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May 11, 2016 - Phenothiazine-diaminomalenonitrile based Colorimetric and. Fluorescence “Turn-off-on” Sensing of Hg2+ and S2−. K. Muthu Vengaiana, ...

Sensors and Actuators B 235 (2016) 232–240

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Phenothiazine-diaminomalenonitrile based Colorimetric and Fluorescence “Turn-off-on” Sensing of Hg2+ and S2− K. Muthu Vengaian a , C. Denzil Britto a , Karuppannan Sekar a,∗ , Gandhi Sivaraman b , Subramanian Singaravadivel c,∗ a

Department of Chemistry, Anna University—University College of Engineering, Dindigul 624622, India Institute for Stem Cell Biology and Regenerative Medicine, Bangalore 560065, India c Department of Chemistry, SSM Institute of Engineering and Technology, Dindigul 624002, India b

a r t i c l e

i n f o

Article history: Received 27 February 2016 Received in revised form 27 April 2016 Accepted 28 April 2016 Available online 11 May 2016 Keywords: Phenothiazine Diaminomalenonitrile Mercury Sensor Fluorescence

a b s t r a c t In this paper a novel, easily available, “on-off-on” fluorescent and colorimetric probe based on Phenothiazine was synthesized and characterized by 1 H NMR, 13 C NMR and ESI–MS. Results showed selective and sensitive recognition towards Hg2+ and S2− with no significant interference with other competitive metal ions and anions. The detection limit of Hg2+ was found to be 17.8 nM. Furthermore the probe has been utilized for fluorescence imaging of Hg2+ in live cells. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Recognition and sensing of heavy and transition metal ions are of considerable current interest in supramolecular chemistry because of their significant importance in chemical, biological, and environmental assays [1]. As one of the most toxic and ubiquitous heavy-metal ions of pollutants, Mercury (Hg2+ ) arisen from a variety of natural and anthropogenic sources, has attracted a particular attentions [2–6]. Mercuric ions can be converted by bacteria into methyl mercury, which is the most common organic mercury and more toxic than inorganic mercury because of its lipophilic nature and easily absorbed by aquatic organisms [7,8]. The biological targets and toxicity profile of mercury species depend on their chemical composition [9,10]. Mercury species that can readily pass through biological membranes [11] are powerful neurotoxicants [12] to fish animal and humans [13,14]. The United States Environmental Protection Agency (EPA) has set a maximum Hg2+ contaminant level in food and drinking water at 0.002 mg L−1 (0.01 M) [15]. Therefore, it is very important to detect the level of mercury in water and develop a simple yet

∗ Corresponding authors. E-mail addresses: [email protected] (K. Sekar), [email protected] (S. Singaravadivel). 0925-4005/© 2016 Elsevier B.V. All rights reserved.

environmentally friendly mercury sensor with high sensitivity and selectivity [1,7,16]. Development of probes for the sensing Hg2+ ions is of great importance due to their implications in broad areas of chemistry, biology, and environment. As fluorescence measurement provides a powerful way for detecting metal ions because of its low detection limit and simple instrumentation, considerable efforts have been devoted to design fluorescent chemical sensors. Many fluorescent probes based on rhodamine [17], coumarin [18], as well as other fluorophores [19–22] have developed for Hg2+ ions detection. The presence of mercury causes quenching in fluorescence because Hg2+ often acts as an efficient fluorescence quencher like many other heavy- and transition-metal (HTM) ions through an effective spin-orbit coupling [23–26] The sensors that exhibit “turn-on” response upon binding with metal ions are less sensitive over “turn-off” sensors due to lack of background signal [27–29]. Phenothiazine and its derivatives are used in different areas such as dyes, probes and electrochemistry[30]. Phenothiazine can provide with strong fluorescence, structural regulation and good hole transport capacity. It is widely used as an electron donor in organic light-emitting diodes (OLEDs) and dye-sensitized solar cells [31–34], that exhibits intense luminescence and high photoresponsivity. To the best of our knowledge, only limited reports of fluorescent probes based on PTZ exist [35–37]. In this work, we designed and synthesized a Phenothiazine derivative as a new colorimetric chemosensor P-1 (Scheme 1),

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Scheme 1. Synthesis of P-1.

in which 10-Hexyl-10H-Phenothiazine-3-Carbaldehyde serves as chromophore core and diaminomaleonitrile was coupled to form Schiff base derivative. The intramolecular charge transfer (ICT) is thus enhanced as a result of the extended -conjugation and the stronger electron-withdrawing ability of the nitrile group. The extended ICT usually exhibits high sensitivity to external perturbations such as the polarity of solution and the electric field in its vicinity, often shows remarkable color change. The new developed imine P-1 act as chelating sites of metallic cations, in particular transition and post-transition metal cations. 2. Experimental

pressure to give the crude product, which was purified by column chromatography on silica gel using ethyl acetate/hexane (2:98, v/v) to afford pure compound 2-((10-hexyl-10H-phenothiazin7-yl)methyleneamino)-3-aminomaleonitrile (P-1) as dark brown powder. Yield: 90%; Mp: 191 ◦ C; IR KBr (cm−1 ): 3407 (NH2 ), 3295 (-NH, str), 3203 ( C H), 2969 (C H, str), 2232 (C N), 1590 (NH, bend), 1472 (C H, bend), 1365 (C H, rock), 1258 (C N, str); 1 H NMR (300 MHz, DMSO-d6 ): 0.90(t, 3H, J = 6 Hz); 1.32(bs, 4H); 1.46(q, 2H, J = 6 Hz); 1.63(bs, 2H); 4.00(t, 2H, J = 6 Hz); 5.14(s, 2 H); 6.85-7.11(m, 2 H); 7.14-7.22(m, 2 H); 7.28(s, 1 H); 7.52(d, 1H, J = 9 Hz); 7.62(bs, 1 H); 8.09(s, 1 H). Mass (ESI–MS): 401.017 Calcd, 401.17.

2.1. Materials and general methods

2.3. Stock solution preparation for spectral detection

All reagents and solvents were used without purification. Phenothiazine and 2,3-diaminomalenonitrile was purchased from Acros Organics. Metal chloride salts procured from Merck were used as the source for metal ions. Absorption measurements were carried out using JASCO V-530 UV–vis spectrophotometer. Fluorescence spectra were recorded on an F-4500Hitachi fluorescence spectrophotometer. The slit width was 5 nm for both excitation and emission. NMR spectra were recorded on a Bruker (Avance) 300 M Hz instrument using TMS as internal standard. ESI–MS spectral analysis was performed in positive ion mode on a liquid chromatography-ion trap mass spectrometer (LCQ Fleet, Thermo Fisher Instruments Limited, US). Fluorescence microscopic images were taken with a Nikon fluorescence microscope using a filter.

The nitrate salts of Ag+ , Al3+ , Ba2+ , Ca2+ , Cd2+ , Co2+ ,Cr3+ , Cu2+ , Hg2+ , K+ , Mg2+ , Mn2+ , Na+ , Ni2+ , Pb2+ and Zn2+ were prepared in ethanol-water (6:4) mixture as stock solutions (1 mMol). The P-1 stock solution (1 mMol) was prepared in ethanol-water (6:4) mixture. The working solutions of P-1 were freshly prepared by diluting the highly concentrated stock solution to the desired concentration prior to spectroscopic measurements. Fe3+ ,

2.4. UV–vis-fluorescence titration studies The absorption and fluorescence responses of the probe P-1 towards various metal ions was investigated by UV–vis spectroscopy and fluorescence spectroscopy respectively in ethanol-water (6:4) mixture.

2.2. Synthesis 2.5. MTT assay 2.2.1. Synthesis of probe P-1 To a mixture of 2, 3 diaminomalenonitrile (0.1 g, 0.93 mmol) and dry Ethanol (10 mL) were added 10-Hexyl-10H-Phenothiazine3-Carbaldehyde 3 (0.3 g, 0.96 mmol) under nitrogen atmosphere. The reaction mixture was refluxed at 78 ◦ C for 6 h. After the reaction was completed, the solvent was removed under reduced

The cell viability of the probe P-1 were tested against HeLa cell lines using the 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. The cells were seeded into a well plate at a density of 1.5 × 104 cells per well and incubated in medium containing RDP-1 at concentrations ranging from 0 to 50 ␮M for


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Fig. 1. UV–vis spectrum of probe P-1 (10 ␮M) upon addition of Hg2+ (0–1 equiv.) in ethanol-water (6:4) mixture.

48 h. To each well, 100 ␮L of MTT was added and the plates were incubated at 37 ◦ C for 4 h to allow MTT to form formazan crystals on reacting with metabolically active cells. The medium with MTT was removed from the wells. Intracellular formazan crystals were dissolved by adding 100 ␮L of DMSO to each well and the plates were shaken for 10 min. The absorbance was recorded using Plate reader.

2.6. Cell culture and fluorescence imaging HeLa cells were grown in modified Eagle’s medium supplemented with 10% FBS (fetal bovine serum) at 37 ◦ C. The HeLa cells were incubated with the probe P-1 (10 ␮M in DMSO/H2 O (8:2, v/v) buffered with HEPES buffer) and imaged through fluorescence microscope. The cells were washed with HEPES three times to remove the excess of the probe P-1 in the extra cellular parts and growth medium. Again the probe treated cells were further incubated with HgCl2 (10 ␮M in H2 O) for 10 min at 37 ◦ C and imaged with Nikon fluorescence microscope.

3. Results and discussion 3.1. Synthesis of P-1 To a mixture of 2,3 diamino malenonitrile (0.93 mmol) and dry Ethanol (10 mL) were added 10-Hexyl-10H-Phenothiazine3-Carbaldehyde (0.96 mmol) under nitrogen atmosphere. The ◦ reaction mixture was refluxed at 78 C for 6 h. After the reaction was completed, the solvent was removed under reduced pressure to give the crude product, which was purified by column chromatography on silica gel using Ethyl acetate/Hexane (2:98, v/v) to afford pure compound P-1 as dark brown powder in good yield (Scheme 1). P-1 was characterized by 1 H NMR, and ESI–MS (Fig. S1-2).

3.2. UV–vis spectral response to metal ions The absorption and fluorescence behaviour response of the probe P-1 towards various metal ions was investigated by UV–vis spectroscopy and fluorescence spectroscopy respectively ethanolwater in EtOH H2 O (6:4 v/v). The absorption spectra of the probe P-1 shows three characteristic peaks around 287, 340, 425 nm. The changes in the photonic properties with the addition of metal ions like Zn2+ , Mg2+ , Co2+ , Cu2+ , Ba2+ , Al3+ , Mn2+ , Pb2+ , Hg2+ , Cd2+ , Fe3+ , Ni2+ , Ca2+ , Na+ and K+ is shown in Fig. S3. Upon addition of Hg2+ (0 − 2 eq) the probe P-1 exhibited red shift of 425 nm peak to 448 nm which clearly indicates the presence of ground state interactions of probe and Hg2+ ion (Fig. 1). There was no spectral changes observed in the presence of other competitive metal ions such as Zn2+ , Mg2+ , Co2+ , Cu2+ , Ba2+ , Al3+ , Mn2+ , Pb2+ , Cd2+ , Fe3+ , Ni2+ , Ca2+ , Na+ and K+ (all the salts studied herein were taken as nitrates to eliminate the effect of anions Fig. S3), indicating that the UV–vis response of probe P-1 is highly specific and selective to Hg2+ .

3.3. Fluorescence titration and selectivity The fluorescent properties of the probe P-1 in the presence of various metal ions were studied in (ethanol–water, 6:4). The probe P-1 displayed emission upon excitation at 425 nm. The addition of Hg2+ ion culminated in an intense emission band quenching at 550 nm (Fig. 2). The fluorescence titration of P-1 with 0–1 equivalent of Hg2+ ions gradually quenches the fluorescence intensity at 550 nm. An excitation at the absorption maximum, ␭ex = 425 nm of the complex, while other competitive metal ions such as Zn2+ , Mg2+ , Co2+ , Cu2+ , Ba2+ , Al3+ , Mn2+ , Pb2+ , Cd2+ , Fe3+ , Ni2+ , Ca2+ , Na+ and K+ (10 ␮M) does not respond to the emission of the probe when coexisted with Hg2+ (Fig. S4, S5). The effect of pH was studied in acidic pH the fluorescence of the probe P-1 is completely quenched whereas in basic pH the probe P-1 remains fluorescent (Fig. S6). Addition of 1 equiv. of Na2 S makes the quenched fluorescence restore at 550 nm. It is found that sulfide ion also increases the fluorescence intensity of [P-1 + Hg2+ ] ensemble in concentration dependence. [P–1 + Hg2+ ] ensemble displays a high sensitivity to

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Fig. 2. Fluorescence emission spectrum of Probe P-1 (10 ␮M) upon addition of Hg2+ (␭ex = 425 nm) (0–1 equiv) in ethanol-water (6:4) mixture.

Fig. 3. Fluorescence response of [P–1 + Hg2+ ] ensemble to Na2 S.

sulfide ions (Fig. 3) to form the stable species compared to that of other anions (Fig. S7). The fluorescence of the probe was not affected with Na2 S, even though sulfide ions are strong nucleophile it does not undergo any reaction with the probe. This is attributed to the fact that mercury is often associated with its high affinity for sulfur, such that it binds effectively with sulfide ions and sulfur containing compounds. Also the formation constant for HgI2 is less when compare to HgS. Hence the probe-Hg (II) ensemble prefers to bind with Sulfide rather than iodide [38,39]. The selectivity of P-1 +Hg(II) with other sulfur containing species were studied. The thiol containing amino acids were checked with the P–1 + Hg(II), the amino acids cys, hcy showed the emission enhancement but not like as sulfide ions. But GSH and Cystine does not show any

fluorescence enhancement. It clearly indicates that the P–1 + Hg(II) ensemble selectively used for detection of sulfide ions (Fig. S8). To confirm the stoichiometry of the binding of probe P-1 with Hg2+ ion Job’s plots analysis was carried out. The plot of the absorbance variation at 425 nm against mole fraction clearly showed the maxima with a mole fraction at 0.6 indicating 1:2 stoichiometry and the proposition is further supported by the peak at 1003.1725 (P–1 + Hg2+ ) in ESI–MS analysis (Fig. S9). Similarly Job’s plots were derived from fluorescence analysis which also conforms to the 1:2 binding of P-1 with Hg2+ ions (Fig. 4). The binding constant of P-1 with Hg2+ was calculated and it is found to be 4.18 × 104 M−1 [40]. A linear response of the fluorescence intensity as a function of [Hg2+ ] was observed from 30 nM to 10 ␮M


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Fig. 4. Job’s plot of mole fraction of Hg2+ vs fluorescence intensity at ␭ex = 550 nm.

Scheme 2. Plausible mechanism for sensing Hg2+ and S2− by P-1.

(R2 = 0.9989) (Fig. S10). The detection limit (DL) was calculated from this, the lower detection limit was found to be 17.8 × 10−9 M [41]. This may be ascribed due to an ICT (internal charge transfer) process, the N Hexyl Phenothiazine is an strong electron-donating group when diaminomalenonitrile moieties were complexed with Hg2+ , weaker electron-withdrawing effect will inhibit the original ICT (internal charge transfer) process from the N Hexyl Phenothiazine group to the diaminomalenonitrile moieties. Thus the emission is quenched by addition of Hg2+ and recovered again with the addition of S2− . All these properties indicates that the capa-

bility of P-1 and [P–1 + Hg2+ ] ensemble for quantitative detection Hg2+ and S2− respectively as an “on-off-on” type probe. Plausible mechanism for sensing Hg2+ and S2− by P-1 is shown in Scheme 2.

3.4. NMR titrations The 1 H NMR titrations experiment were performed to understand the nature of interactions between the P-1 and the Hg2+ ion (Fig. 5). The comparison of the 1 H NMR spectra of P-1 and P-1 with 0.5 equivalent of Hg2+ ion suggest that the addition of Hg2+ to the

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Fig. 5. Partial 1 H NMR (300 M Hz) spectra (a) Probe P-1, (b), Probe P–1 + 0.5equiv Hg2+ in DMSO-d6.

Fig. 6. Bright field image and fluorescence images of HeLa cells. (A) Bright field image of control cells, (B) Fluorescence imaging of probe P-1(10 ␮M), (C) Fluorescence imaging of P-1 after addition of Hg2+ , (D) Fluorescence imaging of probe P–1 + Hg2+ ensemble after addition of S2− , (E) Bright field image of P-1 after addition of Hg2+ , (D) Bright field image of probe P–1 + Hg2+ ensemble after addition of S2− .

P-1 solution caused a down field shift in the signal corresponds to NH2 (5.14 ppm–5.41 ppm) and N CH (8.12 ppm–8.24 ppm) protons, it shows that Hg2+ is bound to the receptor through coordination of Hg2+ to the lone pair electrons of nitrogen atom in amine and imine site [42,43]. Also the protons attached with Phenothiazine aromatic system slightly shift towards down field especially, the protons of imine functionality shift more downfield Ha (7.62 ppm–8.00 ppm), Hb (7.52 ppm–7.83 ppm) and Hc

(7.28 ppm–7.93 ppm) due to strong electron donating ability of Phenothiazine ring to diaminomalenonitrile moiety during complexation with Hg2+ ion. 3.5. Application in living cells The MTT assay was adopted to study cytotoxicity (Ic 50 value is 53.89 ␮mol) of the probe P-1 at varying dose and time dependent


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Fig. 7. Frontier molecular orbitals of P-1 and P–1 + Hg2+ obtained from the DFT calculations using Gaussian 09 program.

assay which shows that the probe P-1 did not exert any adverse effect on cell viability (Figs. S11 and S12). However, exposure of HeLa cells to P-1 + Hg2+ ensemble resulted in a declined in cell viability above a concentration of 20 ␮M. The effect was more pronounced at higher concentrations and showed an adverse cytotoxic effect in a dose dependent manner which is in agreement with previous literature reports suggesting cytotoxic and anti-proliferative effects of P-1 + Hg2+ ensemble on cancer cells [44]. The viability of HeLa cells was not influenced by the solvent (DMSO) leading to the conclusion that the observed cytotoxic effect could be attributed to the Probe P-1 + Hg2+ ensemble formation. To test the capability of P-1 to image in living cells, HeLa Cells were incubated with probe P-1 for 30 min at room temperate and then it was treated with HgCl2 for 10 min. The fluorescence image became dim and quenches, implying that the intracellular uptake of Hg2+ ions complexed with probe P-1 yielded non-fluorescent ensemble (Fig. 6). Upon further incubation of cell with S2− (20 ␮M) for 10 min, green fluorescence image was recovered, indicating that the uptake of S2− resulted in the decomplexation of intracellular [P–1 + Hg2+ ] ensemble to fluorescent P-1. Therefore, the ‘on–off–on’ fluorescence imaging of probe P-1 was accomplished in HeLa cells by the intracellular complexation and decomplexation interaction modulated by Hg2+ and S2− respectively. These findings show that P-1 is biocompatible in nature and can be used for detecting Hg2+ and S2− ions in cells rapidly.

DFT calculations with B3LYP and 6–311G/LANL2DZ basis sets using Gaussian 09 program [45]. Frontier molecular orbitals were derived from the optimized geometries (Fig. S11). In P-1, HOMO is spread over on the whole pi moiety and LUMO on the diaminomaleonitrile unit. In P-1-Hg2+ HOMO is localized on part of the pi moiety and LUMO is spread on Hg2+ only (Fig. 7 and Fig. S13-optimised geometry of probe alone). These results clearly show that the disturbance of internal charge transfer on the appendage of Hg2+ with Probe P-1. 4. Conclusion In summary, we have designed and synthesized a highly selective and ratiometric dual-functional fluorescence probe P-1 and its ensemble for sensing Hg2+ and S2− . Consequently, P-1 appears to be a practical system for colorimetric detection of Hg2+ in aqueous ethanol medium. The sensitivity of probe P-1 to Hg2+ and S2− was demonstrated in living cells, and cell toxicity assay reveals that the probe P-1 can be used for selective imaging of Hg2+ and S2− in living cells. Acknowledgements K.S. thanks the DST-SERB Fast Track programme for financial support (No. SB/FT/CS-062/2013).

3.6. Density functional theory studies

Appendix A. Supplementary data

In order to understand further the absorption and fluorescence behaviour of the P-1 and the Hg2+ complex, we carried out the

Supplementary data associated with this article can be found, in the online version, at

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Biographies K. Muthu Vengaian completed B. Sc. Chemistry in 2009, M.Sc. Chemistry in 2012 and M.Phil chemistry in 2013 from G. T. N. Arts College Dindigul affiliated to Madurai Kamaraj University, Madurai. He is currently working as a research fellow in Anna University, University College of Engineering − Dindigul under the guidance of Dr. K. Sekar. His main area of research is synthesis of Chemosensor for toxic metal ions and molecules. C. Denzil Britto completed B.Sc. Chemistry in 2012, M.Sc. in Chemistry in 2014 from Government Arts College, Ooty, The Nilgiris affiliated to Bharathiar University, Coimbatore. He is currently working as a Project Fellow in DST-SERB project in Anna University, University College of Engineering − Dindigul under the guidance of Dr. K. Sekar. His field of interest is synthesis of cyclodextrin based supramolecules and Chemosensors for toxic metal ions and molecules.


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Karuppannan Sekar is an Assistant Professor in Chemistry at Anna university, College of Engineering Dindigul. He received his Ph.D., in 2008 from the University of Madras under the supervision of Professor Perumal Rajakumar. He worked as a postdoctoral fellow at the University of Bourgogne, France under the guidance of Research Professor Jean-Claude Chambron in 2008–2009. His major research interests are primary in the fields of synthesis of Supramolecules for HostGuest chemistry for small molecules and ions, specifically on the toxic metal ion chemosensors. Gandhi Sivaraman received his, (Chemistry) from thiyagarajar college (Madurai Kamaraj University) in 2007,, (Chemistry) from The American college (Madurai Kamaraj University) in 2009. He received his Ph.d from the same university under the guidance of Prof. D. Chellappa in 2014. He is currently working as a bridging fellow in institute of stem cell biology and regenerative medicine (instem), Bangalore, India. His research interests are Chemosensors, Chemical Biology, and Computational Chemistry.

Subramanian Singaravadivel completed B. Sc. in 2004 and M.Sc. in chemistry in 2006 from G. T. N. Arts College Dindigul affiliated to Madurai Kamaraj University, Madurai. He joined for Ph. D. in 2006 under the supervision of Professor S. Rajagopal at Madurai Kamaraj University, Madurai and received doctoral degree in 2013. He worked as a postdoctoral fellow at Institute of Chemistry; Academia Sinica with Prof. Kuang-Lieh Lu in 2012–2013 in the field of Rhenium based sumpramolecular chemistry for sensor. He is currently working as assistant professor in chemistry at SSM Institute of Engineering and Technology. His field of interest is design and synthesis of fluorescent probes for sensing heavy metal ions, explosives and small molecules.