squarate. The reaction intermediate (3) was conveniently synthesized in good yield by the quaternization of 1,1,2-trimethyl-1H-benz[e]indole (2) using iodoethane (7) under reflux in acetonitrile. Semi-squaraine dye intermediate (4) was prepared by the condensation of (3) and 3,4diethoxysquarate (8) using triethylamine as a base to enhance the deprotonation and reactivity of the benzoindolium intermediates (4), which was then hydrolyzed to give semisquaraine dye (5). The target DSQ dye (1) was finally prepared by the acid-catalyzed self-condensation reaction of (5) in the presence of an excess thionyl chloride (SOCl2) as an acid catalyst in ether. The structural of the final DSQ dye (1) and their corresponding intermediates were verified by Mass and NMR spectral investigations (Supplementary Materials, Figure S1-S15). It is worth to mention here that the overall yield of the target dye by this synthesis route is too low (8.2%). In order to improve the synthetic yield of the double squaraine dye, we adopted an improved synthetic route as shown in Scheme 2. In this improved method, synthetic intermediate (3) was reacted with 0.45 equivalent of squaric acid (9) in toluene and 1-butanol mixture (1:1, v/v) to give the symmetrical mono squaraine dye (6). This symmetrical squaraine dye has a double bond, which can be attacked again by nucleophilic squaric acid (9), since the reactivity of the double bond is reduced due to the presence of squaric acid (9) moiety but reaction conditions, especially the choice of solvent, play a crucial role in the promotion of the condensation reaction. The use of a mixture of toluene and 1-butanol in (5:1, v/v) ratio was important to obtain the target DSQ dye (1) at a 25% yield. This proposed improved method not only improved the overall yield (19%) but also further verified the structure of the DSQ dye (1). At the same time, conversion of the typical mono squaraine dye to double squaraine dye by this improved synthetic route opens the door for the synthesis of a variety of symmetrical and unsymmetrical double squaraine dyes by altering the diverse variety of electron-donating heterocyclic units.  The DSQ dye (1) was successfully synthesized in four steps as shown in Scheme 1. The key precursor (5) was synthesized in three steps from the commercially available starting compound 1,1,2-trimethyl-1H-benz[e]indole and diethyl squarate. The reaction intermediate (3) was conveniently synthesized in good yield by the quaternization of 1,1,2-trimethyl-1H-benz[e]indole (2) using iodoethane (7) under reflux in acetonitrile. Semi-squaraine dye intermediate (4) was prepared by the condensation of (3) and 3,4-diethoxysquarate (8) using triethylamine as a base to enhance the deprotonation and reactivity of the benzoindolium intermediates (4), which was then hydrolyzed to give semisquaraine dye (5). The target DSQ dye (1) was finally prepared by the acid-catalyzed self-condensation reaction of (5) in the presence of an excess thionyl chloride (SOCl 2 ) as an acid catalyst in ether. The structural of the final DSQ dye (1) and their corresponding intermediates were verified by Mass and NMR spectral investigations (Supplementary Materials, Figure S1-S15). It is worth to mention here that the overall yield of the target dye by this synthesis route is too low (8.2%). In order to improve the synthetic yield of the double squaraine dye, we adopted an improved synthetic route as shown in Scheme 2. In this improved method, synthetic intermediate (3) was reacted with 0.45 equivalent of squaric acid (9) in toluene and 1-butanol mixture (1:1, v/v) to give the symmetrical mono squaraine dye (6). This symmetrical squaraine dye has a double bond, which can be attacked again by nucleophilic squaric acid (9), since the reactivity of the double bond is reduced due to the presence of squaric acid (9) moiety but reaction conditions, especially the choice of solvent, play a crucial role in the promotion of the condensation reaction. The use of a mixture of toluene and 1-butanol in (5:1, v/v) ratio was important to obtain the target DSQ dye (1) at a 25% yield. This proposed improved method not only improved the overall yield (19%) but also further verified the structure of the DSQ dye (1). At the same time, conversion of the typical mono squaraine dye to double squaraine dye by this improved synthetic route opens the door for the synthesis of a variety of symmetrical and unsymmetrical double squaraine dyes by altering the diverse variety of electron-donating heterocyclic units.

Solvatochromism in the DSQ Dye
Solvatochromism is basically a change in the color of target molecule by change in the molecular environment created by solvent molecules by differential interactions between target and solvent molecules. This imparts the inherent tendency of sensing to the solvatochromic molecules by the changing molecular environment as external stimulus and the possibility of controlling this interaction by judicious selection of the suitable solvent media. To investigate the solvatochromism, the synthesized DSQ dye was dissolved in different polar and non-polar organic solvents followed by the observation of change in color by naked eye and spectral investigation by the UV-visible-NIR electronic absorption spectroscopy as shown in the Figure 2. It can be seen from the Figure 2a that, under natural light conditions, there was a distinct change in color by the naked eye for the 20 µM solution of DSQ dye in different polar and non-polar organic solvents demonstrating strong solvatochromism. To investigate this qualitative color change observed by naked eye in more detail, electronic absorption spectra of these different solvents was also recorded and shown in Figure 2b. It can be seen from this figure that DSQ dye exhibits strong solvatochromism having absorption spectral features ranging from visible to NIR wavelength regions (450-800 nm) in different solvents. Contrary to this dye, its mono-squaraine dye (6) as well as semi-squaraine dye (4) counterparts exhibits weak solvatochromism with only a small change of 20-30 nm in the absorption maximum (λ max ) (Supplementary Material, Figure S16a,b) demonstrating that our newly proposed double squaraine dye (DSQ dye) functions as a potential solvatochromic dye probe.

Scheme 2.
Improved synthetic route for the synthesis of DSQ dye (1).

Solvatochromism in the DSQ Dye
Solvatochromism is basically a change in the color of target molecule by change in the molecular environment created by solvent molecules by differential interactions between target and solvent molecules. This imparts the inherent tendency of sensing to the solvatochromic molecules by the changing molecular environment as external stimulus and the possibility of controlling this interaction by judicious selection of the suitable solvent media. To investigate the solvatochromism, the synthesized DSQ dye was dissolved in different polar and non-polar organic solvents followed by the observation of change in color by naked eye and spectral investigation by the UV-visible-NIR electronic absorption spectroscopy as shown in the Figure 2. It can be seen from the Figure 2a that, under natural light conditions, there was a distinct change in color by the naked eye for the 20 μM solution of DSQ dye in different polar and non-polar organic solvents demonstrating strong solvatochromism. To investigate this qualitative color change observed by naked eye in more detail, electronic absorption spectra of these different solvents was also recorded and shown in Figure 2b. It can be seen from this figure that DSQ dye exhibits strong solvatochromism having absorption spectral features ranging from visible to NIR wavelength regions (450-800 nm) in different solvents. Contrary to this dye, its mono-squaraine dye (6) as well as semi-squaraine dye (4) counterparts exhibits weak solvatochromism

Scheme 2.
Improved synthetic route for the synthesis of DSQ dye (1).

Solvatochromism in the DSQ Dye
Solvatochromism is basically a change in the color of target molecule by change in the molecular environment created by solvent molecules by differential interactions between target and solvent molecules. This imparts the inherent tendency of sensing to the solvatochromic molecules by the changing molecular environment as external stimulus and the possibility of controlling this interaction by judicious selection of the suitable solvent media. To investigate the solvatochromism, the synthesized DSQ dye was dissolved in different polar and non-polar organic solvents followed by the observation of change in color by naked eye and spectral investigation by the UV-visible-NIR electronic absorption spectroscopy as shown in the Figure 2. It can be seen from the Figure 2a that, under natural light conditions, there was a distinct change in color by the naked eye for the 20 μM solution of DSQ dye in different polar and non-polar organic solvents demonstrating strong solvatochromism. To investigate this qualitative color change observed by naked eye in more detail, electronic absorption spectra of these different solvents was also recorded and shown in Figure 2b. It can be seen from this figure that DSQ dye exhibits strong solvatochromism having absorption spectral features ranging from visible to NIR wavelength regions (450-800 nm) in different solvents. Contrary to this dye, its mono-squaraine dye (6) as well as semi-squaraine dye (4) counterparts exhibits weak solvatochromism Scheme 2. Improved synthetic route for the synthesis of DSQ dye (1).
A perusal of the Figure 2b clearly corroborates that the spectral features can be grouped into two major classes, one exhibiting dominant absorption between 600-800 nm by typical dipolar solvents (DMF, DMSO) and protic solvents (methanol, ethanol) while second where the dominant absorption manly in the 450-650 nm exhibited by acetonitrile, tetrahydrofuran (THF), ethyl acetate, toluene, chloroform, acetone, 2-propanol, n-butanol and acetic acid (AcOH). Blue-shift absorption in the 450-650 nm range is thought to be caused by the production of H-aggregates. It has been widely reported that squaraine dyes exhibit blueshifted spectral features due to H-aggregate formation various efforts has been directed to control this aggregation behavior for controlling the functionality in photovoltaics and sensing [38]. In DMF, DMSO, methanol, ethanol, DSQ dye (1) predominantly exists in monomeric state due to these dipolar solvents have significant dye-solvent dipole-dipole interactions to hampering of the dye-aggregation in the monomeric state. The differential of the spectral features of the DSQ dye in protic solvent of methanol, ethanol, n-butanol, 2-propanol and AcOH could be attributed to the different of their dielectric constant. This argument is further validated by the fact that contrary to methanol (ε = 32.2) and ethanol (ε = 24.5), use of 2-propanol (ε = 19.9), 1-butanol (ε = 17.5) and AcOH (ε = 6.2) leads to not only visual change in the color of the solution but also pronounced decrease in the absorbance around λ max of 710 nm and concomitant increase in the absorbance around λ max of 580 nm [39].
Considering the two dipolar organic solvents acetonitrile (ACN) and DMF with high and nearly similar dielectric constants [40] of 37.5 and 37.8 exhibit contrasting color and absorption spectral features with λ max of 592 nm and 728 nm, respectively. We investigated the spectral changes of the DSQ dye in the different ratio of binary solvent mixtures consisted of ACN and DMF and results are shown in Figure 2c. The excited-state interactions such as change in the conformation, charge (electron/proton) transfer, and non-covalent interactions with solvent molecules such as van der Waals forces have been widely used to explain differential solvatochromic behavior in the functional organic molecules [41][42][43][44][45][46]. This contrasting solvatochromic behavior in these solvents can be explained considering the differential aggregation behavior of the DSQ dye taking dipole-dipole, and hydrogen bonding interactions between the dye and solvent molecules. DMF is a strong hydrogen bond acceptor (HBA), while acetonitrile has a weak HBA tendency [47]. The strong HBA tendency of DMF favors the dye to exist in the monomeric state. On the other hand, weak HBA of ACN leads to the formation of H-aggregates resulting in the hypsochromic shift in the absorption spectrum. In order to further verify the H-aggregation formation in the DSQ solution, we investigated the absorption spectra of the different concentrations of DSQ dye in DMF (Supplementary Materials, Figure S17). At the lower concentration, the peak at 726 nm appears, indicating the absorption of the dye in mono-molecule state. As the concentration of the DSQ dye solution increases, 554 nm and 600 nm peaks appear, suggesting a blue shifted absorption spectral features. These spectral changes at higher concentration validates the formation of H-aggregation. Kim et al. have also emphasized that squaraine dyes easily form H-aggregates leading to blue-shifted absorption spectral features [48]. Another possible reason for this contrasting spectral behavior between the ACN and DMF could be attributed to the differential molecular planarization in these solvents. The possibility of strong coordination between the dye molecule and DMF as compared to ACN leads enhanced molecular planarity leading to extended effective π-conjugation, which is supported by a theoretical molecular orbital calculation (MO) calculation using Gaussian (G09) program [49]. A perusal of the optimized molecular structure of the DSQ dye reveals that, unlike typical mono-squaraine dyes exhibiting planar dye molecular structure, double squaraine dye exhibits a slightly non-planar bent structure in the isolated gaseous state. Interaction of the dye with strong HBA solvents leads to the hydrogen bond-assisted molecular planarization, resulting in the extension of the effective π-conjugation and red-shift in the absorption maximum (Supplementary Materials, Figure S18).

Harnessing the Potentiality of DSQ Dye in Metal Ion Sensing
Metal ion sensing for the environment and health monitoring is a fast-growing research field where solvatochromic dye probes are expected to play a dominant role. Using a series of D-π-A molecular framework-based visible light absorbing solvatochromic dyes, Xie et al. demonstrated sensing of Na + , K + and H + ions and K + ion selective nanosensor [50,51]. Although their proposed dyes exhibited solvatochromic behavior, the extent of the solvatochromic shift was small and exhibited negative solvatochromism with the solvent polarity. Considering the contrasting absorption spectral features of DSQ dye in ACN and DMF, we dissolved DSQ dye in different combinations of solvent mixtures such as DMF/Water (1:1, v/v) and DMF/ACN (1:99, v/v) and explored the differential metal ion sensing behavior. A perusal of the Figure 2b clearly corroborates that the spectral features can be grouped into two major classes, one exhibiting dominant absorption between 600-800 nm by typical dipolar solvents (DMF, DMSO) and protic solvents (methanol, ethanol) while second where the dominant absorption manly in the 450-650 nm exhibited by acetonitrile, tetrahydrofuran (THF), ethyl acetate, toluene, chloroform, acetone, 2-propanol, n-butanol and acetic acid (AcOH). Blue-shift absorption in the 450-650 nm range is thought to be caused by the production of H-aggregates. It has been widely reported that squaraine dyes exhibit blue-shifted spectral features due to H-aggregate formation various efforts has been directed to control this aggregation behavior for controlling the functionality in photovoltaics and sensing [38]. In DMF, DMSO, methanol, ethanol, DSQ dye (1) predominantly exists in monomeric state due to these dipolar solvents have significant dye-solvent dipole-dipole interactions to hampering of the dye-aggregation in the monomeric state. The differential of the spectral features of the DSQ dye in protic solvent of methanol, ethanol, n-butanol, 2-propanol and AcOH could be attributed to the different of their dielectric constant. This argument is further validated by the fact that contrary to methanol (ε = 32.2) and ethanol (ε = 24.5), use of 2-propanol (ε = 19.9), 1-butanol (ε = 17.5) and AcOH (ε = 6.2) leads to not only visual change in the color of the solution but also pronounced decrease in the absorbance around λmax of 710 nm and concomitant increase in the absorbance around λmax of 580 nm [39].
Considering the two dipolar organic solvents acetonitrile (ACN) and DMF with high and nearly similar dielectric constants [40] of 37.5 and 37.8 exhibit contrasting color and absorption spectral features with λmax of 592 nm and 728 nm, respectively. We investigated the spectral changes of the DSQ dye in the different ratio of binary solvent mixtures consisted of ACN and DMF and results are shown in Figure 2c. The excited-state interactions such as change in the conformation, charge (electron/proton) transfer, and non-covalent interactions with solvent molecules such as van der Waals forces have been widely used to explain differential solvatochromic behavior in the functional organic molecules [41][42][43][44][45][46]. This contrasting solvatochromic behavior in these solvents can be explained considering the differential aggregation behavior of the DSQ dye taking dipole-dipole, and hy-   Figure 3. It can be seen from the photographs shown in Figure 3a that blank containing only DSQ dye in this solvent system exhibits cyan color and there was change in the color of the solution in the presence of some metal ions such as Ag + , Al 3+ , Cu 2+ , Fe 3+ , Cr 3+ , Fe 2+ , where this changes in color in the presence of Ag + and Cu 2+ ions were highly conspicuous as visualized by the naked eye. To have an in-depth insight about these visual color changes, absorption spectra solution of the dye in the presence and absence of metals ions were also recorded and shown in Figure 3b. The cyan color of the blank solution is associated with monomeric dye absorption mainly in the NIR wavelength region appearing at the λ max of 706 nm. In the presence of Al 3+ and Cr 3+ , the color of the solution changed from cyan to the bright blue along with clear change absorption spectral feature, where absorbance at 706 nm was found to decrease and there was the appearance of a new and blue-shifted peak appearing at the λ max of 620 nm with the isosbestic point around 648 nm. In the presence of Fe 3+ , Fe 2+ visible change in color of the solution was small and is associated with absorption spectral changes with decrease in peak at 706 nm with enhanced absorption at 620 nm and attributed to the hypochromic effect by the Fe 3+ , Fe 2+ ions to the DSQ dye in DMF/H 2 O (1:1) solvent system. A perusal of Figure 3a indicates an obvious and remarkable change in the color of the DSQ dye (1) in the DMF-water (1:1) solvent system from cyan to light yellow in the presence of Cu 2+ and Ag + ions indicating their very strong interaction with the DSQ dye (1). This strong interaction was further confirmed by the clear absorption spectral changes as shown in the Figure 3b exhibiting highly diminished optical absorption around 706 nm. At the same time, with the increasing concentration of these ions, there was a fast and sharp decrease in the absorption peak associated with the monomeric dye molecules appearing at 706 nm, further validating their very strong interaction with the DSQ 1 dye molecules.
This DMF/H2O (1:1) solvent system exhibits sensing behavior (although not selective for a particular ion) with the Ag + , Al 3+ , Cu 2+ , Fe 3+ , Cr 3+ , Fe 2+ , concentration dependent spectral change with these ions was measured, which is shown in the Figure 3c. It can be seen from this figure that, in the presence of these ions, absorption at 706 nm associated with the monomeric dye molecules decreases as a function of increasing ion concentration this change in the absorbance was more pronounced in the metal ion concentration range of 10 −6 M-10 −4 M). Therefore, it can be concluded that the dye DSQ dye exhibits sensing towards Ag + , Al 3+ , Cu 2+ , Fe 3+ , Cr 3+ , Fe 2+ ions of the DMF/H2O (1:1) solvent system but considering the strong solvatochromism in this newly designed dye, selectivity for a particular ion could be possible by the judicious selection of a suitable solvent or solvent mixture.  This DMF/H 2 O (1:1) solvent system exhibits sensing behavior (although not selective for a particular ion) with the Ag + , Al 3+ , Cu 2+ , Fe 3+ , Cr 3+ , Fe 2+ , concentration dependent spectral change with these ions was measured, which is shown in the Figure 3c. It can be seen from this figure that, in the presence of these ions, absorption at 706 nm associated with the monomeric dye molecules decreases as a function of increasing ion concentration this change in the absorbance was more pronounced in the metal ion concentration range of 10 −6 M-10 −4 M). Therefore, it can be concluded that the dye DSQ dye exhibits sensing towards Ag + , Al 3+ , Cu 2+ , Fe 3+ , Cr 3+ , Fe 2+ ions of the DMF/H 2 O (1:1) solvent system but considering the strong solvatochromism in this newly designed dye, selectivity for a particular ion could be possible by the judicious selection of a suitable solvent or solvent mixture.

Selective Metal Ion Sensing Utilizing DMF/ACN Solvent System
Our exploration for selective metal ion sensing in different solvents resulted in the selection of DMF/ACN solvents owing to their contrasting spectral changes from visible (ACN) to NIR (DMF) wavelength region graduation change in the spectral features upon changing their relative ratio as shown and discussed in the Section 2.2. To accomplish this, 10 µM solution DSQ dye in the (1:99, v/v) DMF/ACN solvent system was treated with the five equivalent different metal ions, such as Na + , K + , Mg 2+ , Ca 2+ , Zn 2+ , Mn 2+ , Ba 2+ , Cs + , Pb 2+ , Ag + , Cd 2+ , Al 3+ , Sr 2+ , Cu 2+ , Fe 3+ , Cr 3+ , Fe 2+ , Ni 2+ , Co 2+ , and photographs of visual color change along with the absorption spectral behavior in the presence and absence of the different metal ions are shown in Figure 4. A perusal of this figure clearly corroborates that DSQ dye in this solvent system light blue color (blank) and there was a conspicuous change in the color of solution from blue to bright red-violet in the presence of Cu 2+ ion only as shown in Figure 3a demonstrating the potential of this dye to work as a selective Cu 2+ ion chemosensor. Although monitoring metal ions play dominant role in controlling the environmental pollution and human health conditions, amongst various metal ions Cu 2+ is the 3rd most abundant metal ion in the human body, controlling various physiological processes [52]. Increased levels of this ion in the human body leads to several diseases such as Menkes syndrome, Wilson's disease, Alzheimer's disease, etc. [53][54][55]. Electronic absorption spectra of the DSQ dye in this solvent system (DMF/ACN, 1:99), as shown in Figure 4b, exhibit λ max at 592 nm corresponding to the observation of the blue color. It is interesting to see that except in the presence of the Cu 2+ ion, presence of other metal ions does not result in any change in the absorption spectral feature, validating the selective detection of Cu 2+ ions by the DSQ dye in this solvent system. It can also be seen that after the addition of Cu 2+ ions, the absorbance of DSQ dye appearing at the 592 nm decreases and a new peak appears at the λ max at the 554 nm, which is responsible for the appearance of the red-violet color. The reason behind this shift peak can be attributed to formation of higher H-aggregation of DSQ-Cu 2+ complex with increase in concentration of Cu 2+ ions. On the contrary, after adding the same concentration of other ions, there was almost no change in the absorption spectra further suggesting that DSQ dye in this solvent system could serve a colorimetric and selective Cu 2+ chemosensor. One can argue that sensitivity of the dye with Cu 2+ ion is not due to aggregation but associated with complex formation without undergoing the molecular aggregation. He at al has reported the formation of complex on addition of metal ion to their dye, which imparts fluorescence [56]. But, in case of DSQ dye, this possibility is ruled out, since the dye is not showing fluorescence in presence of Cu 2+ . This is due to the formation of strong H-aggregation in the dye ion system. These π-π interactions might well be strongly reflected by the overlaps in π-conjugated system, facilitating the development of excimers and, as a result, emission quenching and the aggregation induced quenching effect as reported by Ma et al. [57] and Huang et al. [58]. In this present work, lack of the observed fluorescence in the presence of Cu 2+ ions clearly corroborate that the observed behavior is due to aggregation.   In order to further investigate the selective recognition of DSQ dye in the presence of Cu 2+ ions, concentration dependent titration was conducted. To accomplish this, DSQ dye was dissolved in the DMF/ACN (1:99, v/v) solvent mixture followed by the addition of different concentrations of Cu 2+ ions and measurement of the absorption spectra as shown in Figure 5.    Therefore, the analysis of this Figure 5b reveals that DSQ dye in this solvent system of DMF/ACN (1:99, v/v) exhibits linear detection of the Cu 2+ ion in the concentration range 1 nM to 50 µM with the limit of the detection to be 6.5 × 10 −10 M (Supplementary Materials, Figure S19). It is worth to mention here that World Health Organization have recommended a maximum allowed limit of the Cu 2+ ions in the drinking water to be up 30 µM [55]. Therefore, present DSQ dye based Cu 2+ ion sensor can be easily used for checking the quality of the drinking water owing to the visual color change of dye in this solvent system from blue to the red-violet. By the absorption spectrum-based titrimetric investigation, it was found that at lower concentration of Cu 2+ ions, the DSQ dye-ion binding was guided by a strong electrostatic interactions and quantitative estimation of the binding of Cu 2+ ions to the dye molecules was analyzed by the Benesi-Hildebrand Equation (1).
where A o , A and A 1 are the absorbance in absence, intermediate and at maximum presence of Cu 2+ concentration, respectively, and k a is the association constant. The plot 1/(A o − A) versus 1/[Cu 2+ ] n as shown Figure 5c gives a straight line, when n = 2, indicating 2:1 stoichiometry for DSQ-Cu 2+ complexation. The association constant (k a ) can be determined from the slope of the straight line of this plot, which was found out to be 2.32 × 10 4 M −1 indicating a strong binding of the metal ion with the dye. The value of this k a was further used to calculate Gibb's free energy change (∆G) for the dye complex formation using the Equation (2).
where R and T are universal gas constant and absolute temperature, respectively. The Gibbs free energy change was estimated to be −25.07 kJ mol −1 and such a high and negative value of ∆G indicates the feasibility of the spontaneous DSQ-Cu 2+ complex formation. The 2:1 stoichiometry for dye and Cu 2+ ion complexation estimated by the analysis of the Benesi-Hildebrand equation was further confirmed by using the Job's plot as shown in Figure 5d. It can be seen from this figure that in the case of [Cu 2+ ]/[DSQ-Cu 2+ ] to be 0.6, the value of ∆A was found to be the largest validating the stoichiometric ratio of DSQ dye and Cu 2+ to be the 2:1. In practical applications, the sensor must be highly selective and free of interference from other prospective rivals. The less interference there is, the greater the detecting effect. The DSQ dye was analyzed for a matrix of various cations in order to conduct a competitive experimental study (Supplementary Materials, Figure S20). It was discovered that the DSQ dye did not detect the presence of other cations, however on addition of Cu 2+ , it was recognized by the DSQ dye. This signifies that all the cations did not interfere with the identification of Cu 2+ .

Synthetic Procedure
Synthesis of 3: To the mixture of 1,1,2-trimethyl-1H-benzo[e]indole (6.3 g, 30.0 mmol) in 50 mL acetonitrile was added iodoethane (7) (9.6 mL, 120.0 mmol). The resulting mixture was refluxed 18 h. The TLC showed the start material was consumed completely. The reaction was cooled to room temperature, 150 mL ethyl acetate was added, and the resulting precipitate was filtered and washed with ethyl acetate to obtain the grey solid as titled compound 3, 11.0 g in 100% yield. 1  Synthesis of 4: To the solution of 3 (9.0 g, 24.6 mmol) in 50 mL ethanol was added diethyl squarate (4.2 g, 24.6 mmol) and trimethylamine (10.3 mL, 73.8 mmol). The resulting solution was stirred at room temperature for 15 h giving yellow precipitate. This yellow precipitate was filtered and washed with 100 mL cold ethanol to obtain the titled compound 8.5 g as a yellow solid product in yield 97%. 1   Synthesis of 6: To the solution of 3 (3.65 g, 10.0 mmol) in 40 mL dehydrated toluene/nbutanol (v/v = 1/1) was added squaric acid (9) (0.57 g, 5.0 mmol). The reaction mixture was subjected to azeotrope reflux at 120 • C using Dean-Stark trap for overnight. After the completion of the reaction, the solvent was evaporated, and the crude product was purified by silica-gel flash column chromatography using chloroform-methanol as an eluting solvent. Collected the pure fraction, evaporated the solvent and dried in vacuum to obtain 2.1 g of the titled compound 6 as a blue black solid in 76% yield. 1   Synthesis of 1: Method 1: To the suspension of (5) (0.36 g, 1.0 mmol) in 10 mL anhydrous ether was added thionyl chloride (0.145 mL, 2.0 mmol) and resulting mixture was stirred for 30 min at 0-5 • C. The solvent and excess thionyl chloride was evaporated mildly under reduced pressure and the residue was dissolved in 12 mL of dehydrated toluene/n-butanol (5:1 v/v) mixture. The reaction mixture was subjected to azeotrope reflux using Dean-Stark trap for overnight. After the completion of the reaction, the solvent was evaporated, and the crude product was purified by silica-gel flash column chromatography using hexane-ethyl acetate as an eluting solvent. Collected the pure purple fraction, dried with Nitrogen gas flow to obtain 65 mg of the titled compound (1) as a purple black solid in 10% yield.
Method 2: To the solution of (6) (0.55 g, 1.0 mmol) in 12 mL dehydrated toluene/nbutanol (v/v = 5/1) was added squaric acid (9) (0.11 g, 1.1 mmol) and the reaction mixture was refluxed at 120 • C for overnight. Squaric acid (9) (0.1 g, 1.0 mmol) was added into the above reaction and refluxed for 10 h more. TLC showed the start material was remaining about 30%. Squaric acid (9) (0.1 g, 1.0 mmol) was added into the above reaction and refluxed for 8 h more. TLC showed the starting material not reduced any more, therefore, stopped the heating and cooled to room temperature. The solvent was evaporated, and the crude product was purified by silica-gel flash column chromatography using chloroformmethanol as an eluting solvent. Collected the pure blue fraction, evaporated the solvent and dried in vacuum to obtain 160 mg of the titled compound (1) as a purple black solid in 25% yield. 1

Preparation of Solutions for the Solvatochromism Investigation
Stock solutions were prepared by weighing the solid of DSQ dye on a 5-digital analytical balance and adding ethyl acetate to make a 15 mM solution. Transfer 30 µL of 1.5 mM DSQ dye solution by volumetric pipette into each transparent glass bottle and dried it in vacuum. Then added 3 mL different supper dehydrate solvent into each bottle contained dry DSQ dye and sonicated for 5 min to ensure complete dissolution. The absorption spectra of each sample were measured in duplicate in the wavelength range from 400-800 nm. In the case of compounds 4 and 6, solvatochromism investigation was conducted by the same method as for the DSQ dye.

Metal Ion Sensing by Absorption Spectral Measurement
All the solutions for measurement were prepared by using small volumes (2-20 µL) of different concentrations of each metal ion, adding into 2 mL DSQ dye solution, and mixing well (20 µL Milli-Q water was added into 2 mL DSQ dye solution, the UV-visible spectrum of the DSQ dye has no effect and it was regarded as a reference sample (blank)). The measurements were taken within 10 min of preparation of solutions. The Cl − anion used in the metal ions (LiCl, NaCl, KCl, CaCl 2 , MgCl 2 , MnCl 2 ·4H 2 O, FeCl 2 ·4H 2 O, FeCl 3 , CoCl 2 ·6H 2 O, ZnCl 2 , CdCl 2 , AlCl 3 ) and NO 3 − anion used in the metal ions (Ba (NO 3 ) 2 , Sr(NO 3 ) 2 , Pb(NO 3 ) 2 have no effect to the DSQ dye in the solvent system of this work.

Density Functional Theory (DFT) Calculation
The structures of structure DSQ dye were optimized using density functional theory with the B3PW91 hybrid functional by using a 6-311 basis set. Theoretical MO computation was performed using the Gaussian 16 program package [49].

Conclusions
In summary, we have synthesized and characterized a novel and zwitterionic double squaraine dye with unique D-A-A-D π-conjugated molecular structure. Newly proposed double squaraine dye has demonstrated strong positive solvatochromism having absorp-tion spectral changes encompassing from visible to NIR wavelength region. This opens the door for unique and multiple ion sensing using a single probe in judiciously selected solvent systems. The corresponding double squaraine dye exhibited not only large solvatochromic range of about 200 nm but also strong spectral and color changes in the presence of metal ions. Judicious selection of a binary solvent mixture consisted of DMF and ACN (1:99, v/v), selective detection of Cu 2+ ion with the linearity from 50 µM to 1 nM have been successfully demonstrated. Benesi-Hildebrand and Jobs plot analysis for interaction between the double squaraine dye and Cu 2+ ion clearly corroborated the formation of 2:1 dye-Cu 2+ ion complex with appreciably good association constant of 2.32 × 10 4 M −1 . Considering the WHO allowed limit of Cu 2+ ions intake by human body to be 30 µM, the proposed dye can be used for the simple naked eye colorimetric monitoring of quality of the drinking water.