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Oct 3, 2016 - Department of Chemistry, Institute of Technology (NIT)-Kurukshetra, ... Motilal Nehru National Institute of Technology Allahabad, Allaha...
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Selective detection of cyanide in water and biological samples by an off-the-shelf compound Rahul Kaushik, Amrita Ghosh, Ajeet Singh, Prachi Gupta, Ashwani Mittal, and D. Amilan Jose ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.6b00519 • Publication Date (Web): 03 Oct 2016 Downloaded from http://pubs.acs.org on October 3, 2016

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Selective detection of cyanide in water and biological samples by an off-the-shelf compound Rahul Kaushik1, Amrita Ghosh1, Ajeet Singh2, Prachi Gupta3, Ashwani Mittal3 and D. Amilan Jose*1 1

Department of Chemistry, NIT-Kurukshetra, Kurukshetra, Haryana- 136119, India Email: [email protected] Tel: +91-1744233559 2

Department of Physics, Motilal Nehru National Institute of Technology Allahabad, 211004 Allahabad, India

Skeletal Muscle Lab, Biochemistry Department, University College, Kurukshetra University, Haryana – 136119, India. 3

KEYWORDS: Coumarin, Copper complex, Cyanide, Molecular sensing and Fluorescent sensor

ABSTRACT: Simple off-the-shelf chemical 6,7-dihydroxycoumarin (1) based copper complex(1.Cu²⁺) has been used for the selective detection of toxic cyanide in aqueous medium. The DFT calculation confirms the binding behavior between 1 and Cu2+ (2:1) and red shift in UV-Vis spectrum with copper ion was confirmed by the decrease in energy between HOMOLUMO band gaps. The cyanide sensing in water was confirmed by both absorption and emission spectral studies. Cyanide ion showed 13 fold increments in fluorescent intensity in emission spectrum via displacement of copper from 1.Cu²⁺. The limit of detection of CN- in water is 5.77 M, 1.Cu²⁺ also applicable for the detection of cyanide in fresh mouse serum with detection limit of 14.4 M. The cell images showed that 1.Cu²⁺ could be used to detect intracellular CN- .

The extreme toxicity of cyanide at low concentration demands its detection and determination. Hence, recognition of trace amount of cyanide in biological and environmental samples by simple and sensitive methods is the area of immense interest.1-4 Cyanides occur naturally both in the geologic and biologic samples. The binding of cyanide to the active sites of cytochrome C oxidase inhibits the oxygen transfer in human body which leads to hypoxiation.1,5 Cyanide poisoning affects the humans and animals cardiovascular, respiratory and central nervous systems.1 According to the World Health Organization (WHO) cyanide limit in drinking water is 70 μg/L and the maximum contaminant level for cyanide set by the US EPA in drinking water is 200 μg/L. Therefore it is highly important to detect the trace amount of cyanides in different matrices such as water, food, soil, air, living cells and biological fluids like serum, blood and urine. Traditional methods such as electrochemical6, potentiometry7, polarography8, flow Injection amperometric9 and simple titrations10 are time consuming and need skilled persons for the operation11-14. Therefore, alternative and more sensitive methods that can directly measure cyanide at μg/L level in different matrices are competitive. In this regard fluorescent and colorimetric measurements of cyanide are more popular.2,15 Various methods such as nucleophilic attacks on carbonyl carbon 16-18, hydrogen bonding mechanism19, and cyanide addition on alkenes20,21 are the few approaches widely reported for cyanide sensing. However, due to their poor solubility in aqueous media, they have their own draw backs with respect to detection limit and detection time in physiological conditions.

To resolve this problem, the potential use of a metal displacement approach has drawn much attention22,23. Metal complexes have been effectively used due to high affinity between metal and cyanide ion. They work under the similar mechanism of Anslyn’s Indicator displacement approach24-27. That is, at first, Emission intensity of the receptor is quenched by copper and then on addition of cyanide the emission intensity is recovered. Here copper or metal act as an indicator. Generally complex of cyanide is less toxic than free cyanide. Many metal complexes are known in literature for the detection of cyanide using colorimetric and fluorescent approach. But most of these reported complexes needed expensive reactants28-30 multistep synthesis of ligand31-33, purification by different separation technique34 and different characterization techniques were required and they are very time consuming. To avoid the multistep synthesis and characterization, we have found that an off-the-shelf compound 6,7-dihydroxycoumarin(1) a suitable compound to make complex with copper in water and thereby binding with cyanide ion in water. Here for first time we have observed that compound 1 effectively bind with copper in (DMSO-Water, 1% - 99%) mixture to form 1.Cu²⁺ and this simple copper complex used for the efficient detection of cyanide in water and biological samples. EXPERIMENTAL SECTION Cary eclipse fluorescence spectrophotometer was used to record the fluorescence spectra. Evolution 201 UV-Vis Spectrophotometer was used to obtain UV-Vis absorption spectra using quartz cells of 1.0 cm path length. Deionized water was used to prepare buffer solution and the required pH were

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maintained using HACH sension 2. Nikon Eclipse Ti-S Fluorescence Inverted Microscope was used for cell imaging. Materials and reagent 6,7-dihydroxycoumarin(1), tetra-butylammonium cyanide and all metal perchlorate salts were purchased from SigmaAldrich. Other anion salts and chemicals were purchased from Loba Chemie, Merck limited unless otherwise mentioned. Mouse serum was obtained from Hi Media Chemical limited. Solvents used were analytically pure and used without further purification. Cell culture and Fluorescence microscopic imaging The C2C12 mouse skeletal muscle cell lines were grown in Dulbecco’s modified eagle’s medium (DMEM) supplemented with 20% fetal bovine serum in an ambient atmosphere of 5% CO2 and 95% humidified air at 37 °C for about 24 h. Cells at 60% confluency were fixed with paraformaldehyde and then the cells were treated with the relative compounds for fluorescence microscopic imaging. The fluorescence microscopy experiments were performed on fluorescence microscope (Nikon Eclipse Ti-S Fluorescence Inverted Microscope) with the excitation between 420 and 495 nm using FITC filter and the total magnification was 100X. RESULT AND DISCUSSION

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The compound 1 shows intense absorption band centered at 380 nm (ϵ = 11578 mol-1 L cm-1). Upon addition of Cu2+ this band red shifted to 395 nm (ϵ = 15263 mol-1 L cm-1) (Figure 1a), with a color change from light yellow to dark yellow easily detectable through naked eyes. This can be explained by the stabilization of charge in the excited state after the formation of 1.Cu2+ complex which enhances the ICT mechanism and gives a red shift. However, no such spectral or visual change was observed with different competitive metal cations such as Ca2+, Ni2+, Zn2+, Pb2+, Cd2+, Fe3+, Sr2+, Ag+, Mg2+, Hg2+, K+, Na+, Li+, Al3+ (Figure S1). These results indicated that the selective nature of 1 towards Cu2+ in water, thereby formation of 1.Cu2+ ensemble (2:1) via coordination of Cu2+ with adjacent –OH groups as shown in scheme 1. The sensitivity of compound 1 towards Cu2+ was studied by UV-Vis titration as shown in Figure 1b. Upon addition of Cu2+ maximum absorption at 380 nm gradually shows a red shift along with enhancement and gives a new absorbance band at 395 nm (Figure 1b) with an isosbestic point at 370 nm. The two adjacent –OH groups present on 1 are prone to deprotonation at neutral pH (pH= 7 - 7.4) and this deprotonation enhanced in the presence of metal ions due to internal charge transfer (ICT) or chelation enhanced fluorescence (CHEF) mechanisms which stabilizes the excited state and shows a red shift in the UV-Vis spectra.23,35

(A)

Scheme 1: Schematic representation of 1 and 1.Cu2+. Metal ion binding behavior of compound 1 was examined by UV-Vis absorption spectra in HEPES-DMSO (99:1, 10 mmol, pH = 7.4).

(B)

(A)

(C) (B) Figure 2: A) Emission spectra of 1 (7.6 ×10⁻5 M) with different metal cations (3.8 ×10⁻5 M). Wavelength Ex = 380nm, Wavelength emission = 470 nm, Slit Width – 2.5/2.5 nm. B) Emission titration of 1 (1.52× 10⁻4 M), Cu2+ (0- 7.6 ×10⁻5 M); Wavelength Ex = 380 nm, Wavelength emission = 470 nm, Slit Width– 5/5 nm. C) Under UV light, photograph of 1 with different metals: 1) 1, 2) 1+ Cu2+, 3) Ca2+, 4) Ni2+, 5) K+, 6) Na+, 7) Li+, 8) Al3+ 9) Zn2+, 10) Pb2+, 11) Fe3+, 12) Cd2+, 13) Sr2+, 14) Ag+, 15) Mg2+, 16) Hg2+. Figure 1: A) UV-Vis absorbance spectra of 1 (7.6 ×10⁻5 M) with different metal cations (3.8 ×10⁻5 M). B) UV-Vis absorbance titration of 1 (7.6 ×10⁻5 M) with Cu2+ (0 - 3.8 ×10⁻5 M).

Compound 1 showed an emission maximum at 475 nm (exc = 380 nm) in HEPES: DMSO (99:1, 10 mmol, pH = 7.4). In the

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emission spectra, 1 shows almost 15 fold quenching of the fluorescence intensity in presence of Cu2+ (Figure 2a). The quenching observed is due to the paramagnetic nature of Cu2+ and formation of 1.Cu2+ ensemble.36 Other metals such as Ca2+, Ni2+, Cd2+, Sr2+, Ag+, Mg2+, Hg2+, K+, Na+, Li+ and Al3+ do not show any significant change. However, a considerable change in fluorescence intensity was observed with Zn2+, Pb2+ and Fe3+. The decrease in fluorescence intensity follows the order: Cu2+> Pb2+> Fe3+> Zn2+. The interference studies shows that that there is no such effect on Cu2+ sensing in presence of different competitive common metal ions on the emission intensity (Figure S2). The stoichiometry of 1.Cu²⁺ was calculated as 2:1 (1: Cu2+) using the Jobs’ plot, wherein the total concentration of both 1 and Cu2+ taken as (1.52 ×10⁻4 M) and mole fraction varied from 0 to 1 (Figure S3). Emission titration for probe 1 was carried out with different concentration of Cu2+. Upon successive addition of Cu2+ to 1, the fluorescence intensity at 470 nm was quenched almost 15 folds due to the formation of 1.Cu2+ ensemble (Figure 2b). The detection limit of Cu2+ was found out to be 120 nM (Figure S4). The association constant (Ka) for the 1.Cu2+ ensemble was calculated using the fluorescence titration and found out to be 4.09 × 104 M-1 (Figure S5). Quantum Mechanical Calculation

Effect of Cyanide with 1.Cu2+: Cyanide ions known to coordinate with Cu2+ ions to form a very stable complex Cu(CN)x, 38-40 We have investigated selectivity of the 1.Cu2+ complex toward CN− using the copper–cyanide affinity. The effects of anions with 1.Cu²⁺ were examined by UV- Vis and emission spectroscopy with different competitive anions such as F¯, Cl¯, Br¯, I¯, NO2¯, NO3¯, H2PO4¯, HPO4¯, CH3COO¯, N3¯, SO32¯, S2O32¯ and HCO3¯. The absorption spectroscopy result shows that the peak of 1.Cu2+ ensemble at 395 nm is shifted almost 15 nm to the lower wavelength in the presence of CN¯.

(A)

(B)

To confirm the binding behavior of 1 and Cu2+ i.e. 2:1 and the red shift observed in the absorbance spectra, we have performed quantum mechanical calculations by using Gaussian 0937.

(C)

Figure 4: A) UV-Vis absorbance spectra of 1.Cu2+ with different anions (1.3 ×10⁻4 M). (B) Emission spectra of 1.Cu2+ with different anions (3.5 ×10⁻4 M). Wavelength Ex = 380 nm, Wavelength emission = 470 nm, Slit Width – 5/5. C) Emission photograph of 1.Cu2+ with different anions: 1) blank, 2) CN¯, 3) F¯, 4) Cl¯, 5) Br¯, 6) I¯, 7) NO2¯, 8) H2PO4¯, 9) HPO4¯, 10) CH3COO¯, 11) N3¯, 12) SO32¯, 13) S2O32¯,14) NO3¯, 15) NO., 16) HCO3¯.

Figure 3: Quantum mechanical HOMO and LUMO calculations of 1 and 1.Cu2+ using LANL2DZ for Cu2+ and 6-31G(d) basis set for C, H, O. Calculations of frontier molecular orbitals HOMO (highest occupied molecular orbital) and LUMO (lowest unoccupied molecular orbital) energy and band gap were performed in gas phase. The structure were optimized using LANL2DZ for Cu2+ and 6-31G(d) basis set for C, H, O. The results confirmed that the band gap between HOMO and LUMO of 1 was found out to be 4.15 eV. However, after the formation of 1.Cu2+ ensemble the band gap decreases to 1.03 eV (Figure 3). These results confirm that there is a decrease in energy and bathochromic shift in the wavelength, which corroborate the spectral shift in the experimental absorption spectra.

However, no such spectral change was observed with other competitive anions (Figure 4a). This shows the selective nature of the ensemble 1.Cu2+ towards CN¯ in water. In the fluorescence spectrum only in the presence of CN¯ ion the emission intensity of 1.Cu2+ enhanced to 13 fold and recovered the original fluorescence emission of 1 due to displacement of copper from 1.Cu2+ ensemble. Other anions do not show any increase in emission intensity (Figure 4b). The effect of 1.Cu2+ ensemble with CN¯ was systematically investigated by the absorption and emission spectral titration method as shown in Figure 5. With the successive addition of CN¯ to the 1.Cu2+, the hypsochromic shift (from 395 nm to 380 nm) was observed due to the decomplexation and formation of Cu(CN)n-(n.2) (n = 2 or 4) and the regeneration of 1 in the solution41. The detection limit of detection (LOD) of CN¯ using UV-Vis spectral data was found out to be 6.42 µM (Figure S8a). LOD using emission spectral titration data was found

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out to be 5.77 µM (Figure 5c). LOD calculated by both UV-Vis and fluorescence titration closely matching. Emission studies shows that the initially 1.Cu2+ is an OFF state with low fluorescence intensity in presence CN¯ ion it becomes ON state with 13 folds increase in the fluorescence intensity (Figure 5b). Therefore based on the above experiments results we can state that the probe 1.Cu2+ could be used as a fluorescent OFF-ON sensor for the selective detection of CN¯ in water. Here, Cyanide exhibits metal displacement mechanism and displace copper from the ensemble 1.Cu2+ and destabilize the excited state by blocking the ICT or CHEF mechanisms and from this reversible process 1 is reproduced.

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common ion effect, which inhibits binding of copper metal. The absorbance and emission spectrum does not show considerable change with Cu2+ at pH less than 6 (Figure S10). However, sensing of CN¯ depends upon the stability of 1.Cu2+ at different pH. We have studied the effect of pH on cyanide detection using UV-Vis and emission spectroscopy techniques by taking 1.Cu2+ at different pH such as 4, 6, 7.4, 8, 10 and 12. Higher stability of 1.Cu2+ ensemble at pH 6, 7.4, 8, 10, 12 enable to detect the cyanide ion. (Figures S11a and 11b), but cyanide detection is not effective at pH less than 6 (Figure S11b). These results indicate that the probe can be useful for the detection of cyanide in the pH range of 6.0–12.0 (Figure 6).

(A)

(B)

Figure 6: Change in UV-Vis absorbance intensity of 1.Cu2+ with CN¯ (2 ×10⁻4 M) at 395 nm at different pH values. Detection of cyanide in mouse serum Cyanide measurement is the great interest of research field not only from analytical chemists, but also researchers from diverse medical and clinical arena42,43. Thus, we have explored the cyanide detection behavior of 1.Cu2+ in the mouse serum spiked with cyanide ions. UV-Vis and fluorescence spectral experiments in fresh mouse serum obtained from Hi-media chemical limited, Mumbai were performed.

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(A)

Figure 5: (A) UV-Vis absorbance titration of 1.Cu2+ with CN¯ (0-1.3 ×10⁻4 M). B) Emission titration of 1.Cu2+ with CN¯ (0 3.5 ×10⁻4 M). C) Detection limit plot of CN¯. Wavelength Ex = 380nm, Wavelength emission = 470 nm, Slit Width – 5/5.)

(B)

pH dependence on sensing mechanism The role of Cu2+ and CN¯ in the biological systems and industrial processes are extremely important and undeniable. Therefore, it is important for a sensing probe to be efficient and applicable for detection over a wide range of pH. To check the pH dependence, 1 and 1.Cu2+ were checked over a wide range of from pH 4- 12. Initially the pH effect of 1 with copper was recorded using both UV-Vis and emission spectra and we found that the binding of copper is effective from pH 6 to 12, due to presence of high concentration of hydroxide ions which enhances the deprotonation of –OH group. This facilitates the Cu2+ to bind with the naked adjacent oxygen atoms in 1 (Figure S9). At pH values less than 6, the competition between the protons of -OH group and the metal ion could occur. Therefore, 1 in its deprotonated form becomes less stable due to

Figure 7: A) UV-Vis absorbance spectra of 1.Cu2+ {1 (1×10⁻4 M), Cu2+ (0.5 ×10⁻4 M)} with CN¯ spiked mouse serum (0, 4×10⁻5M, 8×10⁻5M, 1.2×10⁻4M, 1.6×10⁻4M, 2.0×10⁻4M). B) Emission spectra of 1(1×10⁻4 M), Cu2+ (0.5 ×10⁻4 M) with CN¯ spiked mouse serum (0, 4×10⁻5M, 8×10⁻5M, 1.2×10⁻4M, 1.6×10⁻4M, 2.0×10⁻4M).

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The absorbance and emission studies of 1.Cu2+ were performed in fresh mouse serum spiked with cyanide (2.0×10⁻2M) ion in buffer, followed by centrifugation at 3000 rpm for 5 minutes. The supernatant liquid of different concentration (0, 4×10⁻5 M, 8×10⁻5 M, 1.2×10⁻4 M, 1.6×10⁻4M, 2.0×10⁻4M) of cyanide spiked mouse serum was added to 1.Cu2+ in buffer. The UVVis spectrum shows that the absorbance intensity decreases along with the hypsochromic shift at 395 nm and color change was observed light green to light yellow with an isosbestic point at 370 nm (Figure 7a). In emission spectra, fluorescence intensity increases gradually at 470 nm after addition of cyanide spiked mouse serum of different concentration (0, 4×10⁻5M, 8×10⁻5M, 1.2×10⁻4M, 1.6×10⁻4M, 2.0×10⁻4M) to 1 (1×10⁻4M) (Figure 7b). The detection limit of CN¯ using emission spectra was found out to be 14.4 M (Figure S13). However, reported quantity of cyanide in fire victim is about 26 M.44 Usually CN concentrations of blood in fire victims (both survivors and fatalities) are raised after inhaling the smoke containing HCN. This result shows that the probe 1.Cu2+ can be used to detect the CN¯ in real biological samples. Fluorescence cell imaging Encouraged by the results obtained from mouse serum experiment, we have also explored the biological application of 1 and 1.Cu2+ at cellular level (Figure 8). Cell image studies were carried out by earlier reported procedures23 with some modifications (See SI). (A)

Control (bright-field) 10X

(B)

1

of 1 and Cu2+. Then the cell lines were supplemented with 1 mM of Cyanide in the medium and incubated for 30 min at RT. On imaging cyanide treated cells showed high fluorescence in the intracellular area as shown in Figure 8. This high fluorescence is due to the removal of copper from the 1.Cu2+ that is already present in the intracellular area. These experiments revealed that the probe 1.Cu²⁺ could be used to track the trace amount of cyanides in living cells. CONCLUSION: In summary, we have reported a simple off-the-shelf chemical 6, 7-dihyroxycoumarin (1) shows significant change in the absorbance and emission spectral (turn off fluorescence) behavior in the presence of copper in aqueous medium. 2:1 complex formation of 1 and Cu2+ was confirmed by DFT calculation and binding studies. This simple easy to prepare 1.Cu2+ ensemble shows a considerable fluorescence and UV-Vis change in presence only in cyanide anion as compared to other anions. It shows emission ON signal in emission spectrum and the detection limit is in micromolar range and effective over a wide range of pH. Ensemble 1.Cu2+ is also applicable for the detection of cyanide in biological fluid ie, fresh mouse serum. Furthermore the probe also has been utilized for fluorescence imaging of cyanide ion in C2C12 cells under physiological conditions. Overall the ensemble 1.Cu2+ is having the following advantages over other cyanide sensor a) very cheap and easily available, no complicated synthesis is required. b) It could sense Cu2+ and simultaneously CN¯ in almost pure aqueous medium. c) LOD is very low. d) Effective over a wide range of pH. e) 1.Cu2+ can detect CN¯ in real biological samples like mouse serum and C2C12 cell lines with detection limit of permissible perimeter. ASSOCIATED CONTENT

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1.Cu2+

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1.Cu2+ with CN¯

Supporting Information Available: The following file is available free of charge. Final SI, the contents of which are: General procedures: of UV-Visible absorption and Fluorescence experiments, Cell culture and Fluorescence microscopic imaging; Interference studies; Jobs’ plot; Detection limit and Binding study plots; Studies at different pH; Studies with spiked mouse serum; Reversibility in the presence of CN-; Gas phase optimize cartesian for 1 and 1.Cu2+. AUTHOR INFORMATION

Figure 8: A) Bright field image of 1 (500 M). B) Fluorescence image of 1 (500 M) only. C) Fluorescence image of 1 with Cu2+ (250 M) only. D) Fluorescence image of 1.Cu2+ after treatment with CN¯ (1000 M) using C2C12 mouse skeletal muscle fixed cell lines with 50-60% confluency using FITC UV filter with total magnification of 100 x. Initialy, the C2C12 cells lines were incubated with ligand 1 (0.5 mM) in buffer at RT for 45 mins and imaged through fluorescence microscope (Nikon Eclipse Ti-S Fluorescence Inverted Microscope) after washing the live cells with buffer for several times to remove the 1 present in the extracellular medium. It is found that cells incubated with 1 display a strong fluorescence (Figure 8a). Further, Cu2+ (0.025 mM) was added into the ligand 1 treated cells and the images were recorded. It was found that the images display weak fluorescence in presence

Corresponding Author Email: [email protected] Notes No competing financial interests have been declared. ACKNOWLEDGEMENTS: We acknowledge the Department of Chemistry, NIT Kurukshetra for the research facilities. DAJ acknowledge the financial support for the SERB-DST, New Delhi project grant SB/FT/CS-195/2013 and CSIR grant No.01 (2855)/16/EMR-II. AG thankful to the financial support of DST, India for the SERB-DST young scientist research project grants (SB/FT/CS193/2013). AS thanks to SERB, New Delhi (SB/FT/CS-127/2014) for the financial support REFERENCES

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(1) Wang, F.; Wang, L.; Chen, X.; Yoon, J. Recent progress in the development of fluorometric and colorimetric chemosensors for detection of cyanide ions. Chem. Soc. Rev. 2014, 43, 4312-4324. (2) Xu, Z.; Chen, X.; Kim, H. N.; Yoon, J. Sensors for the optical detection of cyanide ion. Chem. Soc. Rev. 2010, 39, 127-137. (3) Cho, D.-G.; Sessler, J. L. Modern reaction-based indicator systems. Chem. Soc. Rev. 2009, 38, 1647-1662. (4) Mohr, G. J. New chromogenic and fluorogenic reagents and sensors for neutral and ionic analytes based on covalent bond formation–a review of recent developments. Anal. Bioanal. Chem. 2006, 386, 1201-1214. (5) Ensafi, A. A.; Kazemifard, N.; Rezaei, B. Label-free and turn-on fluorescent cyanide sensor based on CdTe quantum dots using silver nanoparticles. RSC Adv. 2015, 5, 40088-40093. (6) Kaloo, M. A.; Sankar, J. Reusable and specific proton transfer signalling by inorganic cyanide in solution and solid phase. Chem. Commun. 2015, 51, 14528-14531. (7) Shan, D.; Mousty, C.; Cosnier, S. Subnanomolar Cyanide Detection at Polyphenol Oxidase/Clay Biosensors. Anal. Chem. 2004, 76, 178-183. (8) Bohrer, D.; do Nascimento, C. P.; Garcia Pomblum, S.; Seibert, E.; Machado de Carvalho, L. Polarographic determination of cyanide as nickelcyano complex in blood plasma after selective extraction in a methylene blue impregnated polyethylene column. Fresenius J. Anal. Chem. 1998, 361, 780-783. (9) Dadfarnia, S.; Haji Shabani, M. A.; Tamadon, F.; Rezaei, M. Indirect determination of free cyanide in water and industrial waste water by flow injection-atomic absorption spectrometry. Microchim. Acta 2007, 158, 159-163. (10) Recalde-Ruiz, D. L.; Andres-Garcia, E.; Diaz-Garcia, M. E. Fluorimetric flow injection and flowthrough sensing systems for cyanide control in waste water. Analyst 2000, 125, 2100-2105. (11) Suzuki, T.; Hioki, A.; Kurahashi, M. Development of a method for estimating an accurate equivalence point in nickel titration of cyanide ions. Anal. Chim. Acta 2003, 476, 159-165. (12) Fillaut, J.-L.; Akdas-Kilig, H.; Dean, E.; Latouche, C.; Boucekkine, A. Switching of Reverse Charge Transfers for a Rational Design of an OFF–ON Phosphorescent Chemodosimeter of Cyanide Anions. Inorg. Chem. 2013, 52, 4890-4897. (13) Sukato, R.; Sangpetch, N.; Palaga, T.; Jantra, S.; Vchirawongkwin, V.; Jongwohan, C.; Sukwattanasinitt, M.; Wacharasindhu, S. New turn-on fluorescent and colorimetric probe for cyanide detection based on BODIPY-salicylaldehyde and its application in cell imaging. J. Hazard. Mater. 2016, 314, 277-285.

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(14) Jeong, Y.-H.; Lee, C.-H.; Jang, W.-D. A Diketopyrrolopyrrole-Based Colorimetric and Fluorescent Probe for Cyanide Detection. Chem. Asian J. 2012, 7, 1562-1566. (15) Kang, N.-Y.; Ha, H.-H.; Yun, S.-W.; Yu, Y. H.; Chang, Y.-T. Diversity-driven chemical probe development for biomolecules: beyond hypothesisdriven approach. Chem. Soc. Rev. 2011, 40, 3613-3626. (16) Goswami, S.; Paul, S.; Manna, A. Carbazole based hemicyanine dye for both "naked eye" and 'NIR' fluorescence detection of CN- in aqueous solution: from molecules to low cost devices (TLC plate sticks). Dalton Trans. 2013, 42, 10682-10686. (17) Lee, H. J.; Park, S. J.; Sin, H. J.; Na, Y. J.; Kim, C. A selective colorimetric chemosensor with an electron-withdrawing group for multi-analytes CN- and F. New J. Chem 2015, 39, 3900-3907. (18) Wang, L.; Zhu, L.; Cao, D. A colorimetric probe based on diketopyrrolopyrrole and tert-butyl cyanoacetate for cyanide detection. New J. Chem 2015, 39, 7211-7218. (19) Saha, S.; Ghosh, A.; Mahato, P.; Mishra, S.; Mishra, S. K.; Suresh, E.; Das, S.; Das, A. Specific Recognition and Sensing of CN− in Sodium Cyanide Solution. Org. Lett. 2010, 12, 3406-3409. (20) Park, S.; Kim, H.-J. Highly activated Michael acceptor by an intramolecular hydrogen bond as a fluorescence turn-on probe for cyanide. Chem. Commun. 2010, 46, 9197-9199. (21) Yuan, L.; Lin, W.; Yang, Y.; Song, J.; Wang, J. Rational Design of a Highly Reactive Ratiometric Fluorescent Probe for Cyanide. Org. Lett. 2011, 13, 3730-3733. (22) Kaushik, R.; Ghosh, A.; Jose, D. A. Simple terpyridine based Cu(II)/Zn(II) complexes for the selective fluorescent detection of H2S in aqueous medium. J. Lumin. 2016, 171, 112-117. (23) Kaushik, R.; Kumar, P.; Ghosh, A.; Gupta, N.; Kaur, D.; Arora, S.; Jose, D. A. Alizarin red Szinc(ii) fluorescent ensemble for selective detection of hydrogen sulphide and assay with an H2S donor. RSC Adv. 2015, 5, 79309-79316. (24) Nguyen, B. T.; Anslyn, E. V. Indicatordisplacement assays. Coord. Chem. Rev. 2006, 250, 31183127. (25) Wiskur, S. L.; Ait-Haddou, H.; Lavigne, J. J.; Anslyn, E. V. Teaching Old Indicators New Tricks. Acc. Chem. Res. 2001, 34, 963-972. (26) Joyce, L. A.; Shabbir, S. H.; Anslyn, E. V. The uses of supramolecular chemistry in synthetic methodology development: examples of anion and neutral molecular recognition. Chem. Soc. Rev. 2010, 39, 3621-3632. (27) Sakakibara, K.; Joyce, L. A.; Mori, T.; Fujisawa, T.; Shabbir, S. H.; Hill, J. P.; Anslyn, E. V.; Ariga, K. A Mechanically Controlled Indicator Displacement Assay. Angew. Chem. Int. Ed. 2012, 51, 9643-9646.

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(28) Reddy G, U.; Das, P.; Saha, S.; Baidya, M.; Ghosh, S. K.; Das, A. A CN- specific turn-on phosphorescent probe with probable application for enzymatic assay and as an imaging reagent. Chem. Commun. 2013, 49, 255-257. (29) Park, G. J.; You, G. R.; Choi, Y. W.; Kim, C. A naked-eye chemosensor for simultaneous detection of iron and copper ions and its copper complex for colorimetric/fluorescent sensing of cyanide. Sens. Actuators, B 2016, 229, 257-271. (30) Jose, D. A.; Elstner, M.; Schiller, A. Allosteric Indicator Displacement Enzyme Assay for a Cyanogenic Glycoside. Chem. Eur. J. 2013, 19, 14451-14457. (31) Long, L.; Zhou, L.; Wang, L.; Meng, S.; Gong, A.; Du, F.; Zhang, C. A highly selective and sensitive fluorescence ratiometric probe for cyanide and its application for the detection of cyanide in natural water and biological samples. Anal. Methods 2013, 5, 6605-6610. (32) Singh, P.; Mittal, L. S.; Kumar, S.; Bhargava, G.; Kumar, S. Perylene Diimide Appended with 8-Hydroxyquinoline for Ratiometric Detection of Cu2+ Ions and Metal Displacement Driven "Turn on" Cyanide Sensing. J. Fluoresc. 2014, 24, 909-915. (33) Long, L.; Wang, L.; Wu, Y.; Gong, A.; Da, Z.; Zhang, C.; Han, Z. Reaction-Based Fluorescent Probe for Detection of Endogenous Cyanide in Real Biological Samples. Chem Asian J. 2014, 9, 3291-3298. (34) Khairnar, N.; Tayade, K.; Bothra, S.; Sahoo, S. K.; Singh, J.; Singh, N.; Bendre, R.; Kuwar, A. Novel fluorescent chemosensing of CN- anions with nanomolar detection using the Zn2+isonicotinohydrazide metal complex. RSC Adv. 2014, 4, 41802-41806. (35) Zhang, L.; Dong, S.; Zhu, L. Fluorescent dyes of the esculetin and alizarin families respond to zinc ions ratiometrically. Chem. Commun. 2007, 18911893. (36) Li, J.; Chen, S.; Zhang, P.; Wang, Z.; Long, G.; Ganguly, R.; Li, Y.; Zhang, Q. A Colorimetric and Fluorimetric Chemodosimeter for Copper Ion Based on the Conversion of Dihydropyrazine to Pyrazine. Chem. Asian J. 2016, 11, 136-140. (37) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennucci, B.; Petersson, G. A.; Nakatsuji, H.; Caricato, M.; Li, X.; Hratchian, H. P.; Izmaylov, A. F.; Bloino, J.; Zheng, G.; Sonnenberg, J. L.;

Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.; Montgomery Jr., J. A.; Peralta, J. E.; Ogliaro, F.; Bearpark, M. J.; Heyd, J.; Brothers, E. N.; Kudin, K. N.; Staroverov, V. N.; Kobayashi, R.; Normand, J.; Raghavachari, K.; Rendell, A. P.; Burant, J. C.; Iyengar, S. S.; Tomasi, J.; Cossi, M.; Rega, N.; Millam, N. J.; Klene, M.; Knox, J. E.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Martin, R. L.; Morokuma, K.; Zakrzewski, V. G.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Dapprich, S.; Daniels, A. D.; Farkas, Ö.; Foresman, J. B.; Ortiz, J. V.; Cioslowski, J.; Fox, D. J.; Gaussian, Inc.: Wallingford, CT, USA, 2009. (38) Jung, H. S.; Han, J. H.; Kim, Z. H.; Kang, C.; Kim, J. S. Coumarin-Cu(II) Ensemble-Based Cyanide Sensing Chemodosimeter. Org. Lett. 2011, 13, 5056-5059. (39) Liu, Y.; Lv, X.; Zhao, Y.; Liu, J.; Sun, Y.Q.; Wang, P.; Guo, W. A Cu(ii)-based chemosensing ensemble bearing rhodamine B fluorophore for fluorescence turn-on detection of cyanide. J. Mater. Chem. 2012, 22, 1747-1750. (40) Bhalla, V.; Singh, H.; Kumar, M. Triphenylene based copper ensemble for the detection of cyanide ions. Dalton Trans. 2012, 41, 11413-11418. (41) Das, S.; Biswas, S.; Mukherjee, S.; Bandyopadhyay, J.; Samanta, S.; Bhowmick, I.; Hazra, D. K.; Ray, A.; Parui, P. P. A cyanide selective off-on fluorescent chemosensor with in vivo imaging in 100% water: solid probe preferred over in situ generation. RSC Adv. 2014, 4, 9656-9659. (42) Lindsay, A. E.; Greenbaum, A. R.; O’Hare, D. Analytical techniques for cyanide in blood and published blood cyanide concentrations from healthy subjects and fire victims. Anal. Chim. Acta 2004, 511, 185-195. (43) Baud, F. J.; Barriot, P.; Toffis, V.; Riou, B.; Vicaut, E.; Lecarpentier, Y.; Bourdon, R.; Astier, A.; Bismuth, C. Elevated Blood Cyanide Concentrations in Victims of Smoke Inhalation. N Engl J Med 1991, 325, 1761-1766. (44) Kumari, N.; Jha, S.; Bhattacharya, S. A Chemodosimetric Probe Based on a Conjugated Oxidized Bis-Indolyl System for Selective Naked-Eye Sensing of Cyanide Ions in Water. Chem. Asian J. 2012, 7, 2805-2812.

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