Fluorescence “on–off–on” chemosensor for selective detection of Hg²⁺ and S²⁻: application to bioimaging in living cells

Abstract

A phenothiazine based diamino-malenonitrile-linked chromogenic and fluorogenic probe (P-1) was synthesized and characterized for the specific detection of Hg²⁺ and S²⁻. In the presence of Hg²⁺, the probe produces “switch-off” along with a color change from yellow to brown, allowing colorimetric detection of Hg²⁺ by the naked eye. The sensitivity of probe P-1 towards Hg²⁺ and S²⁻ was demonstrated in living cells, and cell toxicity assay also reveals that the probe P-1 can be used for selective imaging of Hg²⁺ and S²⁻ in living cells.

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1. Introduction

There is increasing interest in the determination of heavy metal ions and anions owing to their huge impact on biology and the environment, therefore the development of chemo receptors for selective sensing and recognition of biologically important anions and heavier metals is of major concern.^1,2 Mercury pollution has sparked interest in the design of new tactics to monitor Hg²⁺ in biological and environmental samples.³ As a strong neurotoxin, methyl mercury ions can cause human health problems due to their easy absorption through the skin, respiratory and cell membranes, leading to digestive, cardiac, kidney and DNA damage, mitosis impairment and especially permanent damage to the central nervous system.^4–7 Therefore, developing highly efficient sensors to detect Hg²⁺ ions is important for human health and environmental protection. The Environmental Protection Agency (EPA) standard for the maximum allowable level of inorganic Hg²⁺ in drinking water is 2 ppb.⁸ Design of such receptors depends primarily on the nature of the binding site for achieving the desired specificity for the Hg²⁺ ion as well as on the nature of the optical response that is preferred for probing the receptor–analyte binding process. Various photophysical pathways like Internal Charge Transfer (ICT),⁹ Electron Transfer (ET),¹⁰ Energy Transfer (eT),¹¹ Förster Resonance Energy Transfer (FRET),¹² Through Bond Energy Transfer (TBET),¹³ are generally utilized for reporting the binding induced phenomena; while photoactive moieties like 1,8-napthalimide, coumarin, pyrene, anthracene, BODIPY, squaraine, xanthanes, cyanine, rhodamine, fluorescein etc. are commonly being used as the reporter moiety.¹⁴ The use of fluorometric sensors has emerged as one of the most promising alternatives to the conventional methods owing to their high sensitivity, selectivity and operational simplicity. Likewise, colorimetric chemosensors have been attracting a great deal of interest for “naked-eye” detection in an uncomplicated and economical manner, offering qualitative and quantitative information.^15–21 A variety of fluorescent small-organic-molecule-based chemosensors that bind selectively to Hg²⁺ have been designed and developed. 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.^22–26 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 Internal Charge Transfer (ICT) mechanism based phenothiazine donor–acceptor molecules and its derivatives are used in different areas such as dyes, probes, and electrochemistry.³⁰ Phenothiazine donor–acceptor molecules can provide strong fluorescence, structural regulation and good hole transport capacity. However there are only few metal ion fluorescent sensors based on phenothiazine donor–acceptor molecules.³¹ Recently Yang et al.^31c reported a fluorescent probe, which deals with the attachment of N-ethyl phenothiazine moiety to aminothiourea which serves as a dual probe for Hg²⁺ and I⁻ and this N-ethyl phenothiazine-aminothiourea-Hg(II) ensemble act as sensor for iodide in aqueous media. In the present work we have developed a new phenothiazine probe based on 2,3-diaminomalenonitrile as sensor for Hg²⁺ and S²⁻ advantage of the present work is this sensor works in red region compared to that of other systems reported so far, and may have promising application in cell-imaging due to its low auto-fluorescent interferences from cells but this system suffers from low aqueous solubility.

2. Results and discussion

To a mixture of 2,3-diaminomaleonitrile (1.36 mmol) and dry ethanol (60 ml), were added 10-hexyl-10H-phenothiazine-3,7-dicarbaldehyde 3 (0.68 mmol) under nitrogen atmosphere. The reaction mixture was refluxed at 70 °C for 6 hours. 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 to afford pure compound of 3,3′-(((1Z,1′Z)-(10-hexyl-10H-phenothiazine-3,7-diyl)bis(methanylylidene))bis(azanylylidene))bis(2-aminomaleonitrile) (P-1) as dark brown powder in 80% yield (Scheme 1). The probe P-1 was characterized by ¹H-NMR, and ESI-MS (ESI Fig. S1 and S2†).

Scheme 1 Synthesis of probe P-1 (a) 1-hexyl bromide, NaH, DMF, N₂ atm, reflux 12 hours. (b) DMF/POCl₃ (1 : 1), DCE, N₂ atm, reflux 24 hours. (c) 2,3-Diaminomaleonitrile, EtOH, N₂ atm, 70 °C, 6 hours.

The binding properties of P-1 with Hg²⁺ were studied by UV-vis titration experiments (Fig. 1). The probe P-1 exhibited strong absorption bands at λ_max 290, 346, 446 nm. Upon addition of Hg²⁺ (0–1 eq.) to a solution of receptor P-1 (20 μM) in ethanol–H₂O (7 : 3), it exhibited a red-shift of 50 nm in the absorption spectrum. This red shift indicates the significant stabilization of the intramolecular charge transfer achieved through the interaction P-1 with Hg²⁺ and the presence of Hg²⁺ could be easily monitored by the naked eye.³² Moreover, no discernible spectral alterations were observed in the presence of various competitive metal ions such as Na⁺, K⁺, Ag⁺, Mn²⁺, Cd²⁺, Cu²⁺, Fe³⁺, Al³⁺, Cr³⁺, Pb²⁺, Co²⁺, Mg²⁺, Zn²⁺ and Ni²⁺ (all the salts studied herein were taken as nitrates to eliminate the effect of anions) (ESI Fig. S3†), indicating that the UV-vis response of probe P-1 is highly Hg²⁺ specific.

Fig. 1 UV-vis spectrum of probe P-1 (10 μM) upon addition of Hg²⁺ (0–1 eq.) in ethanol–water (7 : 3) mixture.

Fluorescence based detection of analyte is preferred over the UV-vis absorption method because of the capability of detecting even very low concentration of analyte. The fluorescent properties of the probe P-1 in the presence of various metal ions were studied in (ethanol–water 7 : 3). The probe P-1 displayed emission at 575 nm upon excitation at 450 nm. Upon addition of Hg²⁺ ion an intense emission band at 575 nm is quenched (90%) (Fig. 2). The fluorescence titration of P-1 with 0–1 equivalent of Hg²⁺ ions gradually quenches the fluorescence intensity at 575 nm. Upon excitation at the absorption maximum of the complex (λ_ex = 450 nm), other competitive metal ions for instance Na⁺, K⁺, Ag⁺, Mn²⁺, Cd²⁺, Cu²⁺, Fe³⁺, Al³⁺, Cr³⁺, Pb²⁺, Co²⁺, Mg²⁺, Zn²⁺ and Ni²⁺ (10 μM) have hardly responded to the emission of the probe when coexisted with Hg²⁺ (ESI Fig. S4†).

Fig. 2 Fluorescence emission spectrum of probe P-1 (10 μM) upon addition of Hg ²⁺ (λ_ex = 450 nm) (0–1 equiv.) in ethanol–water (7 : 3) mixture.

Addition of 1 equiv. of Na₂S makes the quenched fluorescence restore at 575 nm. It is found that sulphide ion also increases the fluorescence intensity of [P-1 + Hg²⁺] ensemble in concentration dependence. [P-1 + Hg²⁺] ensemble displays a high sensitivity to sulphide ions to form the stable species compared to that of other anions (ESI Fig. S5†). This is may be attributed to the fact that mercury is often associated with its high affinity for sulfur, such that it binds effectively with sulfide ions and sulphur containing compounds. The formation constant for HgS is high whereas 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.^31d,31e

To confirm the stoichiometry of the binding of probe P-1 with Hg²⁺ ion Job's plots (continuous-variation plots) analysis was carried out. The plot of the absorbance variation at 450 nm against mole fraction clearly showed the maxima with a mole fraction at 0.5 indicating 1 : 1 stoichiometry and the proposition is further supported by the peak at 718.12 (P-1 + Hg²⁺) in ESI-MS analysis (Fig. S6†). Similarly Job's plots were derived from fluorescence analysis which also conforms to the 1 : 1 binding of P-1 with Hg²⁺ ions (Fig. 3). The linear plot was obtained when plotting fluorescence intensity vs. concentration of Hg²⁺ ions indicating that the probe P-1 can be used to determine Hg²⁺ ion concentration. The binding constant of P-1 with Hg²⁺ was calculated and it is found to be 1.84 × 10⁴ M⁻¹.³³

Fig. 3 Job's plot of mole fraction of Hg²⁺ vs. fluorescence intensity at λ_ex = 575 nm.

The titration profile shows a steady decrease of fluorescence intensity with increasing concentration of Hg²⁺, after addition of 1.2 equivalents it reaches the saturation level. For the practical quantitative detection, fluorescence spectral changes should vary linearly with the concentration of Hg²⁺. To test this, we carried out fluorescence titration studies of P-1 (0.2 μM) with Hg²⁺. A linear response of the fluorescence intensity as a function of [Hg²⁺] was observed from 50 nM to 0.2 μM (R² = 0.9989) (Fig. S7†). The detection limit (DL) was calculated from this, the lower detection limit was found to be 3.5 × 10⁻⁸ M.³⁴

The fluorescence response of probe with other metal ions have been investigated (Fig. 4), the miscellaneous competitive cations lead to no considerable absorption and fluorescence changes, confirmed that the probe retained its binding ability with Hg²⁺ in the presence of a range of interfering metal ions encountered.

Fig. 4 Fluorescence response of 10 μM P-1 with various metal ions. The red bars represent the addition of the corresponding metal ion to P-1. The violet bars represent the change of the emission that occurs upon the subsequent addition of Hg²⁺ to the above solution.

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. The emission is almost quenched by addition of Hg²⁺ and recovered again with the addition of S²⁻ (Fig. 5). All properties mentioned above indicates that the capability of P-1 and [P-1 + Hg²⁺] ensemble for quantitative detection Hg²⁺ and S²⁻ as an “on–off–on” type probe. Plausible mechanism for sensing Hg²⁺ and S²⁻ by P-1 is shown in Fig. 6.

Fig. 5 Fluorescence response of [P-1 + Hg²⁺] ensemble to Na₂S.

Fig. 6 Plausible mechanism for sensing Hg²⁺ and S²⁻ by P-1.

Furthermore, ¹H NMR titrations experiment was performed to understand the nature of interactions between the P-1 and the Hg²⁺ ion, and the selected spectra are given in (Fig. 7). The comparison of the ¹H NMR spectra of P-1 and P-1 mixed with 1 equivalent of Hg²⁺ ion suggest that the addition of Hg²⁺ to the P-1 solution caused a down field shift in the signal corresponding to –NH₂ (1.71 to 1.90) and N=CH– (8.22 to 8.31) protons, it shows that Hg²⁺ is bound to the receptor through coordination of Hg²⁺ to the lone pair electrons nitrogen atom of amine and imine nitrogen.³⁵ Also the protons attached with phenothiazine aromatic system slightly shift towards down field, due to strong electron donating ability of phenothiazine ring to diamino-malenonitrile moiety after complex with Hg²⁺ ion.³⁶

Fig. 7 Partial ¹H NMR (300 MHz) spectra (a) probe P-1, (b) probe P-1 + 1 equivalent Hg²⁺ in DMSO-d₆.

The MTT assay was adopted to study cytotoxicity of the probe P-1 at varying dose and time dependent assay which shows that the probe P-1 did not exert any adverse effect on cell viability (ESI Fig. S8 and S9†). However, exposure of HeLa cells to P-1 + Hg²⁺ ensemble resulted in a declined in cell viability above a concentration of 20 μM. 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.³⁷ 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 + Hg²⁺ ensemble formation.

The results obtained in the in vitro cytotoxic assay suggested that, in order to pursue fluorescence imaging studies of P-1 in live cells, it would be prudent to choose a working concentration of 20 μM. Hence, to assess the effectiveness of P-1 as a probe for intracellular detection of Hg²⁺ by confocal microscopy, HeLa cells were treated with 5 μM of P-1 for 30 min which revealed a red fluorescence in HeLa cells (Fig. 8). Upon incubation with probe followed by Hg²⁺ striking switch-off fluorescence was observed inside HeLa cells, which indicated the formation of P-1 + Hg²⁺ ensemble, as observed earlier in solution studies. The P-1 + Hg²⁺ treated cells are further incubated with sodium sulphide showed the enhanced red fluorescence in cells. These findings show that P-1 is biocompatible in nature and can be used for detecting Hg²⁺ and S²⁻ ions in cells rapidly.

Fig. 8 Bright field image and fluorescence images of HeLa cells. (A) Bright field image of probe. (B) Fluorescence imaging of probe P-1. (C) Fluorescence imaging of P-1 after addition of Hg²⁺. (D) Fluorescence imaging of probe + Hg²⁺ ensemble after addition of S²⁻.

In order to understand further the absorption and fluorescence behaviour of the P-1 and the Hg²⁺ complex, we carried out the DFT calculations with B3LYP and 6-311G/LANL2DZ basis sets using Gaussian 09 program.³⁸ Frontier molecular orbitals were derived from the optimized geometries. 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. 9). These results clearly shows that the disturbance of internal charge transfer on the appendage of Hg²⁺ with probe P-1.

Fig. 9 Frontier molecular orbitals of P-1 and P-1 + Hg²⁺ obtained from the DFT calculations using Gaussian 09 program.

3. Conclusion

In summary, we have synthesized phenothiazine based diamino-malenonitrile-linked (P-1) chromogenic and fluorogenic probe which exhibits as highly selective, sensitive and colorimetric chemosensor for Hg²⁺ ion in an aqueous ethanol solution. 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.

4. Experimental methods

The precursor and compounds 2 and 3 were prepared by earlier literature procedures.³⁹

Synthesis of 3,3′-(((1Z,1′Z)-(10-hexyl-10H-phenothiazine-3,7-diyl)bis(methanylylidene))bis(azanylylidene))bis(2-aminomaleonitrile) (P-1)

To a mixture of 2,3-diamino-malenonitrile (1.36 mmol) and dry ethanol (60 ml) were added 10-hexyl-10H-phenothiazine-3,7-dicarbaldehyde 3 (0.68 mmol) under nitrogen atmosphere. The reaction mixture was refluxed at 70 °C for 6 hours. 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) as eluent afford pure compound (P-1) as dark-brown powder. Yield: 80%; mp: 185 °C; IR KBr (cm⁻¹): 3442 (NH₂), 3313 (–NH, str), 3200 (=C–H), 2225 (C≡N), 1602 (NH, bend), 1471 (C–H, bend); ¹H NMR (300 MHz, DMSO-d₆): 0.82 (bs, 3H); 1.24–1.38 (m, 8H); 1.71 (4H); 3.94 (bs, 2H); 7.07 (d, 2H, J = 8 Hz); 7.76 (d, 2H, J = 9 Hz); 7.91 (s, 2H); 8.13 (s, 2H); mass (ESI-MS): 519.57.

MTT assay

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 96-well plate and incubated in medium containing the probe P-1 at different concentrations for different time intervals. 100 μL of MTT was added and the plates were incubated at 37 °C for 4 h to allow MTT to form formazan crystals. Intracellular formazan crystals were dissolved by adding 100 μL of DMSO to each well and the plates were shaken for 10 min. The absorbance was read at 570 nm and 630 nm using plate reader. The percentage of survival was calculated using the formula: % survival = [live cell number (test)/live cell number (control)] × 100.

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 P-1 (10 μM in DMSO/H₂O (7 : 3, v/v) buffered with PBS, pH = 7.54) and imaged through the fluorescence microscope. After washing with PBS three times to remove the excess of the probe P-1 in the cells, the cells were further incubated with Hg(NO₃)₂ (10 μM in H₂O) for 10 min at 37 °C and imaged with Nikon fluorescence microscope. Then P-1 and Hg(NO₃)₂ (10 μM in H₂O) again incubated with sodium sulphide and imaged through the fluorescence microscope.

Acknowledgements

K. S. thanks the DST-SERB Fast Track programme for financial support (No. SR/FT/CS–062/2013).

Notes and references

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra26281j

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