Development of a pyrene based “turn on” fluorescent chemosensor for Hg²⁺

Abstract

We have designed, synthesized, and evaluated a new pyrene-based “turn on” fluorescent chemosensor which showed remarkable enhanced fluorescence intensity in the presence of Hg²⁺ ions and a high selectivity towards Hg²⁺ ions over a wide range of metal ions in aqueous acetonitrile (ACN). The probe shows selectivity and sensitivity with a 12-fold enhancement in fluorescence towards Hg²⁺. Upon the addition of Hg²⁺ ions, the probe displayed an apparent color change, which could be observed by the naked eye under a UV lamp. It's in vitro sensitivity to Hg²⁺ was demonstrated in candida albicans cells with the use of confocal microscopy.

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Introduction

The development of artificial receptors for the sensing and recognition of environmentally and biologically important species has been actively investigated in recent years.¹ In this regard, chemosensors that can highly sensitively and selectively detect heavy and transition metal ions (HTM) such as Hg²⁺, Pb²⁺, Cd²⁺, and Cu²⁺ are especially important.² Mercury is considered as a prevalent toxic metal in the environment because both elemental and ionic mercury can be converted by bacteria in the environment to methyl mercury, which subsequently bio accumulates through the food chain.³ Mercury can accumulate in the human body and affects a wide variety of diseases even in a low concentration, such as digestive, kidney, and especially neurological diseases.⁴ As a result, developing new and practical multi signaling chemosensors for Hg²⁺ is still a challenge.⁵ The U.S. Environmental Protection Agency (EPA) standard for the maximum allowable level of inorganic mercury in drinking water is 2 ppb.⁶ Sensors based on metal ion-induced changes in fluorescence appear to be particularly attractive and are one of the first choices because of their simplicity and efficiency in even very low concentrations.⁷ Thus, designing fluorescent sensors for mercury⁸ has drawn worldwide attention. Generally, mercury is known to cause fluorescence quenching of fluorophores via the spin–orbit coupling effect.⁹ Fluorescence quenching is not only disadvantageous for a high signal output upon recognition but also hampers temporal separation of spectrally similar complexes with time-resolved fluorometry.¹⁰ However, the sensors that show fluorescence enhancement on binding to the cation of interest are preferred because these allow a lower detection limit and high-speed spatial resolution via microscope imaging.^11,12 Turn on fluorescent chemosensors¹³ for heavy metal ions, particularly for mercury have remained so far limited because these metal ions are known to quench fluorescence¹⁴ emission via enhanced spin–orbit coupling,¹⁵ energy, or electron transfer. The simplicity, effectiveness and inexpensive nature spurred many to put continuous effort into developing new selective fluorescent sensors for different analytes.¹⁶ In particular imine based sensors have drawn attention due to simplicity and sensitivity.¹⁷ Most of the studies about fluorescent chemosensors of Hg²⁺ are based on a fluorescence quenching mechanism,¹⁴ only a few reports are present in the literature on fluorescence “turn on” chemosensors for Hg²⁺.¹⁸ Herein we report a new “turn on” selective fluorescent chemosensor.

Results and discussion

Chemosensor PTE-1 was synthesised by a one step condensation of pyrene-1-carboxaldehyde with 2-(methylthio) aniline in 61% yield (Scheme 1). ¹H and ¹³C-NMR spectra and ESI-MS of PTE-1 are given in Fig. S1–S3†. The molecular framework was designed as a platform for the construction of efficient ionophores via its thioether S and imino N atom.¹⁹ This Schiff base was stable in neutral acetonitrile and water–acetonitrile solution for at least 3 days. The fluorescence emission intensity of the probe PTE-1 is unaffected by pH values between 4.5–12. This observation indicates that the chemosensor PTE-1, which is pH-stable and also organic solvent-stable, may be useful as a potential chemosensor material.

The absorption spectrum of PTE-1 shows the typical pyrene absorption bands at 230(sh), 290, 375 and also a low energy band centred at 404 nm is attributed to the imino bridge. Upon addition of 1–1.2 equiv. of Hg²⁺ ions to the PTE-1, the most significant changes were broadening of the absorption bands around 350–450 nm observed with hypochromism (Fig. 1). This is also responsible for the change of colour, which is perceptible to the naked eye, from pale yellow to colorless. The absorption spectra with several metal cations (Na⁺, K⁺, Ca²⁺, Mn²⁺, Fe³⁺, Co²⁺, Ni²⁺, Cu²⁺, Zn²⁺, Cd²⁺, Hg²⁺, Pb²⁺, and Sn²⁺) using their chloride salts in H₂O–CH₃CN (30 : 70,v/v) are shown in Fig. S4†.

Scheme 1 Synthesis of compounds PTE-1 and PTE-2.

Fig. 1 UV–vis spectra of PTE-1 (10 μM) in the presence of Hg²⁺ ions (1.0 equiv. and 1.2 equiv.) in H₂O–CH₃CN (30 : 70, v/v).

The fluorescence spectra of PTE-1 with several metal cations (Na⁺, K⁺, Ca²⁺, Mn²⁺, Fe³⁺, Co²⁺, Ni²⁺, Cu²⁺, Zn²⁺, Cd²⁺, Hg²⁺, Pb²⁺, and Sn²⁺) using their chloride salts in H₂O–CH₃CN (30 : 70,v/v) are shown in (Fig. 2). The probe PTE-1 is very weakly fluorescent compared to pyrene, due to the photo induced electron transfer from the imine nitrogen to the pyrene fluorophore and/or C=N isomerisation. Both being the primary processes involved in the deactivation of the excited state, which lead to the fluorescence quenching of PTE-1. By virtue of the ligating atoms N and S, PTE-1 readily forms a chelate with the Hg²⁺ ion thereby both the photo induced electron transfer (PET) and C=N isomerisation are inhibited and the fluorescence from the probe is enhanced.

Fig. 2 Fluorescence spectra in the presence of different metal chlorides in H₂O–CH₃CN (30 : 70, v/v). The inset shows the response of the other metal ions with PTE-1. Excitation was performed at 365 nm.

The fluorescent properties of PTE-1–Hg²⁺ were surveyed in typical organic solvent systems as well as aqueous solutions. It exhibits weak monomer emission around 390–410 nm with a characteristic, but a very intense emission of pyrene centered at 462 nm. The pyrene excimer region increases as the water content is increased partially in the vicinity of 30% water composition, and then changes were not so significant up to 40% aqueous solution. This observation implies that the complexation of PTE-1 with Hg²⁺ ions presumably leads to stacking of pyrene moieties from PTE-1 + Hg²⁺ resulting in the switch of the pyrene monomer emission to an excimer emission in solution state but ESI-MS provides information for 1 : 1 complexation. From this we came to understand that there may be a possibility of intermolecular excimer formation.

A 12-fold enhancement of fluorescence was observed upon addition of Hg²⁺ compared to that of PTE-1 in the H₂O–CH₃CN mixture. Upon interaction with various metal ions of alkali (Na⁺, K⁺), alkaline earth (Ca²⁺) and transition-metal ions (Mn²⁺, Fe³⁺ , Co²⁺ , Ni²⁺ , Cu²⁺, Zn²⁺, Cd²⁺, Pb²⁺, and Sn²⁺), a much weaker response is observed relative to Hg²⁺ at the same concentration, and the fluorescence signal of PTE-1–Hg²⁺ in the presence or absence of these contrast ions also exhibited only a mild difference (Fig. S5†).

To test the reversibility of the probe, we added Na₂S (0.1 M) to the solution of PTE-1–Hg²⁺. The addition of 0.52 mL of Na₂S could restore the initial value of free probes due to the K_d value of 10⁻⁵⁰ for Hg²⁺ at standard conditions in the form of [HgS₂].^20,21 Thus the probe could be revived on addition of Hg²⁺ (Fig. S6†).

The titration curves showed a smooth and steady increase until a plateau was reached (15 μM Hg²⁺) with a 12-fold increase at the plateau (Fig. 3). The chemosensor PTE-1 exhibited a very efficient fluorescent response, and over 70% of the total fluorescent intensity increase was observed with 1 equivalent of Hg²⁺. The association constant (K_a) of PTE-1 with Hg²⁺ is 1.02 × 10⁴ M⁻¹ (error < 10%), obtained by a nonlinear curve fitting of the fluorescent titration results.²² Fig. S7†, exhibits the dependence of the intensity ratio of emission at 472 nm (F_f/F_i) on Hg²⁺. This curve served as the calibration curve for detecting Hg²⁺. The detection limit²³ was calculated from titration results and it was found to be 2.2 × 10⁻⁸ M.

Fig. 3 Fluorescence titration of PTE-1 (1 μM) in the presence of different amounts of Hg²⁺ in H₂O–CH₃CN (30 : 70, v/v). Excitation was performed at 365 nm.

To understand the binding of Hg²⁺ with PTE-1 a Job plot was constructed by measuring the fluorescence at different mole fractions of Hg²⁺. It exhibits a maximum at 0.5 mol fraction of Hg²⁺ thus indicating formation of a 1 : 1 complex between PTE-1 and the Hg²⁺ ion (Fig. S8†). It was further supported by the peak at 655.35 in the ESI-MS spectrum corresponding to a molecular weight of PTE-1 + HgCl₂ + CH₃OH + H⁺ (Fig. S9†).

To understand the effect of sulfur in PTE-1 the Schiff base PTE-2 was synthesised (Scheme 1) and its fluorescence behaviour was investigated. The PTE-2 shows a very weak fluorescence with the metal ions but the enhancement is not so significant compared to that of PTE-1 with an excitation of 340 nm (Fig. S10†). This ascertains that the chelate formation of PTE-1 with Hg²⁺ plays a crucial role in the fluorescence and sensing of metal ions.

To further understand the absorption and fluorescence behaviour of the probe PTE-1 and PTE-1 + Hg²⁺ complex, we carried out DFT calculations with the 6-31G basis set using the Gaussian 03 program.²⁴ The dihedral angle −123.685 for C=N–C–C reveals the s-trans conformation of PTE-1 whereas that for PTE-1 + Hg²⁺ is found to be 138.252. The charges on the N atom for PTE-1 and PTE-1 + Hg²⁺ are found to be −0.430, −0.248 respectively thus revealing a significant reduction in the electron density on the N atom upon coordination of the probe with Hg²⁺. This may inhibit fluorescence quenching by the photo induced electron transfer from nitrogen to the fluorophore pyrene. From natural bond orbital analysis, it is evident that non-bonding orbitals containing a lone pair of electrons localised on nitrogen are compatible with the HOMO-1 and HOMO respectively of PTE-1 and PTE-1 + Hg²⁺. The TDDFT calculations focused on oscillator strength indicate three strong transitions for the probe and one strong and two weak transitions for the PTE-1 + Hg²⁺ complex. The oscillator strengths for the n–π* transition of the probe and its mercury complex are 0.4329, 0.5446 corresponding respectively to the wavelengths 388 and 397.14 nm. As we use excitation source at 340 nm, a n–π* transition is possible for the probe but for the complex it is not viable as a wave length of 397 nm is away from the excitation wave length. From the DFT/TDDFT results we thus concluded that the fluorescence enhancement in the complex is due to the inhibition of photo induced electron transfer (Fig. S11, 12 and Table. S1 and S2†).

The sensitivity of probe PTE-1 to Hg²⁺ was examined in candida albicans cells by using confocal microscopy. The qualitatively in vitro results are exhibited in Fig. 4. After the cells were incubated with 10 μM of PTE-1 for 30 min at 37 °C, no obvious fluorescence could be imaged (Fig. 4a). At the same experimental conditions, strong fluorescence was imaged 10 min after introduction of 10 μM of Hg²⁺ to the same cells, displaying enhanced blue fluorescence as in Fig. 4b. The cell imaging experiments demonstrate the good cell-membrane permeability of PTE-1 as a probe for imaging Hg²⁺ within candida albicans cells and no changes in the cell morphology and cell viability (Fig. S13†) were observed.

Fig. 4 Intracellular Hg²⁺ imaged in candida albicans cells at 37 °C with the use of confocal microscopy. (a) Candida albicans cells incubated with PTE-1 for 30 min. (b) The candida albicans cells in part a 10 min after being treated with 10 μM of Hg²⁺. (c) A bright field image of probe treated candida albicans cells.

Conclusion

In conclusion, we have designed, synthesized, and evaluated a new pyrene based “turn on” fluorescent sensor which showed remarkable enhanced fluorescence intensity in the presence of Hg²⁺ ions and a high selectivity towards Hg²⁺ ions over a wide range of metal ions in aqueous acetonitrile. Background metal ions showed only a small interference with the detection of Hg²⁺ ions, indicating that the receptors could be used as efficient Hg²⁺ selective “turn on” fluorescent sensors. This significantly enhanced fluorescence is due to the formation of a 1 : 1 complex PTE-1–Hg²⁺ in which the rotation of acyclic C=N is frozen. The sensitivity of PTE-1 to Hg²⁺ was demonstrated in candida albicans cells, indicating its potential application for imaging of Hg²⁺ in living cells.

Materials and methods

Pyrene-1-carboxaldehyde, 2-(methylthio) aniline and aniline were obtained from Aldrich and used as such. Metal chloride salts were procured from Merck. Absorption spectra were recorded using a JASCO V-530 spectrophotometer while fluorescence analyses were done using a JASCO spectrofluorimeter. The excitation and emission slit widths were kept constant as 5 nm. NMR spectra were recorded on a Bruker (Avance) 300 MHz NMR instrument. Electrospray ionisation mass spectral (ESI-MS) analysis was performed in the positive ion mode on a liquid chromatography-ion trap mass spectrometer (LCQ Fleet, Thermo Fisher Instruments Limited, US). Microanalysis (C, H, and N) was performed using a Perkin-Elmer 4100 elemental analyzer.

Synthesis of PTE-1

Pyrene-1-carboxaldehyde was mixed with 2-(methylthio) aniline in acetonitrile. The reaction mixture was stirred and refluxed for 3 h in the presence of a drop of acetic acid. The purification of the crude product obtained after distillation of the solvent by column chromatography using hexane/ethyl acetate yields a yellow solid. (61% yield). ¹H-NMR (300 MHz, CDCl₃) 2.56(s, 3H), 7.18–7.29(m, 4H), 8.01–8.17(m, 3H), 8.23–8.26(m, 4H), 8.75(d, 1H), 9.17(d, 1H), 9.40(s, 1H). ¹³C-NMR (75 MHz, CDCl₃) 14.94, 117.5, 122.9, 124.6–134.3, 150.04, 158.4. MS (ESI): 352.17 (M+H⁺). Elemental analysis for C₂₄H₁₇NS: calculated: C, 82.02; H, 4.88; N, 3.99 and found: C: 81.94; H, 4.81; N, 3.94.

Synthesis of PTE-2

PTE-2 was synthesised using aniline instead of 2-(methylthio) aniline by adopting same procedure as that of PTE-1. ¹H-NMR (300 MHz, CDCl₃) 7.33–7.26(m, 1H) 7.40–7.38(m, 2H), 7.51–7.46(m, 2H), 8.27–8.03(m, 7H), 8.77(d, 1H), 9.04(d, 1H), 9.49(s, 1H). MS (ESI): 306.47 (M+H⁺).

Computational details

We carried out density functional theory (DFT) calculations with the 6-31G* basis set using the Gaussian 03 program in order to understand the turn on fluorescence behaviour of PTE-1 on complexation with Hg²⁺. Initially the geometries of PTE-1 and the PTE-1 + Hg²⁺ complex were optimized by DFT-B3LYP using 6-31G and LANL2DZ basis sets respectively. The ground state optimized geometries absorption behaviour and corresponding transitions of PTE-1 and the PTE-1 + Hg²⁺ complex were obtained from TDDFT using the above basis sets.

In vitro cellular imaging

Candida albicans cells were incubated with the probe PTE-1 (10 μM) in PBS buffer (1% DMSO) for 30 min, then the cells were centrifuged and washed with PBS buffer three times. PTE-1 treated cells were incubated with Hg²⁺ (10 μM) for 10 min and the cells were imaged using a confocal fluorescence microscope (Zeiss LSM 510 META).

Acknowledgements

G.S. would like to thank UGC for a senior research fellowship. G.S., T.A. and D.C. acknowledge DST-IRHPA, FIST for instrumental facilities.

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Footnote

† Electronic supplementary information (ESI) available: Computational details, NMR, MS spectra. See DOI: 10.1039/c2ra21202a

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