Triarylamine Rhodanine Derivatives as Red Emissive Sensor for

May 9, 2019 - mRA, dRA, and tRA senses Ag+and Hg2+ ions in a buffer-free aqueous solution with the lowest detection ... The yellow color of free senso...
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Cite This: ACS Sustainable Chem. Eng. 2019, 7, 9865−9874

Triarylamine Rhodanine Derivatives as Red Emissive Sensor for Discriminative Detection of Ag+ and Hg2+ ions in Buffer-Free Aqueous Solutions Pichandi Thamaraiselvi,† Natarajan Duraipandy,‡,§ Manikantan Syamala Kiran,‡,§ and Shanmugam Easwaramoorthi*,†

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†

Inorganic and Physical Chemistry Laboratory, ‡Biological Materials Laboratory, CSIR-Central Leather Research Institute, Sardar Patel Road, Adyar, Chennai 600020, India § Academy of Scientific and Innovative Research, New Delhi 201002, India S Supporting Information *

ABSTRACT: A completely water-soluble, red emitting, multibranched sensor based on the electron-rich triphenylamine and electron-deficient rhodanine-3-acetic acid has been developed. The sensors mRA, dRA, and tRA, respectively, have one, two, and three rhodanine-3-acetic acid groups, responsible for the interaction with the metal ions as well as the solubility of the probe in water. mRA, dRA, and tRA senses Ag+ and Hg2+ ions in a buffer-free aqueous solution with the lowest detection level of 0.06 and 0.02 ppm, respectively. The yellow color of free sensor turns into purple and colorless in the presence of Ag+ and Hg2+ ions, respectively, which can be witnessed even by the naked eye. The metal ion preferentially binds with electron-deficient rhodanine-3-acetic acid owing to the presence of an ideal coordination environment. The distinctly different signals for Ag+ and Hg2+ ions occur because of the dissimilar binding modes, wherein the former extends and the latter breaks the π-electron conjugation that results in a different signaling mechanism. Nevertheless, the additional binding sites of dRA and tRA influences the binding constant and sensitivity; however, additional metal ion binding does not occur because of the changes in the electronic properties after initial binding. The applicability of these sensors in the biological medium was also tested using HaCaT cells using Ag+ and Hg2+ ions, which demonstrated the quenching of intense red fluorescence of the probe, and thus, these probes can be also be used as a potential biosensor. KEYWORDS: Fluorescent sensor, Ag(I), Hg(II), donor−acceptor, triphenylamine, discriminative detection of metal ions

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of various metabolites which inactivate sulfhydryl enzymes.9 Thus, it is extremely important to detect silver ions in the aqueous media through the development of selective and sensitive methods. Efforts are being made by several research groups to develop fluorescent sensors toward silver ion detection using peptides,10 luminescent carbon nanomaterials,11 and organic and inorganic molecules and nanoparticles.12−18 Silver detection by the chromophores is based on either the coordination with the receptors, chemical reaction, aggregation-to-deaggregation,19 or interaction with the nanomaterials.20 Another important heavy metal is mercury that is widespread in soil, air, and water because of its usage in chloralkali industries, thermal power plants, thermometers, and barometers. Mercury is known to affect the central nervous system, and its accumulation in the human body leads to cognitive and motor disorders.21 Hg2+ ion can be

INTRODUCTION Developing simple molecular-level tools for the specific detection of several cations, anions, and neutral molecules that have significant roles in biological, environmental, and industrial processes is highly demanded as it offers a costeffective solution to the environmental remediation, forensic, and toxicity analysis. In this regard, detection based on fluorescent and optical methods has been explored widely as it offers ease of use, low cost, selectivity, and sensitive detection up to ppm/ppb level.1−5 Despite attempts by several researchers, the sensors for detection of silver are comparatively less effective when compared with detection of other metal ions. Silver is extensively being used in electrical, imaging, pharmaceutical,6 and cosmetic applications, and its use has resulted in the release of thousands of tons of silver waste into the environment annually, thereby causing ecological contaminations to the water. In addition, the exposure to silver has resulted in bioaccumulation and toxicity, thereby causing adverse effects in living organisms.7,8 Silver is known to combine with amine, imidazole, and carboxyl groups © 2019 American Chemical Society

Received: January 21, 2019 Revised: April 17, 2019 Published: May 9, 2019 9865

DOI: 10.1021/acssuschemeng.9b00417 ACS Sustainable Chem. Eng. 2019, 7, 9865−9874

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ACS Sustainable Chemistry & Engineering Chart 1

Figure 1. UV−visible absorption spectra of the receptors (a) mRA, (b) dRA, (c) tRA and fluorescence spectra of (d) mRA, (e) dRA, (f) tRA in the presence of different metal ions (1 × 10−5 M, 10 equiv) in aqueous solutions. The photographs of the solution in the presence of Ag+ and Hg2+ ions in absence (top panel) and presence of UV light (bottom panel) are also given.

detected using fluorescent sensors, ratiometric fluorescence, gated photochromism, and triple signal readout colorimetric sensing with organic chromophores and nanoparticles as a probe molecules.2,4,22−28 Most of the reported receptors are insoluble in water, and thus, the sensing needs to be carried out in the mixture of organic and aqueous solution with the percentage varying from 50:50 to 10:90 (volume/volume) ratio, which limits applications in biological microenvironments. Since most of the sensors involve complexation, an electrostatic interaction between analyte and receptors, the testing medium plays a crucial role in influencing the receptor−analyte interactions.29 Another important aspect of a sensor has been the molecules that show fluorescent emission in the red to near infrared (NIR) 600−900 nm region, owing to the good tissue penetration, low autofluorescence, avoidance of light-induced biological damage, and hence favorability for in vivo studies. The reports on the NIR emitting sensors for Ag(I) are very limited except the ones based on expanded porphyrins30 and

cyanine,31,32 which work, respectively, in methanol/water and ethanol/HEBES buffer solutions. For instance, the cyanine probe shows fluorescence at 546 and 731 nm, which shows, respectively, enhanced and quenched emission in the presence of Ag(I) ions, thereby exhibiting ratiometric detection owing to the metal-induced aggregation in aqueous medium. The combination of squaraine dye and the protein bovine serum albumin (BSA) has been used as a turn-on NIR sensor for Ag(I) detection.33 Probes include boron-dipyrromethane,34,35 tricarbocyanine,36 triphenylamine derivatives,37 and dyes that selectively sense the Hg(II) with the red-shifted absorption, emission spectra, and turn-off/on fluorescence mechanism. Because of its versatile electronic properties and ease in functionalization with the judicious choice of substituents, triphenylamine has widely been used as one of the molecular building blocks to construct the sensor for different species.28,37−39 In the present work, we have developed three red-emitting probes based on electron-rich triphenylamine (D) and electron-deficient rhodanine-3-acetic acid (A) as a donor 9866

DOI: 10.1021/acssuschemeng.9b00417 ACS Sustainable Chem. Eng. 2019, 7, 9865−9874

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with the seemingly broad fluorescence emission with the maximum at 625 ± 3 nm, which is in fact tailing over 800 nm. The red emission tailing over NIR region from the watersoluble receptors based on the simple triphenylamine skeleton is interesting and would have originated from the combination of strong electron-rich and -deficient substituents connected through the dominant π-conjugation pathway. The sensing ability of the receptor were tested by the addition of 1 × 10−5 M (10 equiv) of various metal ions such as Na+, K+, Al3+, Cu2+, Fe2+, Fe3+, Hg2+, Ag+, Mg2+, Mn2+, Li+, Ni2+, Cr3+, and Zn2+ as their perchlorate salts to the receptors in aqueous solution. As can be seen in Figure 1, Ag+ and Hg2+ turn the yellow -colored solution of mRA, respectively, into a purple and colorless solution, which can even be visualized by the naked eye. The yellow/orange fluorescence of mRA turned to intense purple fluorescence in the presence of Ag+ ion when the solution is kept under the 365 nm UV lamp. The selective and sensitive detection of Ag+ and Hg2+ ions aqueous solution over the other cations was also evident from the UV−visible absorption, and fluorescence spectra are shown in Figure 1. The lowest energy absorption peak of mRA becomes slightly red-shifted from 476 to 486 nm in the presence of Ag+ ions, whereas it becomes blue-shifted to 451 nm with Hg2+ ions. On the other hand, Au3+ ions decrease the intensity of the peak at 476 nm to a significant extent with broadened absorption spectra, but the extent of spectral changes is comparatively smaller with respect to Ag+ and Hg2+ ions (SI, Figure S2). Ag+ ion quenches the fluorescence of mRA along with the 88 nm red-shifted fluorescence (624 to 712 nm) and the emission spectra tailing over 850 nm. On the other hand, Hg2+ also quenches the receptor fluorescence with only marginal 12 nm red-shifts in the fluorescence maximum. Thus, on the basis of the above facts, it can be concluded that the receptor mRA selectively detects both Ag+ and Hg2+ ions, however, with two distinctly different signals for both species. Indeed, the remarkable fluorescence changes in the red to NIR region is quite intriguing. The spectral changes originates from the interaction between metal ions and the S and O atoms of rhodanine-3-acetic acid (RA). In order to further understand the mechanism of binding as well to assess the cooperativity in binding, if any, we have synthesized another two sensors dRA and tRA, respectively, having two and three rhodanine-3-acetic acid substituted at the para-phenylene position of triphenylamine. The molecular design would aid to understand the metal ion binding to the molecules substituted with the multiple receptors coupled with the single signaling unit. The pale brown aqueous solution of dRA and tRA turned to dark brown and purple, respectively, in the presence of Ag+ ions and become colorless for Hg2+ ions. A marginal red-shifted absorption ca. 15 nm was observed for dRA in the presence of Ag+ ions; however, the fluorescence becomes quenched drastically with a 37 nm red-shifted fluorescence along with the appearance of a new peak at 708 nm. In contrast, for tRA, Ag+ ion decreases the intense lowest energy absorption and fluorescence band with no significant shift in the spectral maximum wavelengths. Invariably, Hg2+ ion blue shifts the absorption and fluorescence maxima characteristic to the number of rhodanine-3-acetic acid groups. All the sensors reported here were found to sense Ag+ and Hg2+ ions selectively; however, the extent of color and spectral changes depends upon the number of acceptor moieties. The interference from other metal ions over the selectivity in sensing is tested by measuring the fluorescence spectra of

and acceptor, respectively. The donor−acceptor combination reduces the electronic band gap to enable them to show fluorescence in the red-to-NIR region as well as to incorporate the water solubility without the presence of even a trace amount of any organic solvents or buffer solution. The bufferfree solution avoids the interaction between the receptor, analyte, and the buffer solution, which in some cases leads to the precipitation. Further, the buffer-free solution does not require sample preparation, and the contaminated samples can be used as such for the analysis. It should also be noted that two of the three probes reported here possess more than one binding site for the metal ion and are expected to have a strong influence on the binding constants and water solubility.

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RESULTS AND DISCUSSION The triphenylamines substituted with one, two, and three rhodanine-3-acetic acids at the para-phenylene positions given

Figure 2. Plot of fluorescence intensity of the receptors (a) mRA, (b) dRA, and (c) tRA in the presence of Ag+ (red) and Hg2+ (blue) ions in the presence of different metal ions.

in Chart 1 were synthesized via a two-step simple chemical reaction as shown in Scheme S1 (Supporting Information, SI). Briefly, the triarylamines aldehydes synthesized by using the Vilsmeier−Haack formylation reaction with different equivalents of POCl340 were further subjected to Knoevenagel condensation using the requisite amount of rhodanine-3-acetic acid41 to afford the target compounds in quantitative yields. The final products were characterized by FT-IR, 1H and 13C NMR and mass spectroscopic techniques. The receptors mRA, dRA, and tRA, respectively, possess dipolar (DA), quadrupolar (DA2), and octupolar (DA3) configurations, which have exhibited electronic properties characteristic to that of the number of acceptor groups as can be seen from the UV−visible absorption and fluorescence spectra provided in Figure S1 (SI). All the receptors showed intense absorption centered at 474, 503, and 495 nm, respectively, for mRA, dRA, and tRA 9867

DOI: 10.1021/acssuschemeng.9b00417 ACS Sustainable Chem. Eng. 2019, 7, 9865−9874

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Figure 3. Fluorescence titration spectra of Ag+ and Hg2+ ions in aqueous solution. The spectra were measured by exciting the samples at the respective isosbestic point observed in the UV−visible absorption titration spectra.

fluorescence spectra for all the molecules were observed at ∼625 nm and was tailing over 750 nm (i.e. in the near-infrared region), which has definitely been advantageous when considering their applications in biological samples. The significantly larger Stokes shift value of 5070, 3930, and 4350 cm−1 is an indication of the involvement of intramolecular charge transfer from the donor TPA to the acceptor RA moiety. Thus, binding of the metal ions perturb the intramolecular charge transfer interactions that would result in the selective signaling pathway toward Ag+ and Hg2+ ions. The possibility of aggregation-induced emission (AIE) has also been probed using tetrahydrofuran and water solvent mixtures in different proportions. The increased water content from 20 to 97% (volume/volume) in the THF solution resulted in the gradually red-shifted fluorescence maximum by 70 nm along with the changes in the intensity (SI, Figure S6). The spectra become broadened with full width at half-maximum value increased from 2400 to 3830 cm−1 at higher water percentage in THF. Notably, no suspension or precipitate is obtained by the addition of water to THF, a characteristic feature often observed for AIEgens. Since a significant change in the absorption spectra was also noted, we have calculated the relative fluorescence quantum yield (Φf) to ascertain the fluorescence enhancement quantitatively by using the Φf of mRA in THF as 0.46. Indeed, the relative Φf were measured to be 0.44, 0.46, 0.44, 0.47, and 0.61, respectively, for 20, 40, 60, 80, and 97% water, and except for the one with 97% water, the remaining values are similar within the experimental limits. On the other hand, dRA exhibits drastic fluorescence quenching along with the red-shifted fluorescence maximum from 543 to 593 nm with increased water percentage. Thus, on the basis of the above facts, we believe that no aggregation-induced emission behavior is observed for these molecules because

sensor-Ag+/Hg2+ ions in the presence of other metal ions in aqueous solution, and the corresponding bar chart is given in Figure 2. Notably, the presence of metal ions such as Na+, K+, Al3+, Cu2+, Fe2+, Fe3+, Mg2+, Mn2+, Li+, and Zn2+ does not have any impact and suggests that all the molecules reported here selectively senses the Ag+ and Hg2+ ions in aqueous solution. Interestingly, simultaneous addition of Hg2+ and Ag+ ions resulted in the spectra (SI, Figure S3) similar to that of the former, ascribing that Hg2+ ions preferentially binds with the receptor. Another important aspect of the sensor molecules, the lowest detection limit of the Ag+ ions, was calculated using 3σ which corresponds to 7.59, 5.12, and 2.71 μM (Figure S4) for mRA, dRA, and tRA respectively. On the other hand, mRA, dRA, and tRA shows the LOD for Hg2+ ions as 0.35, 2.49, 1.96 μM (Figure S5). Interestingly, the detection limit shows an opposite trend for Ag+ and Hg2+ ions with the number of binding sites, but the reason for the observed behavior is still elusive and needs further investigation to unravel the underlying mechanism. The detection limit of the sensor is adequate to measure the presence of Ag+ and Hg2+ ion in water above permissible limits.42 It is imperative to understand the UV−visible absorption and fluorescence spectra of the receptors in the aqueous solution given in Figure S1 (SI) as the binding was assessed by the changes in their electronic properties. Sensors mRA, dRA, and tRA exhibit an intense, broad absorption that spreads over the ultraviolet and most of the visible region with lowestenergy peaks having the maximum at 474, 503, and 493 nm, respectively. The lowest energy absorption was attributed to the transition from lowest-energy singlet state (S0) to the first excited singlet state (S1), which has been significantly influenced by the number of RA groups attached with the TPA core through extended π-conjugation. On the other hand, 9868

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of Ag+ ion. The sensors with two (dRA) and three (tRA) binding sites were also found to show similar absorption and fluorescence spectral changes as that of mRA with the addition of Ag+ ions. The stoichiometry of the sensor−Ag+ complex was calculated using the Jobs continuous variation method (Figure S8), wherein, interestingly, all the sensors reported here were found to exhibit a 1:1 ratio.43 These observations revealed that despite the availability of more than one metal ion binding sites, only one Ag+ ion binds with dRA and tRA, and the other binding sites seemingly remain intact. The initial Ag+ binding to dRA/tRA presumably alters the electron density distribution in the chromophoric unit, thereby influencing the unbound rhodanine-3-acetic acid groups affinity in such a way that no second metal ion is bound with the free receptor. Nevertheless, the first binding events provides the negative cooperativity to the sensor, and therefore, the 1:1 complex between the receptor and sensor has only been observed despite the availability of the binding sites. The binding constants for Ag+ ions calculated using Benesei−Hildebrand equation44 correspond to 8.2 × 105, 3.32 × 106, and 1.05 × 105 M−1, respectively, for mRA, dRA, and tRA (Figure S9). Surprisingly, dRA shows nearly 180 times higher binding constant than mRA, while tRA shows ∼95 times higher value. Albeit negative cooperativity was observed for the second Ag+ ion binding, the binding constant and thereby the signal could be amplified several fold using the optimum number of additional receptor sites. It has been reported by Li et al. that the multibranched triphenylamine-rhodamine forms only the 1:1 complex with Hg2+ and Cu2+ in aqueous acetonitrile and aqueous ethanol solution, and no cooperative effect was found for the additional metal ion binding.45 Another analyte, Hg2+, also resulted in gradually decreased the absorbance of lower energy electronic transition and the fluorescence becomes quenched for all the sensor molecules as shown in Figures 3 and S10 in SI). The stoichiometry of sensor−Hg2+ complex calculated using the Job’s continuous variation method corresponds to a 2:1 ratio and reveals a negative cooperative effect for the binding of Hg2+ to the sensors with more than one RA moiety (SI, Figure S11). The ESI mass spectra (SI, Figure S12) of the mRA-Ag+ complex was observed at m/z 555 corresponding to 1:1 ratio and that of mRA-Hg2+ was observed at m/z 856 corresponding to a 2:1 binding ratio, which confirms the stoichiometry obtained from the Job’s method. The binding constants using the Benesi−Hildebrand equation was calculated to be 2.4 × 105, 4.6 × 105, and 2.5 × 105 M−1 for mRA, dRA, and tRA respectively (SI, Figure S13). An almost identical binding constant was observed for dRA with both Ag+ and Hg2+ ions, but mRA and tRA distinctly showed different binding constants with respect to analytes. The isothermal calorimetric (ITC) experiments at 25 °C were performed using the TAM III (TA Instruments) isothermal calorimeter using Ag+ ion as an example. The aqueous solution of the receptor was taken in the calorimeter cell, and Ag+ ion was gradually added to the receptor solution (titration spectra is given in Figure S14 SI). The ITC data were analyzed using the software provided with the instrument, and the stoichiometry of the complex was found to be a 1:1 ratio for the sensors.46 The binding constants were calculated to be 3 × 107, 2 × 107, and 3 × 107 M−1, respectively, mRA, dRA, and tRA. The free energy change ΔG for the binding event is calculated as −42.1 ± 0.4 kJ mol−1, but notable difference in other associated thermodynamic parameters ΔS and ΔH with respect to the receptors were noted, and thus, it can be

Figure 4. 1H NMR titration spectra of (a) mRA, (b) dRA, and (c) tRA with Hg2+ ions in D2O.

the probes possess good solubility both in THF and water and the spectral changes originated from the solvent mixture can better be explained by the effect of solvent polarity on the intramolecular charge transfer processes. The mechanistic details of the metal ion−probe interaction were studied using UV−visible absorption and fluorescence titration experiments performed in aqueous solution. The UV−visible absorption titration spectra of mRA with the gradual addition of Ag+ ions given in Figure S7 reveals that the absorption band centered at 474 nm gradually decreased along with the slightly red-shifted absorption ca. 12 nm. The binding event was accompanied by the appearance of isosbestic point at 510 nm, indicating the presence of only two species (i.e., bound and unbound sensor). The fluorescence titration spectra shown in Figure 3 evince the fluorescence quenching along with the red-shifted spectra with increased concentration 9869

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ACS Sustainable Chemistry & Engineering Scheme 1. Schematic Representation of the Binding of Metal Ions with the Receptor mRA

experiments performed in D2O and is shown in Figure 4 and Figures S15−17. 1H NMR chemical shift values of triphenylamines remain intact by the addition of Ag+ ions, except for the slight shift in the protons of rhodanine-3-acetic acid moiety which are in close proximity to the metal ions. For Hg2+ ions, changes in 1H NMR signals of dRA are more apparent with the appearance of the new peak around 7.90 and 4.31 ppm when the analyte concentration is 1 equiv, and subsequently, it becomes gradually intensified with further additions. Owing to the precipitation, we were unable to continue the experiment beyond 2 equiv of Hg2+ ions. The new signals are assigned to the proton attached to the carbon that bridges triphenylamine and rhodanine-3-acetic acid and methylene proton of acetic acid, which are located in the vicinity of Hg2+ binding site. The downfield shift of ∼1 ppm for the methine proton reveals the comparatively decreased electron density around it after metal binding, suggesting that most of the π-electron density would have been shifted to the carbonyl carbon owing to the extended π-conjugation and thereby introducing more single bond character between the methine and rhodanine carbon. Furthermore, this leads to the break in π-electron conjugation between the triphenylamine and rhodanine-3-acetic acid. Thus, on the basis of the above facts, the binding mechanism for both Ag+ and Hg2+ ions were given in Scheme 1, and indeed, this hypothesis is also supported by the colorless solution of the sensor in the presence of Hg2+ and reduced absorbance of the visible absorption band. For mRA and tRA, new resonance signals for the methine proton appear at 7.89/8.46 ppm and the methylene proton appear at 4.37/4.27 ppm, which reveals that the same binding mechanism is observed for all the sensors. Time-dependent density functional theory (DFT) calculations were performed for all the molecules using Gaussian 09 software to unravel the influence of metal ions on the electronic properties.47 Initially, the geometries of the sensors were optimized at B3LYP-6/31G* level, and then the molecular orbitals (MO) were calculated at the same level of theory. All the receptors exist in propeller-like structure owing to the central triphenylamine moiety (SI, Figure S18). The

Figure 5. HOMO−LUMO energy diagrams of mRA, dRA, and tRA with Ag+ and Hg2+ ions.

concluded that the binding event is driven by the entropy and enthalpy factors. A molecular-level understanding about the metal ion binding with the sensor was further gleaned from the 1H NMR titration 9870

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Figure 6. Fluorescence microscopic images of live HaCaT cells treated with sensors mRA, dRA, and tRA in the presence of various concentrations of silver ions. The scale bar represents 25 μm.

lowest energy transitions are due to the HOMO → LUMO transition; however, it has a charge transfer character to a significant extent. A close examination of the MO diagram shown in Figure S19 (SI) reveals that the electron density localized on TPA core in highest occupied molecular orbital (HOMO) is localized on the RA moiety in the lowest unoccupied molecular orbital (LUMO). While the electron density remains on TPA core for dRA and tRA, the LUMO depicts the electron density on both the phenylene linked RA moieties and two of the three RA units, respectively. Nevertheless, tRA possesses degenerate LUMO and LUMO +1 orbitals. The geometry of mRA with the Ag+ and Hg2+ ions were also calculated at B3LYP/6-31G (d,p) and LANDLZ level, and the optimized geometry is given in Figures S20 and 21. Examination of the frontier molecular orbitals given in Figure

5 suggests that the electron density in the HOMO is entirely localized on the silver-ion-bound RA moiety, while the TPA possesses the electron density in LUMO. A similar observation was made for the dRA and tRA with the free receptor group(s) left with no electron density in HOMO after the initial Ag+ ion binding, thus rendering the additional binding sites unavailable for the subsequent binding of positively charged metal ions. On the other hand, for Hg2+ ions, the Job’s plot reveals the 2:1 binding for the sensor:Hg2+ ion, and the frontier orbitals obtained in this stoichiometric ratio are given in Figure 5. After Hg2+ ion binding with mRA, the electron density localized on the triphenylamine moiety in the HOMO becomes shifted to rhodanine-3-acetic acid during transition to LUMO evenly in both the mRA units. In contrast, for dRA and tRA, HOMO is localized on one of the sensor units in 2:1 complex of Hg2+ ion, which then shifted to another unit in LUMO. In fact, the free 9871

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different signaling outputs with the lowest detection level of these metal ions in subppm level. The different signaling pathway arises probably from the binding of silver ion with thiocarbonyl and mercury with carbonyl group, while the carboxyl group is expected to facilitate the binding of both metal ions. Nevertheless, more than two binding sites are available for metal ion binding and have a significant influence on the sensor−metal ion interactions. Additionally, all the compounds were found to bind with only one metal ion because the initial binding causes perturbations in the electron density distribution with no or significant electron density available with the rhodanine-3-acetic acid for further binding. The present work demonstrates that sensors with more than one binding sites aid the metal-ion-binding interactions; however, no additional metal ion binding occurs despite the availble free receptor moietites. The applicability of the probe as a paper strip and in biological medium has also been demontrated for the detection of metal ions.

rhodanine-3-acetic acid units in the receptor possess no electron density and thus render them not available for the binding with the another metal ion. These calculated results further support our experimental observation based on spectrophotometric and calorimetric results, and thus, it can be concluded that the presence of additional receptors connected to the same reporter unit does not aid the binding of multiple metal ions due to the noncooperativity effect. In addition, the workability of the sensor in different pH range in the presence of Ag+ and Hg2+ ions was tested, and it was found that the fluorescence intensity has not been perturbed while changing the from pH 1.4 to 8.2 (SI, Figure S22). This feature would be an added advantage that the sensors could be used to detect the metal ions with the wider pH window. In addition, we have prepared the test strips by coating the THF solution of the selected compounds, mRA and dRA, on Whatman filter paper, and the test strips were found to exhibit intense green and yellow fluorescence, respectively (SI, Figure S23). Addition of a small amount of water sample spiked with Ag+ and Hg2+ ions quenches the fluorescence except that of Ag+ ion with mRA, wherein slight but notable changes in fluorescence were observed. On the other hand, the dRA test strip does not show any significant difference among the metal ions, and it might have been due to the comparatively low quantum yields for dRA in water (vide supra). The application of the probe in a biological environment has been tested by taking Ag+ ions as an example. The cytotoxicity of the sensors in the presence of the metal ions were evaluated using MTT assay and is shown in Figure S24 (SI). At concentrations above 10 μM, the cell viability of 86%, 96%, and 88% were observed for mRA, dRA, and tRA respectively. The IC50, for mRA, dRA, and tRA were found to be 80, 91, and 140 μM. Various concentrations of mRA, dRA, and tRA below 80, 91, and 140 μM and different concentrations of Ag+ below 200 μM were used for the bioimage application. Figure 6 shows the in vitro fluorescence study of mRA, dRA, and tRA on HaCaT cells. No fluorescence was observed in cells when treated with Ag+ alone; however, intense red fluorescence was observed on treatment with mRA, dRA, and tRA. When the cells were exposed to Ag+ ions in the presence of sensors mRA, dRA, and tRA, a reduction in the red fluorescence was observed in a concentration-dependent manner. The results showed that the concentration of Ag+ ion is inversely proportional to the intensity of the red fluorescence. A similar observation was also made for the Hg2+ ions with HaCaT cells, as shown in Figure S25. The results are consistent with the fluorescence quenching data of solution-phase studies. The inherent fluorescence from the cells did not interfere with the analysis since the receptors exhibited fluorescence in the NIR region.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssuschemeng.9b00417.

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Synthesis characterization, MO diagrams, UV−visible, fluorescence spectral details (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail for S.E.: [email protected]. ORCID

Manikantan Syamala Kiran: 0000-0003-3736-4775 Shanmugam Easwaramoorthi: 0000-0001-5314-2422 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The work was supported by the SERB project, EMR/2017/ 000149. P.T. acknowledges DST for INSPIRE fellowship. The work is partly supported by DST project, Water IC for SUTRAM of EASY WATER (DST/TM/WTI/WIC/2K17/ 82(C))

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CONCLUSIONS Triphenylamine-based sensors having mono, di, and trirhodanine-3-acetic acid moeity have been synthesized following simple synthetic steps. They are denoted as mRA, dRA, and tRA, respectively, and have an intense absorption in the visible region and emission in the NIR region. The rhodanine-3-acetic acid group is responsible for the interaction with the metal ion binding and also provides excellent solubility in 100% aqueous solution, which is essential in realizing the pracaticality in testing the environmental samples and biological samples. These sensors were found to be highly sensitive and selective in sensing Ag+ and Hg2+ ions with 9872

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DOI: 10.1021/acssuschemeng.9b00417 ACS Sustainable Chem. Eng. 2019, 7, 9865−9874

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DOI: 10.1021/acssuschemeng.9b00417 ACS Sustainable Chem. Eng. 2019, 7, 9865−9874