sensors Article

A Highly Selective Sensor for Cyanide in Organic Media and on Solid Surfaces Belygona Barare 1 , Ilknur Babahan 1,2 , Yousef M. Hijji 3 , Enock Bonyi 1 , Solomon Tadesse 1 and Kadir Aslan 1, * 1 2 3

*

Department of Chemistry, Morgan State University, Baltimore, MD 21251, USA; [email protected] (B.B.); [email protected] (I.B.); [email protected] (E.B.); [email protected] (S.T.) Department of Chemistry, Adnan Menderes University, Aydin 09010, Turkey Department of Chemistry and Earth Sciences, Qatar University, P.O. Box 2713, Doha, Qatar; [email protected] Correspondence: [email protected]; Tel.: +1-443-885-4257

Academic Editor: W. Rudolf Seitz Received: 12 January 2016; Accepted: 13 February 2016; Published: 24 February 2016

Abstract: The application of IR 786 perchlorate (IR-786) as a selective optical sensor for cyanide anion in both organic solution (acetonitrile (MeCN), 100%) and solvent-free solid surfaces was demonstrated. In MeCN, IR-786 was selective to two anions in the following order: CN´ > OH´ . A significant change in the characteristic dark green color of IR-786 in MeCN to yellow was observed as a result of nucleophilic addition of CN´ to the fluorophore, i.e., formation of IR 786-(CN), which was also verified by a blue shift in the 775 nm absorbance peak to 430 nm. A distinct green fluorescence emission from the IR-786-(CN) in MeCN was also observed, which demonstrated the selectivity of IR-786 towards CN´ in MeCN. Fluorescence emission studies of IR-786 showed that the lower detection limit and the sensitivity of IR-786 for CN´ in MeCN was 0.5 µM and 0.5 to 8 µM, respectively. The potential use of IR-786 as a solvent-free solid state sensor for the selective sensing and monitoring of CN´ in the environment was also demonstrated. On solvent-free solid state surfaces, the sensitivity of the IR-786 to CN´ in water samples was in the range of 50–300 µM with minimal interference by OH´ . Keywords: IR-786; anion sensing; cyanide sensing; cyanine dyes; near-infrared fluorophores; optical sensors; chemical sensors

1. Introduction Detection of anions in biological systems and the environment within the desired concentration range in a timely manner is central to federal and state regulations imposed on commercial chemical and biological processes [1–5]. Among the common anions are cyanide, hydroxide, phosphate and fluoride; while cyanide is an important industrial chemical, it is known to be extremely toxic to human and animals even when present in insignificant concentrations between 0.5 and 3.5 mg per kilogram of body weight [3]. Cyanide is more lethal in two forms: free cyanide and hydrogen cyanide (HCN) [6,7]. Cyanide anion (CN´ ) has the detrimental impact of forming complexes in the cell mitochondrion, interfering with the capacity of a cell to produce energy, consequently leading to accumulation of chemicals in the bloodstream [6,7]. Consequently, the U.S. Environmental Protection Agency (EPA) has set a CN´ limit in drinking water of 2 ppm (76 µM) [7]. Conventional methods of testing for cyanide such as titration, distillation, chromatography or potentiometry are expensive, laborious and time consuming [5]. Therefore, there is still a need to develop more robust, inexpensive and less laborious testing methodologies for cyanide [5]. In an effort to make anion quantitative detection and monitoring inexpensive, easy and straight-forward, a number

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of colorimetric detection methods were developed [7–9]. For cyanide detection, these sensors rely on either displacement or a chemodosimeter principle [7]. Some of these sensors are synthesized in several steps, which could result in the escalation of the overall costs as well as environmental pollution [10]. In addition, several of the available cyanide sensors are limited as they work best in organic solvents, suffer interference by other anions (OH´ , F´ or AcO´ ), and/or have low sensitivity due to their short absorption and emission wavelength, which interferes with other natural processes [9,11]. For example, Isaad et al. have developed a new chemodosimeter-sensitized starch film [12], cellulose [13] and water soluble and fluorescent copolymers [14]. Immersion of these functionalized films in an aqueous solution of cyanide induced a color change that can be used for the detection of cyanide. These easy-to-use materials also showed also a high degree of cyanide selectivity in aqueous media [12–14]. Near-infra red (NIR) chemical sensors in the form of cyanine dyes, independent of their operational principle, have the potential to overcome these difficulties by employing long-wavelength absorbance, large extinction coefficients and emission spectra in the visible to near infrared region [9,11]. These dyes have found use as additives to photographic emulsions, laser dyes, optical recording media, solar cells and biological applications such as non-invasive alternatives to radionuclides [11,15–17]. In addition, these dyes can be synthesized and purified by simple methods that do not rely on the use of excessive solvents, such as microwave and solid phase protocols [15,18–20]. These developments have fueled the development of new NIR dyes with desirable photophysical properties as well as synthesized on a large scale [15,17]. NIR chemical sensors such as IR-786 operate in a longer wavelength range (absorption peak between 700 and 900 nm) [21], and exhibit minimal background interferences affording for high sensitivity for anions compared to the short wavelength organic sensors [17,20–22]. Moreover, these dyes are relatively inexpensive and can operate in both aqueous and organic media [9,18,19]. For example, a naturally occurring dye (i.e., 2-hydroxy-1,4-naphthoquinone or lawsone) was used to detect acetate and cyanide in aqueous media [23]. However, lawsone operates in the short-wavelength (visible) region, is prone to background interferences and scattering [9] and therefore is not best suited for biological applications [22,24]. In this work, we demonstrate the use of a cyanine dye, IR-786, which operates in the NIR region, as an anion sensor for the qualitative and quantitative sensing of cyanide in water. IR-786 has a heptamethine bridge with a peak absorption and emission in the range of 760–780 nm and 795–815 nm, respectively [21]. We demonstrated that IR-786 can detect CN´ in 100% MeCN with interference from OH´ and CN´ on solvent-free solid surfaces in a selective manner using absorbance and fluorescence spectroscopy. The proposed chemical sensor, IR-786, is capable of exhibiting NIR absorptions as well emission in both organic and aqueous media, properties not common to other anion probes. 2. Materials and Methods 2.1. Materials and Instrumentation All anions, in the form of sodium or tetrabutylammonium salts, all solvents and IR 786 dye, 98% (Catalog No: 102185035) were purchased from Sigma-Aldrich Chemical Company (St. Louis, MO, USA) and used without further purification. Solid surfaces (chromatography paper, 3 mm, Catalog Number: 3030–6187) were purchased from VWR International Ltd. (Bridgeport, NJ, USA). All UV/vis spectroscopy experiments were carried out in solution phase recorded using a Cary UV/vis-NIR spectrophotometer 5000 (Varian, Walnut Creek, CA, USA) equipped with a quartz cuvette (path length = 1 cm). Fluorescence emission spectra experiments were measured using a Cary 60 series spectrometer (Agilent, Walnut Creek, CA, USA). Spectrophotometric titrations were performed at various concentrations of the sensors in MeCN and water: MeCN mixtures. The 1 H- and 13 C-NMR spectra were recorded using an Avance™ 400 MHz spectrometer (Bruker, Billerica, MA, USA). FT-IR spectra were obtained from an Agilent Cary 630 FTIR spectrometer and reported in cm´1 units.

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2.2. Preparation of IR-786 Stock Solutions Stock solutions of IR-786 (50 µM) (100:0, 75:25, 50:50, 25:75 = H2 O/MeCN) and organic (100% MeCN) media were prepared at room temperature. Aliquots of fresh sodium and tetrabutylammonium salt standard solutions of the anions were added to the IR-786 stock solutions, mixed and the UV/vis and fluorescence spectra of the mixture were recorded as described in the following sections. 2.3. Qualitative Response of IR-786 to Anions in Solution Phase and on Solid Surfaces Solution phase experiments were carried out by addition of 1.0 mL of anions from tetrabutylammonium salts (up to 50 µM in MeCN) to 1.0 mL of IR-786 (50 µM in MeCN). Solid surfaces were cut into 6 mm pieces and incubated in IR-786 in MeCN until the surfaces were completely dry at room temperature. A drop (20 µL) of anion solutions from tetrabutylammonium salts (CN´ and OH´ up to 1000 µM in deionized water) was incubated on the dry solid surfaces with dried IR-786 for 2 min at room temperature. Color changes in solution phase and on solid surfaces were observed visually under normal light and under a hand held UV lamp (λ = 365 nm) upon addition of various anions at room temperature. 2.4. Quantitative Response of IR-786 to Anions in Solution Phase and on Solid Surfaces Standard solutions of IR-786 (5.0 µM) and sodium/tetrabutylammonium salt solutions (1.0 ˆ 10´2 M) of the anions (F´ , AcO´ , H2 PO4 ´ , Br´ , Cl´ , ClO4 ´ and HSO4 ´ ) were prepared in MeCN and in aqueous media. We note that the concentration of IR-786 was reduced to 5.0 µM to obtain a maximum absorbance value less than 1.0 and fluorescence spectroscopy studies were carried out using the identical concentrations for the sake of consistency. The UV-visible titrations were carried out by adding up to 8.0 or 10 µM of selective anions to 2.0 mL solution of IR-786 solution (Note: the total volume of anions added was less than 3.0 µL). After mixing for a few seconds, UV-vis, fluorescence spectra of the samples were recorded. Modifications of UV-vis or fluorescence spectra data were processed through a plot of inverse changes in absorbance/emission band against the inverse of anion concentration. Binding constants were determined by use of Benesi-Hildebrand plots [25] and reported as an average of at least three trials. In this regard, IR-786 (5.0 µM) and each selective anions (5.0 µM) were mixed in the ratio of 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1 in MeCN. After mixing for a few seconds, UV-vis spectra of the samples were recorded at room temperature. 2.5. Characterization of IR-786-(CN) Complex Using 1 H-NMR A stock solution of IR-786 (5.0 µM) and 1.0 mL of tetrabutylammonium cyanide (5.0 µM) was mixed and the color change from green to yellow was observed visually under normal light and under a hand held UV lamp (λ = 365 nm). The mixture was characterized using 1 H-NMR and the summary of peaks are given below: 1 H-NMR (400 MHz, CD3 CN): δ = 8.40 (d; 1H), 7.53 (d; 1H), 7.46 (t; 1H), 7.40 (t; 1H), 7.11 (t; 1H), 7.07 (d; 1H), 6.85 (t; 1H), 6.74 (t; 1H), 6.52 (d; 1H), 6.21 (d; 1H), 5.90 (d; 1H), 5.49 (d; 1H), 3.17 (s; 3H), 3.11 (s; 3H), 2.68 (s; 3H), 2.22 (s; 3H), 1.72 (s; 3H), 1.27 (s; 3H), 1.62 (quin; 2H), 1.36 (sext; 2H), 1.06 (d; 2H), and 0.99 (t; 3H). 2.6. Characterization of IR-786-(OH) Complex Using 1 H-NMR A stock solution of IR-786 (5.0 µM) and 1.0 mL of tetrabutylammonium hydroxide (5.0 µM) was mixed and the color change from green to yellow was observed visually under normal light and under a hand held UV lamp (λ = 365 nm). The mixture was characterized using 1 H-NMR and the summary of peaks are given below: 1 H-NMR (400 MHz, CD3 CN): δ = 8.52 (s; 1H), 7.55 (d; 1H), 7.46 (d; 1H), 7.22 (t; 1H), 7.17 (t; 1H), 6.89 (t; 1H), 6.86 (t; 1H), 6.76 (d; 1H), 6.70 (d; 1H), 5.86 (d; 1H), 5.84 (d; 1H), 5.50 (d; 1H), 5.36 (d; 1H), 3.13 (s; 3H), 3.11 (s; 3H), 3.08 (s; 3H), 2.40 (s; 6H), 1.12 (s; 3H), 1.62 (quin; 2H), 1.36 (sext; 2H), 1.11 (d; 2H), and 0.99 (t; 3H).

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2.7. Characterization of IR-786 in the Presence of Cyanide and Hydroxyl Anions IR-786-(CN)-(OH) 2.7.1Characterization of IR-786 in the Presence of Cyanide and Hydroxyl Anions IR-786-(CN)-(OH) Using Using H-NMR 1 H-NMR

A stock solution of IR-786 (5.0 µM), 1.0 mL of tetrabutylammonium cyanide and 1.0 mL of A stock solution of IR-786 (5.0 μM), 1.0 mL of tetrabutylammonium cyanide and 1.0 mL of tetrabutylammonium hydroxide and the thecolor colorchange changefrom from green yellow was tetrabutylammonium hydroxide(5.0 (5.0µM) μM)was was mixed mixed and green to to yellow was observed visually under normal light and under a hand held UV lamp (λ = 365 nm). The mixture observed visually under normal light and under a hand held UV lamp (λ = 365 nm). The mixture was 1 H-NMR and the summary of peaks are given below: 1 H-NMR (400 MHz, wascharacterized characterized using 1H-NMR using and the summary of peaks are given below: 1H-NMR (400 MHz, CDCD δ =δ 8.54 1H), 7.40 7.40(t; (t;1H), 1H),7.11 7.11(t;(t;1H), 1H), 7.07 1H), 6.85 (t; 1H), 3 CN): 3CN): = 8.54(d; (d;1H), 1H),7.53 7.53(d; (d;1H), 1H), 7.46 7.46 (t; (t; 1H), 7.07 (d;(d; 1H), 6.85 (t; 1H), 6.746.74 (t; 1H), 6.52 (d;(d; 1H), 5.49 (d; (d;1H), 1H),3.17 3.17(s;(s;3H), 3H), 3.11 3H), 2.68 (s; 3H), (t; 1H), 6.52 1H),6.21 6.21(d; (d;1H), 1H),5.90 5.90 (d; (d; 1H), 5.49 3.11 (s;(s; 3H), 2.68 (s; 3H), 2.222.22 (s; (s; 3H), 1.72 (s;(s; 3H), 1.27 (quin; 2H), 2H),1.06 1.06(sext; (sext;2H), 2H),and and 0.99 3H). 3H), 1.72 3H), 1.27(s;(s;3H), 3H),1.62 1.62(h; (h;2H), 2H), 1.36 1.36 (quin; 0.99 (t;(t; 3H). 3. 3. Results Results IR-786 is aiscommercially available cyanine dye, traded as as IR IR 786786 perchlorate (2-(2-[2-chloro-3-([1,3IR-786 a commercially available cyanine dye, traded perchlorate (2-(2-[2-chloro-3([1,3-dihydro-1,3,3-trimethyl-2H-indol-2-ylidene]ethylidene)-1-cyclohexen-1-yl]ethenyl)-1,3,3-tridihydro-1,3,3-trimethyl-2H-indol-2-ylidene]ethylidene)-1-cyclohexen-1-yl]ethenyl)-1,3,3-tri-methylindolium methylindolium with a symmetrical heptamethine bridge [21]. IR-786 perchlorate) with aperchlorate) symmetrical heptamethine bridge (Scheme 1) [21]. (Scheme IR-786 1) possesses useful possesses useful beanion considered an anion sensor: absorption spectral near-infra characteristics to becharacteristics considered astoan sensor:asabsorption spectral near-infra red region, Stokes’ red region, Stokes’ shift (25 nm), large extinction coefficient, yields [16] and conjugation ability to shift (25 nm), large extinction coefficient, high quantum yieldshigh [16]quantum and ability to undergo undergo conjugation with selective anions [9,22]. Therefore, we investigated whether IR-786 cansensor be with selective anions [9,22]. Therefore, we investigated whether IR-786 can be used as an anion used as an anion sensor by observing the changes in its color, absorption and emission spectrum by observing the changes in its color, absorption and emission spectrum before and after the addition before and after the addition of anions (F−, AcO−, CN−, OH−, H2PO4−, Br−, Cl−, N3−, ClO4− and HSO4−) in of anions (F´ , AcO´ , CN´ , OH´ , H2 PO4 ´ , Br´ , Cl´ , N3 ´ , ClO4 ´ and HSO4 ´ ) in organic media and organic media and solvent-free solid surfaces. solvent-free solid surfaces.

Scheme 1. Schematic depiction of the nucleophilic addition of CN− ´ to IR-786. Scheme 1. Schematic depiction of the nucleophilic addition of CN to IR-786.

3.1.3.1. Qualitative Assessment inOrganic Organic(MeCN) (MeCN)Media Media Qualitative AssessmentofofSelectivity SelectivityofofIR-786 IR-786 for for Anions Anions in Upon addition ofof 1.0 anionstotoIR-786, IR-786,only onlythree three anions produced Upon addition 1.0equivalents equivalentsof ofall all the the studied studied anions anions produced a discernable color change from green to yellow as observed by the naked eye: in descending order of a discernable color change from green to yellow as observed by the naked eye: in descending order ´ ´ ´ − − − color intensity, thesethese anions werewere CN CN , OH and F F(Figure 1 and Figure S1).addition The addition of changes color changes intensity, anions , OH and (Figures 1 and S1). The of ´ resulted − resulted − and AcO´ 4 in slight discoloration in the IR-786 solution in MeCN and there were no of AcO andHH2PO PO in slight discoloration in the IR-786 solution in MeCN and there were 2 4 ´ Cl−,´ClO ). ,Upon of exposure IR-786 in in thethe other anions tested (Br−, (Br no noticeable noticeablecolor colorchanges changes other anions tested , Cl4´−,,HSO ClO44−´ HSOexposure 4 ). Upon − and OH− resulted ´ ´ solution after the addition of the anions to a UV light at 365 nm, CN in a green and of IR-786 solution after the addition of the anions to a UV light at 365 nm, CN and OH resulted respectively. color change observable for all other anions and in aorange green emission, and orange emission,No respectively. Nowere color change were observable for(Figures all other1 anions S1). The observed color changes (in Figures 1 and S1) were supported by the absorption spectra as by (Figure 1 and Figure S1). The observed color changes (in Figure 1 and Figure S1) were supported − and OH− in terms of appearance shown in Figure 2a, where IR-786 showed selectivity towards CN ´ the absorption spectra as shown in Figure 2a, where IR-786 showed selectivity towards CN and OH´ of an additional absorbance peak at 430 nm (yellow color). The selectivity of IR-786 towards CN− was in terms of appearance of an additional absorbance peak at 430 nm (yellow color). The selectivity further confirmed by´ratiometric plot of the absorbance values of the two prominent peaks at 430 nm of IR-786 towards CN was further confirmed by ratiometric plot of the absorbance values of the and 775 nm (Figure 2a), where the ratiometric value for CN− is ~10 fold larger than the value for OH−´. two prominent peaks at 430 nm and 775 nm (Figure 2a), where the ratiometric value for CN is There were no significant spectral changes for IR-786 upon the addition of Br−, Cl−, N3−, ClO4− and ~10 fold −larger than the value for OH´ . There were no significant spectral changes for IR-786 upon the HSO4 . addition of Br´ , Cl´ , N3 ´ , ClO4 ´ and HSO4 ´ .

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Figure 1. (a) Color changes of IR-786 (5 μM) under normal light (top) and under hand held UV lamp upon the addition ofUV various (λ = 1. 365(a) in (a) 100% MeCN and(5 (b) aqueous solution H2O)and Figure 1.nm) (a) Color changes IR-786 (5µM) μM) under normal(100% light (top) under hand lamp Figure Color changes ofof IR-786 under normal light (top) and under handheld held UV lamp anions. 2 O) upon the addition of various (λ = 365 nm) in (a) 100% MeCN and (b) aqueous solution (100% H (λ = 365 nm) in (a) 100% MeCN and (b) aqueous solution (100% H2 O) upon the addition of various anions. anions.

Figure 2. (a) UV-vis absorptionspectra spectraand and (b) (b) absorbance absorbance ratiometric value at at Figure 2. (a) UV-vis absorption ratiometricvalues values(absorbance (absorbance value divided absorbance valueatat 775nm) nm) before and after addition equivalence of various Figure 2.by (a)by UV-vis absorption spectra and (b) absorbance ratiometric values (absorbance value at 430430 divided absorbance value 775 before and afterthe the additionof of1.01.0 equivalence of various anions to IR-786 (5 μM) 100% MeCN and(c) (c) emission emission spectra and ratiometric values 430 to divided by(5absorbance value at 775and nm) before and after the addition ofemission 1.0 equivalence of various anions IR-786 µM) inin100% MeCN spectra and(d) (d)emission ratiometric values (intensity nm byintensity intensity value at 800 nm) from anionsvalue to value IR-786 (5520 μM) individed 100% MeCN and (c) emission spectra and (d) emission (intensity at at 520 nm divided by value at 800 nm) obtained obtained from(a). (a).ratiometric values (intensity value at 520 nm divided by intensity value at 800 nm) obtained from (a).

In order to investigate whether the presence of water in the solution can affect the sensitivity In selectivity order to investigate water in themixtures solution(100:0, canaffect affect the sensitivity and of investigate the IR-786whether towards anions, severalof water– 75:25, 50:50, 25:75 In order to whetherthe thepresence presence of waterMeCN in the solution can the sensitivity andwater/MeCN) selectivity of the IR-786 towards anions, several water– MeCN mixtures (100:0, 75:25, 50:50, 25:75 used as the sensoranions, medium. The most noticeable visual change the IR-786 was and selectivitywas of the IR-786 towards several water– MeCN mixtures (100:0,in 75:25, 50:50, 25:75 water/MeCN) was used aswater/MeCN The noticeable visual change the IR-786 was observed with the 25:75 mixture S1 noticeable and S2). The increase in the water content water/MeCN) was used asthe thesensor sensormedium. medium.(Figures The most most visual change ininthe IR-786 was observed with the 25:75 water/MeCN mixture (Figures S1 and S2). The increase in the water content of the IR-786 solution result in no color change, which can be partly attributed to the decrease in the observed with the 25:75 water/MeCN mixture (Figures S1 and S2). The increase in the water content of the IR-786 result inin nonocolor which be content partly attributed attributed tothe the decrease solubility ofsolution IR-786 in the mixture due tochange, the increased water (IR-786 is to not soluble in water of the IR-786 solution result colorchange, which can can partly decrease in in thethe or mixture less than 75%:25% MeCN:water). solubility of IR-786 in the mixture due to to thethe increased water solubility of IR-786 in the mixture due increased watercontent content(IR-786 (IR-786isisnot notsoluble solublein inwater water or In addition to visual observations of the color change in the IR-786 solution with the addition of or mixture less 75%:25% than 75%:25% MeCN:water). mixture less than MeCN:water). anions, of anion response ofchange IR-786 in to the anions was evaluated bythe fluorescence In qualitative addition to assessment visual observations thecolor color change IR-786 solution with addition of of In addition to visual observations ofof the IR-786 solution with the addition − emission spectroscopy. When illuminated with a hand held UV lamp at 365 nm, the addition of CN anions, qualitative assessment of anion response of IR-786 to anions was evaluated by fluorescence anions, qualitative assessment of anion response of IR-786 to anions was evaluated by fluorescence − to IR-786 solutions resulted in green and orange fluorescence emission from IR-786 solution− ´ and OH emission spectroscopy. When illuminatedwith witha ahand handheld heldUV UVlamp lamp at at 365 365 nm, emission spectroscopy. When illuminated nm, the theaddition additionofofCN CN − to IR-786 in 100% MeCN and 25:75 water–MeCN, respectively (Figure 1 and S1). The addition of other anions solutions resulted in green and orange fluorescence emission from IR-786 solution and OH ´ and OH to IR-786 solutions resulted in green and orange fluorescence emission from IR-786 solution −, Br−, Cl−, OH−, N3−) did not result in fluorescence emission. (F AcO−,MeCN H2PO4and in−,100% 25:75 water–MeCN,respectively respectively (Figure (Figure 11 and TheS1). addition of other anions in 100% MeCN and 25:75 water–MeCN, and S1). Figure The addition of other −, H2PO4−, Br−, Cl−, OH−, N3−) did not result in fluorescence emission. (F−, AcO ´ ´ ´ ´ ´ ´ ´

anions (F , AcO , H2 PO4 , Br , Cl , OH , N3 ) did not result in fluorescence emission.

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To further investigate the fluorescence-based response of IR-786 solution to anions, fluorescence spectra To of further the mixtures were measured between 460 nm to 820 nm (Figure 2b). IR-786 in MeCN investigate the fluorescence-based response of IR-786 solution to anions, fluorescence solutions single peak nm, which was 460 completely quenched after theIR-786 addition of CN´ spectra show of theamixtures were800 measured between nm to 820 nm (Figure 2b). in MeCN ´ andsolutions slightlyshow decreased bypeak the addition of OHwas. On the other hand, a after newthe emission peak at −520 andnm a single 800 nm, which completely quenched addition of CN ´ in−conjunction with the disappearance of the peak at 800 nm. wasslightly observed after the addition of CN decreased by the addition of OH . On the other hand, a new emission peak at 520 nm was Theobserved ratiometric of the fluorescence emission with values the two prominent peaks at 520 theofdisappearance of the peak at 800 nm.nm Theand afterplot the addition of CN− in conjunction ´ is ~40 fold larger than the value for OH´ . 800ratiometric nm (Figure 2b),ofwhere the ratiometric values for of CN plot the fluorescence emission values the two prominent peaks at 520 nm and 800 − is ~40 nm qualitative (Figure 2b), observations where the ratiometric values for CN fold larger valuein fororganic OH−. These These imply that IR-786 shows selectivity tothan boththe anions media, ´ ´ ´ ´ were qualitative observations that IR-786 shows to both anions media, however, it is more selectiveimply and sensitive to CN thanselectivity OH . Subsequently, only in CNorganic and OH − than OH−. Subsequently, only CN− and − were however, it is more selective and sensitive to CN used in the remainder of the study presented here. It is important to note that OH´ is OH regarded as − is regarded as an ´ used in the remainder of the study presented here. It is important to note that OH an interference for the detection of CN . interference for the detection of CN−.

3.2. Quantitative Assessment of Selectivity of IR-786 for Anions in MeCN 3.2. Quantitative Assessment of Selectivity of IR-786 for Anions in MeCN

The selectivity of IR-786 to CN´ and OH´ in 100% MeCN and 25:75 water–MeCN mixture was The selectivity of IR-786 tomanner CN− andusing OH− absorbance in 100% MeCN and 325:75 mixture was further assessed in a quantitative (Figure and water–MeCN Figure S3a) and fluorescence further assessed in a quantitative manner using absorbance (Figures 3 and S3a) and fluorescence ´ measurements (Figure 4 and Figure S3b). Upon incremental addition of CN (0.0–8.0 µM) and OH´ measurements (Figures 4 and S3b). Upon incremental addition of CN− (0.0–8.0 μM) and OH− (0.0–10 (0.0–10 µM) (separate experiments), the absorbance band at 775 nm gradually decreased accompanied μM) (separate experiments), the absorbance band at 775 nm gradually decreased accompanied by a by a gradual increase in the new peak at 430 nm (Figure 3a). An isosbestic point at 550 nm indicated the gradual increase in the new peak at 430 nm (Figure 3a). An isosbestic point at 550 nm indicated the formation of a new complex between IR-786 and the anions, i.e., IR-786-(CN) or IR-786-(OH) (Figure 3 formation of a new complex between IR-786 and the anions, i.e., IR-786-(CN) or IR-786-(OH) (Figures and3 Figure S3). Job’s plots displayed a 1:1 stoichiometric complex between IR-786 and CN´ (Figure S4), and S3). Job’s plots displayed a 1:1 stoichiometric complex between IR-786 and CN− (Figure S4), and ´ (Figure S5). In addition, the UV-vis absorption spectra measurements were used andIR-786 IR-786and and OH − (Figure S5). In addition, the UV-vis absorption spectra measurements were used to OH ´ and OH´ from the variation of absorbance at λ to calculate = 430 calculate the the binding binding constants constants of of CN CN− and OH− from the variation of absorbance at λmaxmax = 430 nmnm 5 ´ 1 5 ´ 1 ´ ´, (Figure S6).S6). These constants were found toto bebe 2.72.7 ˆ ×1010M M−1, ,for forCN CN and OH 5 M−1 and − and −, and 2.5 2.5 ˆ × 10 105 M OH (Figure These constants were found ´ − respectively. WeWe note thethe pH ofofIR-786 the titration titrationofofstock stocksolution solution CN respectively. note pH IR-786solution solutiondid didnot not change change the ofof CN ´ in − in andand OHOH buffer buffer(Figure (FigureS7). S7).

Figure 3. UV-vis absorption spectra of IR-786 in 100% MeCN (5 μM) before and after the addition of

Figure 3. UV-vis absorption spectra of IR-786 in 100% MeCN (5 µM) before and after the addition of (a) CN− (up to 8 μM) and (b) OH− (up to 10 μM) and (c) absorbance ratiometric values (absorbance (a) CN´ (up to 8 µM) and (b) OH´ (up to 10 µM) and (c) absorbance ratiometric values (absorbance value at 430 divided by absorbance value at 775 nm) obtained from (a). Arrows show the direction of value at 430 divided by absorbance value at 775 nm) obtained from (a). Arrows show the direction of increased amount of anions. increased amount of anions.

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Figure 4. Fluorescence emissionspectra spectra(λex (λex==430 430 nm, nm, excitation excitation slit slitslit = 20 nm), Figure 4. Fluorescence emission slit==20 20nm, nm,emission emission = 20 nm), − (up to 8 μM) and (b) OH− (up to 8 μM) and (c) fluorescence for IR-786 (50 μM) in 100% MeCN (a) CN for IR-786 (50 µM) in 100% MeCN (a) CN´ (up to 8 µM) and (b) OH´ (up to 8 µM) and (c) fluorescence emission ratiometric values (intensity value at 520 nm divided by intensity value at 800 nm) obtained emission ratiometric values (intensity value at 520 nm divided by intensity value at 800 nm) obtained from (a). Arrows show the direction of increased amount of anions. from (a). Arrows show the direction of increased amount of anions.

In order to determine the detectable concentration range for CN− and OH− in solution, the

In order absorbance to determine detectable concentration range for CN´was and OH´(Figures in solution, ratiometric and the fluorescence plots as a function of concentration plotted 3b the and ratiometric plots as3aaand function concentration was plotted 4b) usingabsorbance the spectral and data fluorescence presented in Figures 4a. Theofratiometric absorbance plot (Figures 3b3b) andshows 4b) using spectral dataofpresented in Figures 3a and 4a.detection The ratiometric absorbance (Figure that: the (i) the addition CN− as low as 1.5 μM (lower limit) result in a colorimetric response MeCN, of where level ratiometric response limit) is measured plotdetectable (Figure 3b) shows that: (i) the in addition CN´the assame low as 1.5ofµM (lower detection result in low as 4.0 μM and (ii) detectable for CN− and OH− was 1.5–6.0 for OH− as a detectable colorimetric response inthe MeCN, whereconcentrations the same levelrange of ratiometric response is measured ´ ´ μM, respectively. These imply that the colorimetric detection CN´− in for μM OHandas7.0–10 low as 4.0 µM and (ii) the observations detectable concentrations range for CN andofOH was MeCN carriedµM, outrespectively. for a narrow These concentration range with interference fromdetection OH−, i.e., of 1.5–6.0 µMcan andbe7.0–10 observations implystrong that the colorimetric ratiometric values are out observed anions. On the otherrange hand,with the ratiometric fluorescence´ in MeCN CNsimilar can be carried for a both narrow concentration strong interference from − in MeCN by IR-786 is more promising due to the large ratiometric response based detection of CN ´ OH , i.e., similar ratiometric values are observed both anions. On the other hand, the ratiometric for CN− (up to 120) in the range of 0.5–7.0 μM and minimal interference by OH− (the ratiometric fluorescence-based detection of CN´ in MeCN by IR-786 is more promising due to the large ratiometric response for OH− was 3- to 500-fold less than that for CN− at 5.0–45 μM). Similar results were obtained response for CN´ (up to 120) in the range of 0.5–7.0 µM and minimal −interference by OH´ (the for IR-786 in 25:75 water–MeCN (Figure S3), where the interference by OH was markedly less in the ratiometric response for OH´ was 3- to 500-fold less than that for CN´ at 5.0–45 µM). Similar results ratiometric absorbance and fluorescence plots as compared to those observed in 100% MeCN. This were obtained for IR-786 in 25:75 water–MeCN (Figure S3), where the interference by OH´ was observation can be attributed to the presence of OH− in the water–MeCN mixture. Consequently, IRmarkedly in the absorbance fluorescence plots as compared to those observed in 786 canless be used asratiometric an anion sensor in 25:75 and water–MeCN. 100% MeCN. This observation can be attributed to the presence of OH´ in the water–MeCN mixture. Consequently, IR-786 can beofused astoanAnions anionon sensor in 25:75 water–MeCN. 3.3. Quantitative Response IR-786 Solid Surfaces The use of IR-786 as a solid-state sensor was also demonstrated. In this regard, the detectable 3.3. Quantitative Response of IR-786 to Anions on Solid Surfaces

concentration range for CN− and OH− on a solid surface with dried IR-786 was determined by The use ofand IR-786 as a solid-state sensor (Figure was also this therange detectable colorimetric fluorescence measurements 5).demonstrated. Figure 5a showsIn that theregard, detectable for ´ and OH´ on a solid surface with dried IR-786 was determined by concentration range forby CN CN− in water sample IR-786 solid state sensor is 50–300 μM: an increase in the concentration of −) to yellow-green colorimetric andinfluorescence measurements (Figure 5). original Figure 5a shows that range for a change in color of the sensor from the green color (nothe CNdetectable CN− resulted ´ − μM) and yellow (>500solid μM ).state Similar changes in the fluorescence emission was observed for of CN(50.0–300 in water sample by IR-786 sensor is 50–300 µM: an increase in the concentration ´ resulted − in water sample: blue emission (no CN−), blue-green (50.0–300 μM) and green μM−). In CNCN in a change in color of the sensor from the original green color (no CN´ )(>500 to yellow-green

(50.0–300 µM) and yellow (>500 µM´ ). Similar changes in the fluorescence emission was observed

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´ ´ forSensors CN´ 2016, in water 16, 271sample: blue emission (no CN ), blue-green (50.0–300 µM) and green (>500 8 ofµM 12 ). ´ In addition, the potential interference by OH was also investigated. Figure 5b shows that the addition − was also investigated. Figure 5b shows that the addition ´ (up the addition, potential by OH of OH to 1000 µM) interference on to the solid surface with dried IR-786 did not result in significant changes − (up to 1000 μM) on to the solid surface with dried IR-786 did not result in significant changes of OH in color and fluorescence emission. These results demonstrate the utility of IR-786 dye as a selective ´ color and fluorescence emission. These of IR-786 dye astowards a selective CNin sensor with minimal interference fromresults OH´ .demonstrate We note thatthe theutility sensitivity of IR-786 CN´ − sensor with minimal interference from OH−. We note that the sensitivity of IR-786 towards CN− CN is lower than those novel chemical sensors reported in the literature [12–14]. However, our results is lower than those novel chemical sensors reported in the literature [12–14]. However, our results show that IR-786 is effective in the detection of CN´ within the current limits set by EPA.

show that IR-786 is effective in the detection of CN− within the current limits set by EPA.

Figure 5. Real-colorphotographs photographsof ofcolorimetric colorimetric and from IR-786 dried on on to to Figure 5. Real-color and fluorescence fluorescenceemission emission from IR-786 dried −´ −. ´ and (b) OH solvent-free solid surfaces before and after the addition of (a) CN solvent-free solid surfaces before and after the addition of (a) CN and (b) OH .

Elucidation BindingMechanism Mechanismfor forAnions Anions in in MeCN 3.4.3.4. Elucidation of of Binding MeCN It is important to understand the binding mechanisms between IR-786 and the anions to gain It is important to understand the binding mechanisms between IR-786 and the anions to gain further insights to anion sensing applications of IR-786. In this regard, the characterization of IR-786 further insights to anion sensing applications of IR-786. In this regard, the characterization of IR-786 in in the absence and presence of individual anions and both anions were carried out by FT-IR (Figure the absence and presence of individual anions and both anions were carried out by FT-IR (Figure 6) 6) and NMR spectroscopy (data presented in the Materials and Methods section). The FT-IR spectrum andofNMR (data presented in the Materials andbetween Methods section). Thecm FT-IR spectrum −1 and lacks solid spectroscopy IR-786 has characteristic peaks for aromatic groups 2800 and 2980 ´1 and lacks of solid IR-786 has characteristic peaks for aromatic groups between 2800 and 2980 cm −1 −1 two important bands (–CN: 2200 cm and OH: 3224 cm ) due to the absence of these groups in the ´1 and OH: 3224 cm´1 ) due to the absence of these groups in the twomolecule important bands [26,27]. A (–CN: strong 2200 peakcm for –CN at 2200 cm−1 appeared when IR-786 is dissolved in MeCN − is added molecule A strong for –CN atat2200 cm´1 appeared when IR-786 is dissolved in MeCN and thepeak aromatic peaks 2800–2990 cm−1 became stronger due to the conversion and CN[26,27]. ´ ´1 became stronger andofCN is added the aromatic peaks at 2800–2990 due to the conversion aromatic ringsand to aliphatic groups. A broad peak for cm –OH at 3224 cm−1 was observed when only ´1 was observed when only − −1 of aromatic rings to aliphatic groups. A broad peak for –OH at 3224 cm OH is added to IR-786, where –CN peak at 2200 cm still exists (due to MeCN). The presence of both ´ is − and − in OHto the same solution of IR-786 resulted observation ofto allMeCN). three characteristic peaks: OHCN added IR-786, where –CN peak at 2200 cm´1instill exists (due The presence of both ´ ´ −1 , less pronounced –OH peak 3224 cm−1 and lessthree pronounced aromatic 2200 cmsolution CNstrong and –CN OH peak in the same of IR-786 resulted in observation of all characteristic peaks: −1. ´ − and OH− 1 ´ 1 peaks at 2800–2990 cm There are two possible explanations for these observations: (i) CN strong –CN peak 2200 cm , less pronounced –OH peak 3224 cm and less pronounced aromatic 1 . There ´ and binds the different of IR-786 and/or (ii) a mixture of IR-786-(OH) IR-786-(CN)(i)can exist peaks at to 2800–2990 cm´parts are two possible explanations for theseand observations: CN ´ when both anions are present in the same IR-786 solution. In addition, the pH of the IR-786 solution OH binds to the different parts of IR-786 and/or (ii) a mixture of IR-786-(OH) and IR-786-(CN) can didwhen not change significantly, which in implies the effect of pH on observed fluorescence emission exist both anions are present the same IR-786 solution. In addition, the pH of the was IR-786 negligible. In order to further understand the binding mechanism for CN− and OH−, 1H-NMR solution did not change significantly, which implies the effect of pH on observed fluorescence emission spectroscopy was employed. ´ ´ 1

was negligible. In order to further understand the binding mechanism for CN and OH , H-NMR spectroscopy was employed.

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100 IR + CN in MeCN

Transmittance Transmittance

100 80 IR in MeCN IR ++ CN CN +inOH MeCN 80 IR + OH in MeCN 60 IR + CN + OH in MeCN 60 40 40 20

IR + OH in MeCN IR solid IR solid

20 0 4000 0 4000

3000

2000

1000 -1

Wavenumber (cm 3000 2000

)

1000

-1 Figure 6. FT-IR spectra of IR-786 dye in different forms: solid as purchased, in MeCN and with CN−

Wavenumber (cm ) Figure 6. FT-IR spectra of IR-786 dye in different forms: solid as purchased, in MeCN and with CN´ − alone and in MeCN and with OH− and CN−. alone, in MeCN and with OH alone, in MeCN and with OH´ alone and in MeCN and with OH´ and CN´ . − Figure 6. FT-IR spectra of IR-786 dye in different forms: solid as purchased, in MeCN and with CN

− alone and in MeCN and with OH− and CN−. alone, in MeCNspectrum and with OH The 1H-NMR of IR-786-(CN) indicates the presence of eight aromatic protons (δH: 8.39,

1

The7.46, H-NMR spectrum of IR-786-(CN) indicates the presence of eight protons (δHsix : 8.39, 7.53, 7.40, 7.11, 7.07, 6.85 and 6.74), four CH protons (δH: 6.52, 6.21, 5.90aromatic and 5.49 as doublet), 1H-NMR spectrum of IR-786-(CN) indicates the presence offor eight aromatic (δcyanide Hsix : 8.39, 7.53, 7.46, 7.40, 7.11, 6.85 6.74), protons (δH : 6.52, 6.21, 5.90 and 5.49 asprotons doublet), –CH3 –CH 3The protons (δH7.07, : 3.17, 3.11,and 2.68, 2.22,four 1.72CH and 1.27) and four peaks tetrabutylammonium − 7.53, 7.46, 7.40, 7.11, 7.07, 6.85 and 6.74), four CH protons (δ H : 6.52, 6.21, 5.90 and 5.49 as doublet), six protons (δ : 3.17, 3.11, 2.68, 2.22, 1.72 and 1.27) and four peaks for tetrabutylammonium cyanide (δH: 1.62, H 1.36, 1.06 and 0.99). The addition of CN to IR-786 resulted in disappearance of the polarized(δH : ´ to1.27) –CH 3 protons (δH0.99). : 3.17,The 3.11, 2.68, 2.22, four peaks forAs tetrabutylammonium cyanide 1.62, 1.36, 1.06of and addition of1.72 CNand IR-786 resulted in disappearance of in theScheme polarized C=N C=N bond indolium group and the formation of and a new C-N bond. illustrated 1, the − to IR-786 resulted in disappearance of the polarized H : 1.62, 1.36, 1.06 and 0.99). The addition of CN (δ reaction mechanism can be the explained by the addition of cyanide anion with the bond of indolium group and formation of nucleophilic a new C-N bond. As reaction illustrated in Scheme 1, the reaction C=N bondcan of be indolium and the formation of aobservations newreaction C-N bond. As illustrated in Scheme 1, the polarized C=N bond ofgroup theby indolium group. These clearly indicated the mechanism explained the nucleophilic addition of cyanide anion that with thecyanide polarized reaction mechanism can be explained by the nucleophilic addition reaction of cyanide anion with the . anion was added to the C=N group of IR-786 and suggested 1:1 reaction between IR-786 and CN C=N bond of the indolium group. These observations clearly indicated that the cyanide anion was −added polarized C=N bond of the indolium group. These observations clearly indicated that the6.89, cyanide ´ H : 7.53, 7.46, 7.22, 7.17, 6.86, The spectrum of [IR-786-(OH)] shows eight aromatic protons (δ to the C=N group of IR-786 and suggested 1:1 reaction between IR-786 and CN . −. anion was6.70), added to CH the C=N group IR-786 and suggested 1:1 reaction between and CN :of5.86, 5.84, and 5.36 as doublet), five 7.46, –CHIR-786 3 protons H 3.13, 6.76 and four protons (δHshows The spectrum of [IR-786-(OH)] eight5.50 aromatic protons (δH : 7.53, 7.22, 7.17,(δ6.89, 6.86, H: 7.53, 7.46, The spectrum of [IR-786-(OH)] shows eight aromatic protons (δ H:7.22, 1.62,7.17, 1.36,6.89, 1.116.86, and 2.40 andCH 1.12) and four tetrabutylammonium hydroxide (δ–CH 6.763.11, and3.08, 6.70), four protons (δHpeaks : 5.86,for 5.84, 5.50 and 5.36 as doublet), five 3 protons (δH 3.13, : 5.86, 5.84, 5.50 5.36 as doublet), five –CH3 protons (δH 3.13, 6.76 four CH protons (δHIR-786-(OH) 0.99).and The6.70), signals for–OH bonds of andand tetrabutylammonium hydroxide are observed at 3.11, 3.08, 2.40 and 1.12) and four peaks for tetrabutylammonium hydroxide (δH : 1.62, 1.36, 1.11 and − anion H: 1.62, 1.36, 1.11 and 3.11, 3.08, and 2.40 7.21 and ppm 1.12) as and four peaks for tetrabutylammonium 8.52 ppm a singlet, respectively. As illustrated inhydroxide Scheme 2,(δ OH was added 0.99). The signals for–OH bonds IR-786-(OH) and tetrabutylammonium tetrabutylammoniumhydroxide hydroxide observed 0.99). signals for–OH bondsof IR-786-(OH) and areare observed at at to the The indole group and formed aofnew C-OH bond. An intramolecular charge transfer from the indole ´ anion 8.528.52 ppm and 7.21 ppm as a singlet, respectively. As illustrated in Scheme 2, OH was added − ppm and 7.21 group ppm as singlet, respectively. illustrated in Scheme 2, OH was group. added to group to indolium of a[IR-786-(OH)] occurs asAs a result of ࣊-delocalization fromanion the indole thetoindole group and formed a new C-OH bond. An intramolecular charge transfer from the indole the indole group and formed a new C-OH bond. An intramolecular charge transfer from the indole group to indolium group as aaresult resultofof࣊-delocalization π-delocalization from indole group. group to indolium groupofof[IR-786-(OH)] [IR-786-(OH)] occurs occurs as from thethe indole group.

Scheme 2. Schematic depiction of nucleophilic addition of OH− to IR-786. Scheme 2.in Schematic depiction of nucleophilic addition ofwith OH− that to IR-786. The main difference the 1H-NMR spectrum of IR-786-(OH) of IR-786-(CN) is the Scheme 2. Schematic depiction of nucleophilic addition of OH´ to IR-786. presence of the hydroxyl proton signal at δH 8.32 (1H, s) for IR-786-(OH) and a shift in the signals of − an OH− ions The protons main difference in the H-NMR spectrum of IR-786-(OH) IR-786-(CN) is the are aromatic to up field. The 1chemical shifts for aromatic protonswith showthat thatofCN 1 The main difference in the ofs)IR-786-(OH) with that of IR-786-(CN) presence of the hydroxyl proton H-NMR signal at δspectrum H 8.32 (1H, for IR-786-(OH) and a shift in the signalsisofthe − anin − ions presence of protons the hydroxyl proton at δH 8.32for (1H, s) for IR-786-(OH) shift the signals OH are of aromatic to up field. Thesignal chemical shifts aromatic protons showand thataCN

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aromatic protons to up field. The chemical shifts for aromatic protons show that CN´ an OH´ ions are bound to different carbon atoms. Subsequently, IR-786-(OH) compound displays an intramolecular charge transfer from the indole group to indolium group, which is supported by the change in color of the solution from green to yellow as observed by colorimetric measurements. In addition, the 1 H-NMR spectra of IR-786-(CN)-(OH) and IR-786-(CN) are very similar: the peaks of aromatic protons of IR-786-(CN)-(OH) appear at 8.54, 7.53, 7.46, 7.40, 7.11, 7.07, 6.85 and 6.74 ppm, –CH protons in appear at 6.52, 6.21, 5.90 and 5.50 and –CH3 protons appear at 3.17, 3.11, 2.68, 2.22, 1.72 and 1.27 ppm, which supports the conclusions reached by the FT-IR results. Our research laboratory is currently working on the design of chromogenic biosensors based on cyanine dyes with better solubility in aqueous media by replacing the chlorine group with a high capacity electron-donating or withdrawing groups (such as methyl, nitro, methoxyl and benzo in increasing order) and/or binding with nucleic acids in the heptamethine bridge [16,17]. The results of these investigation will be reported in due course. 4. Conclusions IR-786, a heptamethine cyanine dye was demonstrated to be a highly selective and sensitive sensor for cyanide anions in water–MeCN mixtures and solvent-free solid surfaces. IR-786 exhibited three unique properties: broad spectral range, and a large hypsochromic (blue) shift of about 345 nm (from 775 nm to 430 nm) in the presence of cyanide anions. The mechanism for the binding of cyanide and hydroxide anions to IR-786 was elucidated using FT-IR and NMR spectroscopy, where cyanide and hydroxide are thought to bind to different carbons on the IR-786 backbone. The most probable mechanism is the nucleophilic addition of CN´ to the indolium group of IR-786, as supported by the observed changes in absorption spectrum. The use of IR-786 in a solvent-free solid state sensor for the selective sensing of CN´ in the environment was demonstrated, where CN´ in the range of 50–300 µM in water samples with minimal interference by OH´ was detected using colorimetric and fluorescence based detection methods. Supplementary Materials: The following are available online at http://www.mdpi.com/1424-8220/16/3/271/s1, Figure S1: Color changes of IR-786 (50 µM) under normal light and under hand held UV lamp (λ = 365 nm) in MeCN: water mixtures = 75%:25%, 50%:50%, 25%:75% before and after the addition of various anions. Figure S2: (a) UV-vis absorption spectra and (b) absorbance ratiometric values (absorbance value at 430 divided by absorbance value at 775 nm) before and after the addition of 1.0 equivalence of various anions to IR-786 in 75%:25% MeCN–water mixture (50 µM) and (c) emission spectra and (d) emission ratiometric values (intensity value at 520 nm divided by intensity value at 800 nm) obtained from (a). Figure S3: (a) UV-vis absorption spectra of IR-786 in 75%:25% MeCN–water mixture (50 µM) before and after the addition of CN´ (up to 8.0 µM) and (b) absorbance ratiometric values (absorbance value at 430 divided by absorbance value at 775 nm) obtained from (c) Fluorescence emission spectra (λex = 430 nm, excitation slit = 20 nm, emission slit = 20 nm), for IR-786 (5.0 µM) 75%:25% MeCN–water mixture before and after the addition of OH´ (up to 10 µM) and (d) fluorescence emission ratiometric values (intensity value at 520 nm divided by intensity value at 800 nm) obtained from (c). Arrows show the direction of increased amount of anions. Figure S4: Job’s plots for the determination of the binding stoichiometry between IR-786 (5.0 µM) and CN´ in (a) 100% MeCN (b) 75%:25% MeCN–H2 O mixture based on absorbance at λmax = 430 nm and (c) 100% MeCN (d) 75%:25% MeCN–H2 O mixture based on fluorescence emission at λmax = 520 nm. Figure S5: Job’s plot for the determination of the binding stoichiometry between IR-786 (5.0 µM) and OH´ in 100% MeCN based on absorbance at λmax = 430 nm. Figure S6: Plots for the determination of the binding constants between IR-786 (50 µM) and CN´ in (a) 100% MeCN (b) 75%:25% MeCN–H2 O mixture based on absorbance at λmax = 430 nm and (c) 100% MeCN (d) 75%:25% MeCN–H2 O mixture based on fluorescence emission at λmax = 520 nm. Figure S7: Change in pH of IR-786 solution in MeCN during the titration of stock solution of CN´ and OH´ in buffer as described in the experimental section. Volume of anions correspond to 0–20 µM. Acknowledgments: Authors would like to thank Morgan State University for the funds to cover the costs to publish in open access. Author Contributions: K.A. conceived and designed the experiments; B.B., I.B., E.B., S.T. performed the experiments; Y.M.H. analyzed the data; K.A. and I.B. wrote the paper. Conflicts of Interest: The authors declare no conflict of interest.

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Abbreviations The following abbreviations are used in this manuscript: IR 786: IR 786 perchlorate or 2-(2-[2-Chloro-3-([1,3-dihydro-1,3,3-trimethyl-2H-indol-2-ylidene]ethylidene)1-cyclohexen-1-yl]ethenyl)-1,3,3-trimethylindolium perchlorate MeCN: Acetonitrile References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.

15. 16. 17. 18.

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