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Hexaphenylbenzene appended AIEE active FRET based fluorescent probe for selective imaging of Hg2+ ions in MCF-7 cell lines
Sensors and Actuators B 249 (2017) 311–320
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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Hexaphenylbenzene appended AIEE active FRET based fluorescent probe for selective imaging of Hg2+ ions in MCF-7 cell lines Gurpreet Singh a , Shahi Imam Reja a , Vandana Bhalla a,∗ , Davinder Kaur b , Pardeep Kaur b , Saroj Arora b , Manoj Kumar a,∗ a b
Department of Chemistry, UGC Sponsored Centre for Advanced Studies-II, Guru Nanak Dev University, Amritsar, Punjab, India Department of Botanical and Environmental Sciences, Guru Nanak Dev University, Amritsar, Punjab, India
a r t i c l e
i n f o
Article history: Received 14 September 2016 Received in revised form 11 April 2017 Accepted 12 April 2017 Available online 14 April 2017 Keywords: Hexaphenylbenzene AIEE Mercury FRET Fluorescent aggregates Cell imaging
a b s t r a c t Hexaphenylbenzene (HPB) appended rhodamine derivative 4 has been synthesized which exhibits aggregation-induced emission enhancement (AIEE) characteristics and forms fluorescent aggregates in mixed aqueous media. These aggregates show remarkable fluorescence resonance energy transfer (FRET) in the presence of Hg2+ ions among various metal ions tested. Further these aggregates were also utilized for the ratiometric imaging of Hg2+ ions in MCF-7 cell lines. © 2017 Elsevier B.V. All rights reserved.
1. Introduction Mercury is recognized as one of the most toxic pollutants which has severely contaminated land, water, air and food chain [1,2]. Unfortunately, from environment mercury can easily enter the human body by passing through the biological membranes, thus, causing serious damage to endocrine and nervous systems [3–6]. Further, the environmental mercury level is significantly enhanced through man-made activities. Additionally, lower organisms can easily transform mercury into methylmercury a highly potent neurotoxin that impacts the functioning of central nervous system in living beings [7]. The health and environment aspects provide sufficient impetus for quantification and trace detection of mercury in various biological and environmental samples. Among various approaches for detection of the mercury in biological and environmental systems, fluorescence signalling is one of the first choices due to its high detection sensitivity and selectivity [8–10]. In recent past, a variety of fluorescent probes based on small organic molecules have been reported for the detection of mercury ions, however, most of these reported probes are operational only in
∗ Corresponding authors. E-mail addresses: [email protected] (V. Bhalla), [email protected] (M. Kumar). http://dx.doi.org/10.1016/j.snb.2017.04.074 0925-4005/© 2017 Elsevier B.V. All rights reserved.
organic media [11,12], require high energy excitation [13–15] and exhibit emission at lower wavelength which thereby prevent their in vivo applications due to autofluorescence [16]. Recently, fluorescent micro supramolecular assemblies based on rhodamine derivatives have been developed which exhibit ‘turn on’ response towards mercury ions in aqueous media. In comparison to small molecule based mercury chemosensors, supramolecular assemblies based derivatives exhibit highly sensitive superamplified response towards mercury ions in aqueous media [17]. However, different types of additives were a necessity for the formation of microstructures [18] which decreased the economic and environmental advantages of the strategy. Another limitation of the assemblies based probe is that all the emission changes were centred on a single wavelength [19–24]. Emission changes on a single wavelength are disadvantageous due to interference from fluctuations of the background fluorescence. Thus, development of additive free supramolecular assemblies showing mercury induced emission changes at two different wavelengths in aqueous media is a challenge. Keeping this in mind, we envisaged that if we could design a new probe having a tendency to undergo self-aggregation in mixed aqueous media and having mercury sensing unit which could exhibit sensitive response towards mercury ions based on energy transfer mechanism then it will be possible to overcome above limitations. Recently, aggregation induced emission enhancement
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Chart 1. HPB derivative 1.
phenomenon has emerged as a powerful tool to form fluorescent aggregates in aqueous media [25–28]. Further, literature reports show that the probes based on donor-acceptor system has the potential to exhibit stimuli sensitive response based on energy transfer mechanism [29,30]. In this context, we planned to design a donor-acceptor dyad having aggregation induced emission enhancement active donor unit and mercury sensitive acceptor unit. We envisaged that the designed probe may form aggregates in aqueous media due to its AIEE characteristics and in the presence of mercury ions energy transfer may be triggered from donor moiety to acceptor unit. Thus, mercury induced emission changes could be visible at two different wavelengths. So, we planned to develop a donor- acceptor system having hexaphenylbenzene as the donor unit and rhodamine as the acceptor moiety. Hexaphenylbenzene is the scaffold of our choice as the donor unit due to its known AIEE characteristics [31]. We chose rhodamine as acceptor unit due to its known affinity for mercury ions [32–36]. Recently, from our laboratory, we reported a variety of rhodamine based probes for ‘turn-on’ detection of mercury ions which exhibited emission changes at two different wavelengths based on fluorescence resonance energy transfer (FRET)/through bond energy transfer (TBET) mechanism [37–39]. All these probes were operational in organic media; however, their applicability in aqueous media was not explored. Before designing a new target molecule, we planned to re-examine one of the previously reported probes for the detection of mercury ions in aqueous media. To test the validity of our idea regarding coupling AIEE characteristics with energy transfer mechanism, we chose hexaphenylbenzenerhodamine dyad 1 [40] which exhibits mercury induced through bond energy transfer (TBET) from HPB donor to rhodamine acceptor in methanol (Chart 1). We investigated the AIEE behaviour of this derivative in aqueous media. Derivative 1 exhibits weak aggregation induced emission enhancement in the aqueous media, however, only 45% mercury induced energy transfer phenomenon was observed in aqueous media (vide infra). Based on these results we envisaged that if we could design a new probe with increased number of rotatable carbon-carbon/carbon-nitrogen bonds then the AIEE characteristics of the molecule will be enhanced which in effect will increase the spectral overlap between emission spectrum of donor and absorption spectrum of the acceptor and thus designed and synthesized derivative 4 having rhodamine as acceptor and hexaphenylbenzene as AIEE active donor joined together through imine linkages. To our pleasure, derivative 4 exhibited the AIEE characteristics and formed fluorescent aggregates in mixed aqueous media. Furthermore, the mercury mediated opening of spirolactam ring of rhodamine unit triggered the energy transfer
from donor HPB unit to acceptor rhodamine groups, hence, resulting in emission changes centred at two distinct wavelengths. The work being reported in this manuscript has several advantages; first, for the first time AIEE active hexaphenylbenzene appended rhodamine derivative 4 has been synthesized which forms aggregates in mixed aqueous media. Secondly, in molecular form in organic media derivative 4 exhibits only 25% mercury induced energy transfer whereas in assembled form in mixed aqueous media mercury induced FRET is observed with 97.14% energy transfer. The large Stokes shift shown by these assemblies is very high in comparison to Stokes shift reported in the literature for different probes (Table S1A in the Supplementary Information). These studies highlight the importance of supramolecular assemblies in fluorescence resonance energy transfer mechanism in comparison to other probes reported in the literature (Table S1B in the Supplementary Information). Third, the work being reported in this manuscript demonstrates the practical application of fluorescent assemblies for the ratiometric detection of Hg2+ ions in MCF-7 cell lines. To the best of our knowledge this is the first report where supramolecular aggregates of hexaphenylbenzene appended rhodamine derivative have been utilized for selective cell imaging of Hg2+ ions in MCF-7 cell lines. 2. Experimental 2.1. General experimental methods and materials [41] All the reagents were purchased from Aldrich and were used without further purification. HPLC grade solvents were used in UV–vis and fluorescence studies. UV–vis spectra were recorded on a SHIMADZU UV-2450 spectrophotometer, with a quartz cuvette (path length 1 cm). The fluorescence spectra were recorded with a SHIMADZU 5301 PC spectrofluorimeter. 1 H and 13 C NMR spectra were recorded on a Bruker Avance III HD 500 MHz using CDCl3 as solvent. Data are reported as follows: chemical shift in ppm (d), multiplicity (s = singlet, d = doublet, t = triplet, m = multiplet, br = broad singlet), coupling constants J (Hz). Fluorescence quantum yield [42] was determined by using optically matching solution of diphenyl anthracene (Фfr = 0.90 in cyclohexane) and rhodamine B (Фfr = 0.65 in ethanol) as standard at an excitation wavelength of 373 and 530 nm, quantum yield is calculated using the equation: fs = fr × (1-10−ArLr /1-10−AsLs ) × (Ns 2 /Nr 2 ) × (Ds /Dr )
Фfs and Фfr are the radiative quantum yields of sample and the reference, respectively, As and Ar are the absorbance of the sample and the reference respectively, Ds and Dr the respective areas of emission for sample and reference. Ls and Lr are the lengths of the absorption cells of sample and reference respectively. Ns and Nr are the refractive indices of the sample and reference solutions (pure solvents were assumed respectively). 2.2. UV–vis and fluorescence titrations [41] A 10−3 M stock solution of derivative 4 was prepared by dissolving 16.20 mg of compound 4 in 10 ml of DMSO; 15 l of this stock solution was further diluted with 1485 l CH3 CN and 1500 l water/HEPES buffer (0.05 M, pH = 7.0) to prepare 3 ml solution of derivative 4 and this solution was used for each UV–vis and fluorescence experiment. The aliquots of freshly prepared standard solutions of metal perchlorates [M(ClO4 )X ; M = Hg2+ , Fe2+ , Fe3+ , Pb2+ , Cd2+ , Cu2+ , Zn2+ , Ni2+ , Ag+ , Co2+ , Mg2+ , Li+ , Na+ , and K+ ; X = 13], chlorides (MClY; Y = 1-3) (10−2 M) in distilled water were added to 3 ml solution of derivative 4 taken in quartz cuvette and spectra were recorded.
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Scheme 1. Synthesis of derivative 4.
2.3. Synthesis of compound 4 A clear solution of compound 2 [43] (0.05 g, 0.06 mmol) and 3 [44] (0.07 g, 0.15 mmol) in dry DCM: EtOH (2:8) was stirred at 70 ◦ C. After completion of the reaction in 48 h, precipitates were formed in the reaction mixture which were filtered and recrystallized from methanol to afford the light pinkish coloured compound 4 (0.082 g, 75% yield); mp: >258 ◦ C; 1 H NMR (500 MHz, CDCl3 ) ı = 8.56 (s, 2H, C = NH), 7.98 (d, J = 10 Hz, 2H), 7.48–7.44 (m, 8H), 7.31 (d, J = 10 Hz, 4H), 7.11 (d, J = 10 Hz, 4H), 7.06 (d, J = 5 Hz, 4H), 6.87–6.81 (m, 26H), 6.50 (d, J = 10 Hz, 4H), 6.41 (s, 4H), 6.21 (d, J = 10 Hz, 4H), 3.32–3.28 (q, 16H), 1.13 (t, J = 10 Hz, 24H). 13 C NMR (CDCl3 , 125 MHz) ␦ = 165.02, 153.12, 148.92, 146.94, 140.50, 139.76, 133.97, 131.88, 131.40, 128.23, 127.96, 126.56, 125.09, 123.83, 123.31, 107.99, 106.11, 98.13, 44.32, 29.70, 12.63, Mass, m/z = 1620.6138 [M+1]+ . 2.4. Maintenance of MCF-7 cell lines MCF-7 cells were cultured in RPMI1640 growth medium supplemented with 10% (v/v) FBS (Fetal Bovine Serum), penicillin, streptomycin and gentamycin in CO2 incubator at 37 ◦ C, 5% CO2 and 95% relative humidity in tissue culture flask. Medium was changed on a regular basis and cells were harvested at the log phase of growth for various analysis. 2.5. Confocal imaging Confocal imaging was done according to the method reported by Ramsay et al. [45] MCF 7 cells (100,000 cells/mL) were incubated in 6 well plates having coverslip in each well for 24 h and thereafter treated with different concentrations of test compound for 30 min. Cells were washed twice with chilled phosphate buffer saline (PBS) and then fixed with chilled 4% paraformaldehyde. Then washing of wells was carried out to remove the excessive dye. Finally, images were taken under Nikon Air Laser Scanning Confocal Microscope System. 3. Results and discussion 3.1. Synthesis The compound 2 [43] synthesized by reported method on condensation with rhodamine B hydrazide 3 [44] in dichloromethane
(DCM)/ethanol(EtOH) (2:8) furnished compound 4 in 75% yield (Scheme 1). The structure of compound 4 was confirmed from its spectroscopic data (Figs. S27–S29 in the Supplementary Information). The 1 H NMR spectrum of compound 4 showed one singlet at 6.41 ppm, six doublets at 7.98, 7.31, 7.11, 7.06, 6.50, 6.21 ppm, two multiplets at 7.48–7.44, 6.87-6.81 ppm corresponding to aromatic protons, one quartet at 3.32–3.28 ppm corresponding to methylene protons, one triplet at 1.13 ppm corresponding to methyl protons and one singlet at 8.56 ppm corresponding to imino proton. A parent ion peak for M+H+ was observed at m/z = 1620.6138 in the electrospray ionization mass spectrometry (ESI–MS) spectrum. These spectroscopic data corroborate the structure 4 for this compound. 3.2. Photophysical studies The photophysical behaviour of derivative 4 was studied by UV–vis and fluorescence spectroscopy. The absorption spectrum of derivative 4 (5.0 M) in CH3 CN exhibited two absorption bands at 275 nm and 322 nm (Fig. S1 in the Supplementary Information) corresponding to the S0 -S1 and S0 -S2 transitions with molar extinction coefficients of 8.3 × 104 M−1 cm−1 and 7.8 × 104 M−1 cm−1 , respectively. The absence of any absorption band in 400–600 regions suggests lactonized conformation of rhodamine units in the compound. Upon addition of H2 O (≤50% volume fraction) to CH3 CN, both the absorption bands were red-shifted to 280 and 327 nm, respectively. Further, level off tail was observed in visible region (Fig. 1A). The transmission electron microscopy (TEM) image of compound 4 in H2 O/CH3 CN (1:1) showed the presence of spherical aggregates of average size 450 nm (Fig. 1B). In the fluorescence spectrum, the solution of compound 4 in CH3 CN exhibits a weak emission band at 530 nm ( = 0.019) when excited at ex = 327 nm. The absence of an emission band at higher end suggests that the rhodamine moiety remains in a closed, non-fluorescent spirolactam form, thus, indicating a weak spectral overlap between HPB emission and rhodamine absorption. Upon increasing the volume fraction of water (up to 50%), the emission band shifts from 530 nm to 475 nm along with 10-folds enhancement in the emission intensity (Fig. 2) and quantum yield ( = 0.203). On increasing the water fraction more than 60% a decrease in emission intensity was observed. A decrease in the emission intensity upon the addition of more than a 60% water fraction may be attributed to the low solubility of derivative
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Fig. 1. (A) UV–vis Spectrum of derivative 4 (5.0 M) in different fractions of H2 O/CH3 CN and (B) TEM image of derivative 4 in H2 O/CH3 CN (1:1).
to 0.172 × 109 s−1 whereas a decrease in the rate constant for non-radiative (knr ) pathway was observed from 1.094 × 109 s−1 to 0.677 × 109 s−1 (Table S2 in the Supplementary Information). The increase in radiative rate constant in presence of water is 8-folds; however, decrease in non-radiative rate constant is around two folds [47]. We believe that upon aggregation molecules become more planer and rigid which is the reason behind the emission enhancement (Scheme 2). We also carried out concentration dependent 1 H NMR studies of derivative 4, which show the downfield shift of imino proton and up-field shift of aromatic protons of HPB moiety which suggests intermolecular - stacking between the molecules of derivative 4 (Fig. S4 in the Supplementary Information). On the basis of absorption, fluorescence, TEM and DLS studies, we believe that formation of closely packed ordered aggregates is the reason behind the observed AIEE phenomenon in case of derivative 4. The solution of aggregates is visibly transparent and stable at room temperature for several days. Fig. 2. Fluorescence emission spectra of compound 4 (5.0 M) in different ratios of H2 O/CH3 CN at ex = 327 nm.
4 in the solvent mixture, thus leading to a decrease in the number of emissive molecules per unit volume [46]. The dynamic light scattering (DLS) studies show average size of 443 nm in 50% H2 O/CH3 CN solvent mixture of derivative 4 (Fig. S2 in the Supplementary Information). To get insight into the reason behind the emission enhancement, we investigated the behaviour of the derivative 4 in solvent mixture having different ratio of DMSO–glycerol fractions. It was observed that upon increasing the ratios of glycerol from 0% to 99%, the emission intensity of derivative 4 enhanced by 15-folds (Fig. S3 in the Supplementary Information). This is attributed to high viscosity that restricts the intramolecular rotation, thus, leading to closure of the non-radiative decay channel, thereby making the molecule emissive in its aggregated state. These studies suggest that restriction to motion is one of the important reasons for the AIEE phenomenon observed in this molecule. We also carried out time resolved studies of derivative 4 and determined the rate constants for the radiative (kr ) and non-radiative (knr ) pathways. With increase in fraction of water from 0 to 50%, the rate constant for the radiative (kr ) pathways increased from 0.021 × 109 s−1
3.3. Binding studies In the next part of our work, we studied recognition behaviour of aggregates of derivative 4 toward different analytes viz. Hg2+ , Fe2+ , Fe3+ , Pb2+ , Cd2+ , Cu2+ , Zn2+ , Ni2+ , Ag+ , Co2+ , Mg2+ , Li+ , Na+ , and K+ using UV–vis and fluorescence spectroscopy. The absorption spectrum of probe 4 (5.0 M) in HEPES/CH3 CN (1:1, pH 7.0) shows an absorption band at 327 nm ( = 6.58 × 104 M−1 cm−1 ). Upon addition of Hg2+ ions (0–320 M) to the solution of derivative 4, a new absorption band appears at 564 nm ( = 4.48 × 104 M−1 cm−1 ) (Fig. 3). These absorption changes were accompanied by colour change of the solution from colourless to pink clearly visible to naked eye. No significant change in the absorption band was observed in the presence of the other metal ions viz. Fe2+ , Fe3+ , Pb2+ , Cd2+ , Cu2+ , Zn2+ , Ni2+ , Ag+ , Co2+ , Mg2+ , Li+ , Na+ , and K+ tested (Fig. S5 in the Supplementary Information). In the fluorescence spectrum, a new band appears at 582 nm and the emission intensity at 475 nm gradually decreased upon addition of Hg2+ ions to the solution of aggregates of derivative 4 (Fig. 4). This is attributed to the binding of Hg2+ ions with spirolactam ring of rhodamine, resulting in opening of spirolactam ring which leads to efficient energy transfer from donor (HPB) to acceptor (rhodamine) (Scheme 3). The emission ratio at 475 nm and 582 nm (F475 /F582 ) increased linearly with increase in concentra-
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Scheme 2. Pictorial presentation of proposed mechanism for AIEE and interaction of aggregates of derivative 4 with mercury ions.
Scheme 3. AIEE active Hg2+ induced FRET phenomenon.
tion of Hg2+ ions and plateau was reached upon the addition of 320 M of Hg2+ , with 7-folds enhancement in fluorescence intensity. The fluorescence quantum yield of 4-Hg2+ complex was found to be is 0.622 (at 582 nm). The selectivity of probe 4 for Hg2+ ion was investigated in the presence of different metal ions viz. Fe2+ , Fe3+ , Pb2+ , Cd2+ , Cu2+ , Zn2+ , Ni2+ , Ag+ , Co2+ , Mg2+ , Li+ , Na+ , and K+ but no significant change in fluorescence intensity was noticed, which clearly indicates that derivative 4 is selective for Hg2+ ions among the various metal ions tested (Figs. S6A–B in the Supplementary Information). The detection limit was calculated using equation 3(Sb )/k, where ‘Sb is the relative standard deviation of solution of derivative 4 (5 M) and ‘k’ is the slope of the calibration curve in Fig. 5. The standard deviation was 0.0189 and slope of the calibration curve was
0.56819. So the detection limit of derivative 4 was 100 × 10−9 M which is sufficiently low for monitoring Hg2+ levels in biological samples. In next part of our work, we examined the reversibility of 4Hg2+ complex. Upon addition of potassium iodide to the solution of 4-Hg2+ (5.0 M in CH3 CN/H2 O) complex, the original spectra was restored. This indicates that strong binding between iodide and mercury ions caused the decomplexation of 4-Hg2+ complex, hence the spirolactam ring was closed. Interestingly, on further addition of Hg2+ ions, the energy transfer mechanism was revived (Fig. S7 in the Supplementary Information). These studies show the reversibility of aggregates of derivative 4. We also carried out time resolved fluorescence studies of derivative 4 in the absence and in the presence of Hg2+ ions (Fig. 6). The
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Fig. 3. UV–vis spectra of compound 4 (5 M) in H2 O/CH3 CN (1:1, HEPES, buffer solution pH 7.0) upon addition of Hg2+ ions (320 M). Inset: photograph shows colour change after addition of Hg2+ ions.
Fig. 4. Fluorescence spectra of probe 4 (5.0 M) in response to the presence of Hg2+ ions (320 M) in H2 O: CH3 CN (1:1, v/v); buffered with HEPES, pH 7.0; ex = 327 nm. Inset: photograph shows the change in fluorescence after addition of Hg2+ ions.
Fig. 5. The ratiometric fluorescence responses (I582 /I475 ) of derivative 4 (5 M) to various concentrations of Hg2+ ions in H2 O/CH3 CN (1:1, v/v) mixture. ex = 327 nm.
Fig. 6. Fluorescence lifetime decay profiles of probe 4 in presence Hg2+ ions in H2 O: CH3 CN (1:1, v/v). Arrow indicates the change in decay profile. IRF = instrument response function. ex = 377 nm and emission spectra are recorded at 475 nm with 32 slit width.
fluorescence lifetime of the probe 4 exhibits triexponential decay with life times of 1.89 ns (42.97%), 7.28 ns (37.20%) and 0.34 ns (19.82%) respectively in the absence of Hg2+ ions. Upon addition of Hg2+ ions, shortening of fluorescence decay time were observed. On addition of 320 M of Hg2+ ions the derivative 4 exhibited fast triexponential decay time constants 1.19 ns (51.39%), 5.94 ns (12.65%) and 0.06 ns (19.08%). This shortening of lifetime on addition of Hg2+ ions indicates the energy transfer from donor HPB to acceptor rhodamine moiety. The energy transfer efficiency (ETF) from HPB to rhodamine in the presence of Hg2+ was found to be 97.14%. Further we carried out fluorescence studies of aggregates of derivative 4 at different pH values. At acidic pH (2–3) the derivative 4 shows fluorescence band at 582 nm and the colour of the solution changes from colourless to pink due to opening of the spirolactam ring of rhodamine moiety, however there is no fluorescence band at 582 nm corresponding to ring opened form of spirolactam ring at higher pH values (4–9). From these studies, it is clear that aggregates of derivative 4 exhibit ratiometric response towards Hg2+ ions in the pH range 4–9 (Fig. 7). To get insight into binding interaction between aggregates of derivative 4 and Hg2+ ions, we carried out 1 H NMR studies in D2 O/DMSO-d6 . In the 1 H NMR spectrum, the rhodamine protons of derivative 4 undergo a downfield shift in the presence of Hg2+ ions which proves the interaction between Hg2+ ions and aggregates of derivative 4 through amide linkage of ring opened rhodamine moiety (Fig. S8 in the Supplementary Information). The TEM image of 4 + Hg2+ in CH3 CN/H2 O (1:1) shows the presence of small sized spherical aggregates (Fig. 8). The DLS studies also show the decrease in size of the aggregates after addition of mercury ions and particles of size in the range 140 nm were observed (Fig. S9 in the Supplementary Information). Under the same conditions as used above for derivative 4, we also carried out fluorescence studies of an equimolar mixture of HPB donor 2 and rhodamine acceptor (ring opened form of rhodamine B) and no quenching of emission of HPB donor 2 and no enhancement in the fluorescence emission of the rhodamine acceptor was observed when the mixture was excited at the HPB absorption band, i.e., at 327 nm (Fig. S10 in the Supplementary Information). This study clearly indicates that there is no intermolecular energy transfer between the HPB donor and rhodamine acceptor in the mixture.
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Fig. 7. Fluorescence emission spectrum of compound 4 (5.0 M) in H2 O/CH3 CN (1:1) mixture varies with pH 9–2 (pH of the water is set not the mixture) ex = 327 nm.
Fig. 8. TEM image after adding Hg2+ ions to solution of derivative 4 in CH3 CN/H2 O (1:1) mixture showing small sized aggregates.
After investigating the sensing behaviour of derivative 4 in the aggregated state, we also examined its fluorescence emission response towards mercury ions in molecular form in acetonitrile. The emission spectrum of derivative 4 exhibits weak emission at 530 nm in CH3 CN when excited at 327 nm. The rhodamine moiety is in non-fluorescent spirolactam form, thus, no band at higher wavelength was observed. Upon addition of Hg2+ ions (0–150 M) to the solution of derivative 4 in CH3 CN a band appeared at 582 nm (Fig. S11 in the Supplementary Information) corresponding to ring opened form of rhodamine moiety. The energy transfer is operating through bond, i.e., via conjugated linker which allows energy transfer from donor to acceptor through bond, however, only 25% energy transfer takes place (page S19 in the Supplementary Information). In molecular form, small overlapping between emission spectrum of HPB moiety and absorption spectrum of rhodamine is observed (Fig. S13 in the Supplementary Information). On the other hand, strong overlap between emission spectrum of donor and absorption spectrum of acceptor is observed in assembled form which is responsible for the ratiometric response of derivative 4 towards Hg2+ ions in mixed aqueous media (Fig. S14 in the Supplementary Information). These studies highlight the crucial role of supramolecular assemblies in energy transfer mechanism. To investigate the role of spacer and imine linkages in AIEE induced FRET phenomenon in derivative 4, we prepared derivatives
1 [40], 5 [48] and 6 (Chart 2). Derivatives 1 and 5 were found to be AIEE active in H2 O/CH3 CN solvent mixture (Figs. S15 and S16 in the Supplementary Information). Aggregates of derivative 1 showed small spectral overlap between the donor emission and acceptor absorption and exhibited mercury induced FRET phenomenon in mixed aqueous media with only 45% energy transfer (Figs. S17 and S18 in the Supplementary Information), while in derivative 5 donor unit and acceptor unit exhibited insignificant spectral overlap, thus, no mercury induced energy transfer was observed (Figs. S19 and S20 in the Supplementary Information). On the other hand, derivative 6 was found to be AIEE inactive and did not show any mercury induced ring opening of rhodamine moiety (Figs. S21 and S22 in the Supplementary Information). This study highlights the importance of hexaphenylbenzene scaffold in the designed molecule. In next part of our work, we planned to examine the practical application of aggregates of derivative 4 for detection of mercury ions. The concentration of biothiols is large in the living cells and they are reported to have strong affinity for mercury ions. To investigate the interference of biothiols in detection of mercury, we carried out the competitive experiments in the presence of Hg2+ ions mixed with biothiols (cysteine, homocysteine and glutathione). Interestingly, no significant change in the fluorescence emission was observed in presence of biothiols (Fig. S23 in the Supplementary Information). Thus, from above studies, it is clear that the probe can be safely used for imaging of Hg2+ ions in living cells without any interference from biothiols.
Chart 2. Derivative 5 and 6.
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Fig. 9. Fluorescence images of MCF-7 cell lines. (a and b) Fluorescence image of cells in blue and red channels treated with probe 4 (5.0 M) for 20 min at 37 ◦ C. (c) DIC images of probe 4 only. (d and e) Fluorescence images of cells in blue and red channels upon treatment with probe 4 (5 M) and then Hg(ClO4 )2 (20 M) for 20 min at 37 ◦ C. (f) DIC images of (d) and (e). (g and h) Fluorescence images of cells in blue and red channels upon treatment with probe 4 (5 M) and then Hg(ClO4 )2 (50 M) for 20 min at 37 ◦ C. Fluorescence images are recorded at blue (470 ± 20 nm) and red channels (570 ± 20 nm); ex = 405 nm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
3.4. Cell imaging studies Encouraged by these results, we planned to examine the biological application of the derivative 4. Firstly, we performed MTT [49] (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay (to check the cytotoxicity of the probe) which showed that >86% of MCF-7 cells survived after 24 h (Fig. S24 in the Supplementary Information). Thus derivative 4 was found to be non-toxic to the MCF-7 cells. The potential biological application of the derivative 4 was evaluated for in vitro detection of Hg2+ ions in MCF-7 cell lines. The cell lines were incubated with recep-
tor 4 in an RPMI-1640 medium for 30 min at 37 ◦ C and washed with phosphate buffered saline (PBS) buffer (pH 7.4) to remove excess of receptor 4. Confocal microscope images showed no intracellular fluorescence in red channel and bright fluorescence in blue channel indicate that derivative 4 is cell permeable and acts as imaging agent. The cells were then treated with mercury perchlorate (20.0 and 50.0 M) in the RPMI-1640 medium and incubated again for 30 min at 37 ◦ C and washed with PBS buffer. After treatment with Hg2+ ions the fluorescence emission gradually decreased in blue channel (Fig. 9). These results suggest that receptor 4 is an effective intracellular Hg2+ imaging agent with the ratiometric change
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in fluorescence emission, attributed to the working of the FRET phenomenon within the cellular systems. Moreover, we have calculated the LOD value for detection of Hg2+ ions in MCF-7 cell lines and it was observed that the lowest concentration of Hg2+ ions that can be image using derivative 4 was found to be around 1.5 ± 0.5 M. This study indicates that derivative 4 is a very good candidate for the detection of Hg2+ ions in submillimolar concentration (Fig. S25 and S26 in the Supplementary Information). 4. Conclusion In conclusion, we designed and synthesized AIEE active fluorescent probe 4 which shows fluorescence resonance energy transfer (FRET) in presence of Hg2+ ions in mixed aqueous media. The aggregates of derivative 4 can also be used as a ratiometric fluorescent probe for cell imaging of Hg2+ ions in MCF-7 cell lines. Acknowledgments M.K. and V.B. are thankful to UGC [ref. no. 42-282/2013(SR)] and Science and Engineering Research Board (SERB), New Delhi (ref no. SR/S1/OC-69/2012). Grants under the University with Potential for Excellence (UPE) scheme of the University Grants Commission (UGC) is acknowledged for providing infrastructural facilities to perform the research work. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.snb.2017.04.074. References [1] D.W. Boening, Ecological effects, transport, and fate of mercury: a general review, Chemosphere 40 (2000) 1335–1351. [2] A. Renzoni, F. Zino, E. Franchi, Mercury levels along the food chain and risk for exposed populations, Environ. Res. 77 (1998) 68–72. [3] I. Hoyle, R.D. Handy, Dose-dependent inorganic mercury absorption by isolated perfused intestine of rainbow trout, Oncorhynchus mykiss, involves both amiloride-sensitive and energy-dependent pathways, Aquat. Toxicol. 72 (2005) 147–159. [4] G. Shanker, L.A. Mutkus, S.J. Walker, M. Aschner, Methylmercury enhances arachidonic acid release and cytosolic phospholipase A2 expression in primary cultures of neonatal astrocytes, Mol. Brain Res. 106 (2002) 1–11. [5] J.-D. Park, W. Zheng, Human exposure and health effects of inorganic and elemental mercury, J. Prev. Med. Pub. Health 45 (2002) 344–352. [6] N.K. Mottet, M.E. Vahter, J.S. Charleston, L.T. Friberg, Metabolism of methylmercury in the brain and its toxicological significance, Met. Ions Biol. Syst. 34 (1997) 371–403. [7] W.D. Atchison, M.F. Hare, Mechanisms of methylmercury-induced neurotoxicity, FASEB J. 8 (1994) 622–629. [8] G. Chen, Z. Guo, G. Zeng, L. Tanga, Fluorescent and colorimetric sensors for environmental mercury detection, Analyst 140 (2015) 5400–5443. [9] H.N. Kim, W.X. Ren, J.S. Kim, J. Yoon, Fluorescent and colorimetric sensors for detection of lead cadmium, and mercury ions, Chem. Soc. Rev. 41 (2012) 3210–3244. [10] T.R. Pavase, H. Lin, Q. Shaikh, Z. Li, Rapid detection methodology for inorganic mercury (Hg2+ ) in seafoodsamples using conjugated polymer(1,4-bis-(8-(4-phenylthiazole-2-thiol)-octyloxy)-benzene) (PPT) by colorimetric and fluorescence spectroscopy, Sens. Actuators B 220 (2015) 406–413. [11] R.R. Koner, S. Sinha, S. Kumar, C.K. Nandi, S. Ghosh, 2-Aminopyridine derivative as fluorescence ‘On?Off’ molecular switch for selective detection of Fe3+ /Hg2+ , Tetrahedron Lett. 53 (2012) 2302–2307. [12] M. Kumar, A. Dhir, V. Bhalla, R. Sharma, R.K. Puri, R.K. Mahajan, Highly effective chemosensor for mercury ions based on bispyrenyl derivative, Analyst 135 (2010) 1600–1605. [13] Z.-Q. Hu, C.-S. Lin, X.-M. Wang, L. Ding, C.-L. Cui, S.-F. Liu, H.Y. Lu, Highly sensitive and selective turn-on fluorescent chemosensor for Pb2+ and Hg2+ based on a rhodamine–phenylurea conjugate, Chem. Commun. 46 (2010) 3765–3767. [14] X. Zhang, Y. Xiao, X. Qian, A ratiometric fluorescent probe based on FRET for imaging Hg2+ ions in living cells, Angew. Chem. Int. Ed. 47 (2008) 8025–8029.
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Biographies Gurpreet Singh is working at Department of Chemistry, Guru Nanak Dev University as a PhD student. His research interests include the design, synthesis and evaluation of AIEE active fluorescent probes for detection and imaging of various toxic metal ions.
Shahi Imam Reja received his PhD from the Department of Chemistry, Guru Nanak Dev University in 2017. His research interests include studies in the synthesis and evaluation of the fluorescent probes as chemosensors and imaging agents. He has published 14 research papers in international journals. Vandana Bhalla received her PhD from the Department of Chemistry, Guru Nanak Dev University in 1998. She is presently working as assistant professor at Department of Chemistry, Guru Nanak Dev University. Her current research interests are supramolecular host–guest chemistry and catalysis. Davinder Kaur is working as PhD student in the Department of Botanical and Environmental Sciences, Guru Nanak Dev University. Her research interest is in bioactivity of natural compounds. Pardeep Kaur is working as PhD student in the Department of Botanical and Environmental Sciences, Guru Nanak Dev University. Her research interest is in bioactivity of natural compounds. Saroj Arora is working as Professor at Department of Botanical and Environmental Sciences, Guru Nanak Dev University. Her research interests include Genetic Toxicology, Animal Tissue Culture, and Bioactivity of Natural Compounds. She has published more than 120 research papers in national/international journals. Manoj Kumar is working as professor at the Department of Chemistry, Guru Nanak Dev University, Amritsar. He was conferred with Bronze medal by Chemical Research Society of India, Bangalore (CRSI) in 2011. His researchinterest includes supramolecular host–guest chemistry of cyclic and acyclic receptors, fluorescent probes and their bio imaging applications. He has published more than 164 research papers in international journals.
Contents lists available at ScienceDirect
Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Hexaphenylbenzene appended AIEE active FRET based fluorescent probe for selective imaging of Hg2+ ions in MCF-7 cell lines Gurpreet Singh a , Shahi Imam Reja a , Vandana Bhalla a,∗ , Davinder Kaur b , Pardeep Kaur b , Saroj Arora b , Manoj Kumar a,∗ a b
Department of Chemistry, UGC Sponsored Centre for Advanced Studies-II, Guru Nanak Dev University, Amritsar, Punjab, India Department of Botanical and Environmental Sciences, Guru Nanak Dev University, Amritsar, Punjab, India
a r t i c l e
i n f o
Article history: Received 14 September 2016 Received in revised form 11 April 2017 Accepted 12 April 2017 Available online 14 April 2017 Keywords: Hexaphenylbenzene AIEE Mercury FRET Fluorescent aggregates Cell imaging
a b s t r a c t Hexaphenylbenzene (HPB) appended rhodamine derivative 4 has been synthesized which exhibits aggregation-induced emission enhancement (AIEE) characteristics and forms fluorescent aggregates in mixed aqueous media. These aggregates show remarkable fluorescence resonance energy transfer (FRET) in the presence of Hg2+ ions among various metal ions tested. Further these aggregates were also utilized for the ratiometric imaging of Hg2+ ions in MCF-7 cell lines. © 2017 Elsevier B.V. All rights reserved.
1. Introduction Mercury is recognized as one of the most toxic pollutants which has severely contaminated land, water, air and food chain [1,2]. Unfortunately, from environment mercury can easily enter the human body by passing through the biological membranes, thus, causing serious damage to endocrine and nervous systems [3–6]. Further, the environmental mercury level is significantly enhanced through man-made activities. Additionally, lower organisms can easily transform mercury into methylmercury a highly potent neurotoxin that impacts the functioning of central nervous system in living beings [7]. The health and environment aspects provide sufficient impetus for quantification and trace detection of mercury in various biological and environmental samples. Among various approaches for detection of the mercury in biological and environmental systems, fluorescence signalling is one of the first choices due to its high detection sensitivity and selectivity [8–10]. In recent past, a variety of fluorescent probes based on small organic molecules have been reported for the detection of mercury ions, however, most of these reported probes are operational only in
∗ Corresponding authors. E-mail addresses: [email protected] (V. Bhalla), [email protected] (M. Kumar). http://dx.doi.org/10.1016/j.snb.2017.04.074 0925-4005/© 2017 Elsevier B.V. All rights reserved.
organic media [11,12], require high energy excitation [13–15] and exhibit emission at lower wavelength which thereby prevent their in vivo applications due to autofluorescence [16]. Recently, fluorescent micro supramolecular assemblies based on rhodamine derivatives have been developed which exhibit ‘turn on’ response towards mercury ions in aqueous media. In comparison to small molecule based mercury chemosensors, supramolecular assemblies based derivatives exhibit highly sensitive superamplified response towards mercury ions in aqueous media [17]. However, different types of additives were a necessity for the formation of microstructures [18] which decreased the economic and environmental advantages of the strategy. Another limitation of the assemblies based probe is that all the emission changes were centred on a single wavelength [19–24]. Emission changes on a single wavelength are disadvantageous due to interference from fluctuations of the background fluorescence. Thus, development of additive free supramolecular assemblies showing mercury induced emission changes at two different wavelengths in aqueous media is a challenge. Keeping this in mind, we envisaged that if we could design a new probe having a tendency to undergo self-aggregation in mixed aqueous media and having mercury sensing unit which could exhibit sensitive response towards mercury ions based on energy transfer mechanism then it will be possible to overcome above limitations. Recently, aggregation induced emission enhancement
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Chart 1. HPB derivative 1.
phenomenon has emerged as a powerful tool to form fluorescent aggregates in aqueous media [25–28]. Further, literature reports show that the probes based on donor-acceptor system has the potential to exhibit stimuli sensitive response based on energy transfer mechanism [29,30]. In this context, we planned to design a donor-acceptor dyad having aggregation induced emission enhancement active donor unit and mercury sensitive acceptor unit. We envisaged that the designed probe may form aggregates in aqueous media due to its AIEE characteristics and in the presence of mercury ions energy transfer may be triggered from donor moiety to acceptor unit. Thus, mercury induced emission changes could be visible at two different wavelengths. So, we planned to develop a donor- acceptor system having hexaphenylbenzene as the donor unit and rhodamine as the acceptor moiety. Hexaphenylbenzene is the scaffold of our choice as the donor unit due to its known AIEE characteristics [31]. We chose rhodamine as acceptor unit due to its known affinity for mercury ions [32–36]. Recently, from our laboratory, we reported a variety of rhodamine based probes for ‘turn-on’ detection of mercury ions which exhibited emission changes at two different wavelengths based on fluorescence resonance energy transfer (FRET)/through bond energy transfer (TBET) mechanism [37–39]. All these probes were operational in organic media; however, their applicability in aqueous media was not explored. Before designing a new target molecule, we planned to re-examine one of the previously reported probes for the detection of mercury ions in aqueous media. To test the validity of our idea regarding coupling AIEE characteristics with energy transfer mechanism, we chose hexaphenylbenzenerhodamine dyad 1 [40] which exhibits mercury induced through bond energy transfer (TBET) from HPB donor to rhodamine acceptor in methanol (Chart 1). We investigated the AIEE behaviour of this derivative in aqueous media. Derivative 1 exhibits weak aggregation induced emission enhancement in the aqueous media, however, only 45% mercury induced energy transfer phenomenon was observed in aqueous media (vide infra). Based on these results we envisaged that if we could design a new probe with increased number of rotatable carbon-carbon/carbon-nitrogen bonds then the AIEE characteristics of the molecule will be enhanced which in effect will increase the spectral overlap between emission spectrum of donor and absorption spectrum of the acceptor and thus designed and synthesized derivative 4 having rhodamine as acceptor and hexaphenylbenzene as AIEE active donor joined together through imine linkages. To our pleasure, derivative 4 exhibited the AIEE characteristics and formed fluorescent aggregates in mixed aqueous media. Furthermore, the mercury mediated opening of spirolactam ring of rhodamine unit triggered the energy transfer
from donor HPB unit to acceptor rhodamine groups, hence, resulting in emission changes centred at two distinct wavelengths. The work being reported in this manuscript has several advantages; first, for the first time AIEE active hexaphenylbenzene appended rhodamine derivative 4 has been synthesized which forms aggregates in mixed aqueous media. Secondly, in molecular form in organic media derivative 4 exhibits only 25% mercury induced energy transfer whereas in assembled form in mixed aqueous media mercury induced FRET is observed with 97.14% energy transfer. The large Stokes shift shown by these assemblies is very high in comparison to Stokes shift reported in the literature for different probes (Table S1A in the Supplementary Information). These studies highlight the importance of supramolecular assemblies in fluorescence resonance energy transfer mechanism in comparison to other probes reported in the literature (Table S1B in the Supplementary Information). Third, the work being reported in this manuscript demonstrates the practical application of fluorescent assemblies for the ratiometric detection of Hg2+ ions in MCF-7 cell lines. To the best of our knowledge this is the first report where supramolecular aggregates of hexaphenylbenzene appended rhodamine derivative have been utilized for selective cell imaging of Hg2+ ions in MCF-7 cell lines. 2. Experimental 2.1. General experimental methods and materials [41] All the reagents were purchased from Aldrich and were used without further purification. HPLC grade solvents were used in UV–vis and fluorescence studies. UV–vis spectra were recorded on a SHIMADZU UV-2450 spectrophotometer, with a quartz cuvette (path length 1 cm). The fluorescence spectra were recorded with a SHIMADZU 5301 PC spectrofluorimeter. 1 H and 13 C NMR spectra were recorded on a Bruker Avance III HD 500 MHz using CDCl3 as solvent. Data are reported as follows: chemical shift in ppm (d), multiplicity (s = singlet, d = doublet, t = triplet, m = multiplet, br = broad singlet), coupling constants J (Hz). Fluorescence quantum yield [42] was determined by using optically matching solution of diphenyl anthracene (Фfr = 0.90 in cyclohexane) and rhodamine B (Фfr = 0.65 in ethanol) as standard at an excitation wavelength of 373 and 530 nm, quantum yield is calculated using the equation: fs = fr × (1-10−ArLr /1-10−AsLs ) × (Ns 2 /Nr 2 ) × (Ds /Dr )
Фfs and Фfr are the radiative quantum yields of sample and the reference, respectively, As and Ar are the absorbance of the sample and the reference respectively, Ds and Dr the respective areas of emission for sample and reference. Ls and Lr are the lengths of the absorption cells of sample and reference respectively. Ns and Nr are the refractive indices of the sample and reference solutions (pure solvents were assumed respectively). 2.2. UV–vis and fluorescence titrations [41] A 10−3 M stock solution of derivative 4 was prepared by dissolving 16.20 mg of compound 4 in 10 ml of DMSO; 15 l of this stock solution was further diluted with 1485 l CH3 CN and 1500 l water/HEPES buffer (0.05 M, pH = 7.0) to prepare 3 ml solution of derivative 4 and this solution was used for each UV–vis and fluorescence experiment. The aliquots of freshly prepared standard solutions of metal perchlorates [M(ClO4 )X ; M = Hg2+ , Fe2+ , Fe3+ , Pb2+ , Cd2+ , Cu2+ , Zn2+ , Ni2+ , Ag+ , Co2+ , Mg2+ , Li+ , Na+ , and K+ ; X = 13], chlorides (MClY; Y = 1-3) (10−2 M) in distilled water were added to 3 ml solution of derivative 4 taken in quartz cuvette and spectra were recorded.
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Scheme 1. Synthesis of derivative 4.
2.3. Synthesis of compound 4 A clear solution of compound 2 [43] (0.05 g, 0.06 mmol) and 3 [44] (0.07 g, 0.15 mmol) in dry DCM: EtOH (2:8) was stirred at 70 ◦ C. After completion of the reaction in 48 h, precipitates were formed in the reaction mixture which were filtered and recrystallized from methanol to afford the light pinkish coloured compound 4 (0.082 g, 75% yield); mp: >258 ◦ C; 1 H NMR (500 MHz, CDCl3 ) ı = 8.56 (s, 2H, C = NH), 7.98 (d, J = 10 Hz, 2H), 7.48–7.44 (m, 8H), 7.31 (d, J = 10 Hz, 4H), 7.11 (d, J = 10 Hz, 4H), 7.06 (d, J = 5 Hz, 4H), 6.87–6.81 (m, 26H), 6.50 (d, J = 10 Hz, 4H), 6.41 (s, 4H), 6.21 (d, J = 10 Hz, 4H), 3.32–3.28 (q, 16H), 1.13 (t, J = 10 Hz, 24H). 13 C NMR (CDCl3 , 125 MHz) ␦ = 165.02, 153.12, 148.92, 146.94, 140.50, 139.76, 133.97, 131.88, 131.40, 128.23, 127.96, 126.56, 125.09, 123.83, 123.31, 107.99, 106.11, 98.13, 44.32, 29.70, 12.63, Mass, m/z = 1620.6138 [M+1]+ . 2.4. Maintenance of MCF-7 cell lines MCF-7 cells were cultured in RPMI1640 growth medium supplemented with 10% (v/v) FBS (Fetal Bovine Serum), penicillin, streptomycin and gentamycin in CO2 incubator at 37 ◦ C, 5% CO2 and 95% relative humidity in tissue culture flask. Medium was changed on a regular basis and cells were harvested at the log phase of growth for various analysis. 2.5. Confocal imaging Confocal imaging was done according to the method reported by Ramsay et al. [45] MCF 7 cells (100,000 cells/mL) were incubated in 6 well plates having coverslip in each well for 24 h and thereafter treated with different concentrations of test compound for 30 min. Cells were washed twice with chilled phosphate buffer saline (PBS) and then fixed with chilled 4% paraformaldehyde. Then washing of wells was carried out to remove the excessive dye. Finally, images were taken under Nikon Air Laser Scanning Confocal Microscope System. 3. Results and discussion 3.1. Synthesis The compound 2 [43] synthesized by reported method on condensation with rhodamine B hydrazide 3 [44] in dichloromethane
(DCM)/ethanol(EtOH) (2:8) furnished compound 4 in 75% yield (Scheme 1). The structure of compound 4 was confirmed from its spectroscopic data (Figs. S27–S29 in the Supplementary Information). The 1 H NMR spectrum of compound 4 showed one singlet at 6.41 ppm, six doublets at 7.98, 7.31, 7.11, 7.06, 6.50, 6.21 ppm, two multiplets at 7.48–7.44, 6.87-6.81 ppm corresponding to aromatic protons, one quartet at 3.32–3.28 ppm corresponding to methylene protons, one triplet at 1.13 ppm corresponding to methyl protons and one singlet at 8.56 ppm corresponding to imino proton. A parent ion peak for M+H+ was observed at m/z = 1620.6138 in the electrospray ionization mass spectrometry (ESI–MS) spectrum. These spectroscopic data corroborate the structure 4 for this compound. 3.2. Photophysical studies The photophysical behaviour of derivative 4 was studied by UV–vis and fluorescence spectroscopy. The absorption spectrum of derivative 4 (5.0 M) in CH3 CN exhibited two absorption bands at 275 nm and 322 nm (Fig. S1 in the Supplementary Information) corresponding to the S0 -S1 and S0 -S2 transitions with molar extinction coefficients of 8.3 × 104 M−1 cm−1 and 7.8 × 104 M−1 cm−1 , respectively. The absence of any absorption band in 400–600 regions suggests lactonized conformation of rhodamine units in the compound. Upon addition of H2 O (≤50% volume fraction) to CH3 CN, both the absorption bands were red-shifted to 280 and 327 nm, respectively. Further, level off tail was observed in visible region (Fig. 1A). The transmission electron microscopy (TEM) image of compound 4 in H2 O/CH3 CN (1:1) showed the presence of spherical aggregates of average size 450 nm (Fig. 1B). In the fluorescence spectrum, the solution of compound 4 in CH3 CN exhibits a weak emission band at 530 nm ( = 0.019) when excited at ex = 327 nm. The absence of an emission band at higher end suggests that the rhodamine moiety remains in a closed, non-fluorescent spirolactam form, thus, indicating a weak spectral overlap between HPB emission and rhodamine absorption. Upon increasing the volume fraction of water (up to 50%), the emission band shifts from 530 nm to 475 nm along with 10-folds enhancement in the emission intensity (Fig. 2) and quantum yield ( = 0.203). On increasing the water fraction more than 60% a decrease in emission intensity was observed. A decrease in the emission intensity upon the addition of more than a 60% water fraction may be attributed to the low solubility of derivative
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Fig. 1. (A) UV–vis Spectrum of derivative 4 (5.0 M) in different fractions of H2 O/CH3 CN and (B) TEM image of derivative 4 in H2 O/CH3 CN (1:1).
to 0.172 × 109 s−1 whereas a decrease in the rate constant for non-radiative (knr ) pathway was observed from 1.094 × 109 s−1 to 0.677 × 109 s−1 (Table S2 in the Supplementary Information). The increase in radiative rate constant in presence of water is 8-folds; however, decrease in non-radiative rate constant is around two folds [47]. We believe that upon aggregation molecules become more planer and rigid which is the reason behind the emission enhancement (Scheme 2). We also carried out concentration dependent 1 H NMR studies of derivative 4, which show the downfield shift of imino proton and up-field shift of aromatic protons of HPB moiety which suggests intermolecular - stacking between the molecules of derivative 4 (Fig. S4 in the Supplementary Information). On the basis of absorption, fluorescence, TEM and DLS studies, we believe that formation of closely packed ordered aggregates is the reason behind the observed AIEE phenomenon in case of derivative 4. The solution of aggregates is visibly transparent and stable at room temperature for several days. Fig. 2. Fluorescence emission spectra of compound 4 (5.0 M) in different ratios of H2 O/CH3 CN at ex = 327 nm.
4 in the solvent mixture, thus leading to a decrease in the number of emissive molecules per unit volume [46]. The dynamic light scattering (DLS) studies show average size of 443 nm in 50% H2 O/CH3 CN solvent mixture of derivative 4 (Fig. S2 in the Supplementary Information). To get insight into the reason behind the emission enhancement, we investigated the behaviour of the derivative 4 in solvent mixture having different ratio of DMSO–glycerol fractions. It was observed that upon increasing the ratios of glycerol from 0% to 99%, the emission intensity of derivative 4 enhanced by 15-folds (Fig. S3 in the Supplementary Information). This is attributed to high viscosity that restricts the intramolecular rotation, thus, leading to closure of the non-radiative decay channel, thereby making the molecule emissive in its aggregated state. These studies suggest that restriction to motion is one of the important reasons for the AIEE phenomenon observed in this molecule. We also carried out time resolved studies of derivative 4 and determined the rate constants for the radiative (kr ) and non-radiative (knr ) pathways. With increase in fraction of water from 0 to 50%, the rate constant for the radiative (kr ) pathways increased from 0.021 × 109 s−1
3.3. Binding studies In the next part of our work, we studied recognition behaviour of aggregates of derivative 4 toward different analytes viz. Hg2+ , Fe2+ , Fe3+ , Pb2+ , Cd2+ , Cu2+ , Zn2+ , Ni2+ , Ag+ , Co2+ , Mg2+ , Li+ , Na+ , and K+ using UV–vis and fluorescence spectroscopy. The absorption spectrum of probe 4 (5.0 M) in HEPES/CH3 CN (1:1, pH 7.0) shows an absorption band at 327 nm ( = 6.58 × 104 M−1 cm−1 ). Upon addition of Hg2+ ions (0–320 M) to the solution of derivative 4, a new absorption band appears at 564 nm ( = 4.48 × 104 M−1 cm−1 ) (Fig. 3). These absorption changes were accompanied by colour change of the solution from colourless to pink clearly visible to naked eye. No significant change in the absorption band was observed in the presence of the other metal ions viz. Fe2+ , Fe3+ , Pb2+ , Cd2+ , Cu2+ , Zn2+ , Ni2+ , Ag+ , Co2+ , Mg2+ , Li+ , Na+ , and K+ tested (Fig. S5 in the Supplementary Information). In the fluorescence spectrum, a new band appears at 582 nm and the emission intensity at 475 nm gradually decreased upon addition of Hg2+ ions to the solution of aggregates of derivative 4 (Fig. 4). This is attributed to the binding of Hg2+ ions with spirolactam ring of rhodamine, resulting in opening of spirolactam ring which leads to efficient energy transfer from donor (HPB) to acceptor (rhodamine) (Scheme 3). The emission ratio at 475 nm and 582 nm (F475 /F582 ) increased linearly with increase in concentra-
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Scheme 2. Pictorial presentation of proposed mechanism for AIEE and interaction of aggregates of derivative 4 with mercury ions.
Scheme 3. AIEE active Hg2+ induced FRET phenomenon.
tion of Hg2+ ions and plateau was reached upon the addition of 320 M of Hg2+ , with 7-folds enhancement in fluorescence intensity. The fluorescence quantum yield of 4-Hg2+ complex was found to be is 0.622 (at 582 nm). The selectivity of probe 4 for Hg2+ ion was investigated in the presence of different metal ions viz. Fe2+ , Fe3+ , Pb2+ , Cd2+ , Cu2+ , Zn2+ , Ni2+ , Ag+ , Co2+ , Mg2+ , Li+ , Na+ , and K+ but no significant change in fluorescence intensity was noticed, which clearly indicates that derivative 4 is selective for Hg2+ ions among the various metal ions tested (Figs. S6A–B in the Supplementary Information). The detection limit was calculated using equation 3(Sb )/k, where ‘Sb is the relative standard deviation of solution of derivative 4 (5 M) and ‘k’ is the slope of the calibration curve in Fig. 5. The standard deviation was 0.0189 and slope of the calibration curve was
0.56819. So the detection limit of derivative 4 was 100 × 10−9 M which is sufficiently low for monitoring Hg2+ levels in biological samples. In next part of our work, we examined the reversibility of 4Hg2+ complex. Upon addition of potassium iodide to the solution of 4-Hg2+ (5.0 M in CH3 CN/H2 O) complex, the original spectra was restored. This indicates that strong binding between iodide and mercury ions caused the decomplexation of 4-Hg2+ complex, hence the spirolactam ring was closed. Interestingly, on further addition of Hg2+ ions, the energy transfer mechanism was revived (Fig. S7 in the Supplementary Information). These studies show the reversibility of aggregates of derivative 4. We also carried out time resolved fluorescence studies of derivative 4 in the absence and in the presence of Hg2+ ions (Fig. 6). The
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Fig. 3. UV–vis spectra of compound 4 (5 M) in H2 O/CH3 CN (1:1, HEPES, buffer solution pH 7.0) upon addition of Hg2+ ions (320 M). Inset: photograph shows colour change after addition of Hg2+ ions.
Fig. 4. Fluorescence spectra of probe 4 (5.0 M) in response to the presence of Hg2+ ions (320 M) in H2 O: CH3 CN (1:1, v/v); buffered with HEPES, pH 7.0; ex = 327 nm. Inset: photograph shows the change in fluorescence after addition of Hg2+ ions.
Fig. 5. The ratiometric fluorescence responses (I582 /I475 ) of derivative 4 (5 M) to various concentrations of Hg2+ ions in H2 O/CH3 CN (1:1, v/v) mixture. ex = 327 nm.
Fig. 6. Fluorescence lifetime decay profiles of probe 4 in presence Hg2+ ions in H2 O: CH3 CN (1:1, v/v). Arrow indicates the change in decay profile. IRF = instrument response function. ex = 377 nm and emission spectra are recorded at 475 nm with 32 slit width.
fluorescence lifetime of the probe 4 exhibits triexponential decay with life times of 1.89 ns (42.97%), 7.28 ns (37.20%) and 0.34 ns (19.82%) respectively in the absence of Hg2+ ions. Upon addition of Hg2+ ions, shortening of fluorescence decay time were observed. On addition of 320 M of Hg2+ ions the derivative 4 exhibited fast triexponential decay time constants 1.19 ns (51.39%), 5.94 ns (12.65%) and 0.06 ns (19.08%). This shortening of lifetime on addition of Hg2+ ions indicates the energy transfer from donor HPB to acceptor rhodamine moiety. The energy transfer efficiency (ETF) from HPB to rhodamine in the presence of Hg2+ was found to be 97.14%. Further we carried out fluorescence studies of aggregates of derivative 4 at different pH values. At acidic pH (2–3) the derivative 4 shows fluorescence band at 582 nm and the colour of the solution changes from colourless to pink due to opening of the spirolactam ring of rhodamine moiety, however there is no fluorescence band at 582 nm corresponding to ring opened form of spirolactam ring at higher pH values (4–9). From these studies, it is clear that aggregates of derivative 4 exhibit ratiometric response towards Hg2+ ions in the pH range 4–9 (Fig. 7). To get insight into binding interaction between aggregates of derivative 4 and Hg2+ ions, we carried out 1 H NMR studies in D2 O/DMSO-d6 . In the 1 H NMR spectrum, the rhodamine protons of derivative 4 undergo a downfield shift in the presence of Hg2+ ions which proves the interaction between Hg2+ ions and aggregates of derivative 4 through amide linkage of ring opened rhodamine moiety (Fig. S8 in the Supplementary Information). The TEM image of 4 + Hg2+ in CH3 CN/H2 O (1:1) shows the presence of small sized spherical aggregates (Fig. 8). The DLS studies also show the decrease in size of the aggregates after addition of mercury ions and particles of size in the range 140 nm were observed (Fig. S9 in the Supplementary Information). Under the same conditions as used above for derivative 4, we also carried out fluorescence studies of an equimolar mixture of HPB donor 2 and rhodamine acceptor (ring opened form of rhodamine B) and no quenching of emission of HPB donor 2 and no enhancement in the fluorescence emission of the rhodamine acceptor was observed when the mixture was excited at the HPB absorption band, i.e., at 327 nm (Fig. S10 in the Supplementary Information). This study clearly indicates that there is no intermolecular energy transfer between the HPB donor and rhodamine acceptor in the mixture.
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Fig. 7. Fluorescence emission spectrum of compound 4 (5.0 M) in H2 O/CH3 CN (1:1) mixture varies with pH 9–2 (pH of the water is set not the mixture) ex = 327 nm.
Fig. 8. TEM image after adding Hg2+ ions to solution of derivative 4 in CH3 CN/H2 O (1:1) mixture showing small sized aggregates.
After investigating the sensing behaviour of derivative 4 in the aggregated state, we also examined its fluorescence emission response towards mercury ions in molecular form in acetonitrile. The emission spectrum of derivative 4 exhibits weak emission at 530 nm in CH3 CN when excited at 327 nm. The rhodamine moiety is in non-fluorescent spirolactam form, thus, no band at higher wavelength was observed. Upon addition of Hg2+ ions (0–150 M) to the solution of derivative 4 in CH3 CN a band appeared at 582 nm (Fig. S11 in the Supplementary Information) corresponding to ring opened form of rhodamine moiety. The energy transfer is operating through bond, i.e., via conjugated linker which allows energy transfer from donor to acceptor through bond, however, only 25% energy transfer takes place (page S19 in the Supplementary Information). In molecular form, small overlapping between emission spectrum of HPB moiety and absorption spectrum of rhodamine is observed (Fig. S13 in the Supplementary Information). On the other hand, strong overlap between emission spectrum of donor and absorption spectrum of acceptor is observed in assembled form which is responsible for the ratiometric response of derivative 4 towards Hg2+ ions in mixed aqueous media (Fig. S14 in the Supplementary Information). These studies highlight the crucial role of supramolecular assemblies in energy transfer mechanism. To investigate the role of spacer and imine linkages in AIEE induced FRET phenomenon in derivative 4, we prepared derivatives
1 [40], 5 [48] and 6 (Chart 2). Derivatives 1 and 5 were found to be AIEE active in H2 O/CH3 CN solvent mixture (Figs. S15 and S16 in the Supplementary Information). Aggregates of derivative 1 showed small spectral overlap between the donor emission and acceptor absorption and exhibited mercury induced FRET phenomenon in mixed aqueous media with only 45% energy transfer (Figs. S17 and S18 in the Supplementary Information), while in derivative 5 donor unit and acceptor unit exhibited insignificant spectral overlap, thus, no mercury induced energy transfer was observed (Figs. S19 and S20 in the Supplementary Information). On the other hand, derivative 6 was found to be AIEE inactive and did not show any mercury induced ring opening of rhodamine moiety (Figs. S21 and S22 in the Supplementary Information). This study highlights the importance of hexaphenylbenzene scaffold in the designed molecule. In next part of our work, we planned to examine the practical application of aggregates of derivative 4 for detection of mercury ions. The concentration of biothiols is large in the living cells and they are reported to have strong affinity for mercury ions. To investigate the interference of biothiols in detection of mercury, we carried out the competitive experiments in the presence of Hg2+ ions mixed with biothiols (cysteine, homocysteine and glutathione). Interestingly, no significant change in the fluorescence emission was observed in presence of biothiols (Fig. S23 in the Supplementary Information). Thus, from above studies, it is clear that the probe can be safely used for imaging of Hg2+ ions in living cells without any interference from biothiols.
Chart 2. Derivative 5 and 6.
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Fig. 9. Fluorescence images of MCF-7 cell lines. (a and b) Fluorescence image of cells in blue and red channels treated with probe 4 (5.0 M) for 20 min at 37 ◦ C. (c) DIC images of probe 4 only. (d and e) Fluorescence images of cells in blue and red channels upon treatment with probe 4 (5 M) and then Hg(ClO4 )2 (20 M) for 20 min at 37 ◦ C. (f) DIC images of (d) and (e). (g and h) Fluorescence images of cells in blue and red channels upon treatment with probe 4 (5 M) and then Hg(ClO4 )2 (50 M) for 20 min at 37 ◦ C. Fluorescence images are recorded at blue (470 ± 20 nm) and red channels (570 ± 20 nm); ex = 405 nm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
3.4. Cell imaging studies Encouraged by these results, we planned to examine the biological application of the derivative 4. Firstly, we performed MTT [49] (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay (to check the cytotoxicity of the probe) which showed that >86% of MCF-7 cells survived after 24 h (Fig. S24 in the Supplementary Information). Thus derivative 4 was found to be non-toxic to the MCF-7 cells. The potential biological application of the derivative 4 was evaluated for in vitro detection of Hg2+ ions in MCF-7 cell lines. The cell lines were incubated with recep-
tor 4 in an RPMI-1640 medium for 30 min at 37 ◦ C and washed with phosphate buffered saline (PBS) buffer (pH 7.4) to remove excess of receptor 4. Confocal microscope images showed no intracellular fluorescence in red channel and bright fluorescence in blue channel indicate that derivative 4 is cell permeable and acts as imaging agent. The cells were then treated with mercury perchlorate (20.0 and 50.0 M) in the RPMI-1640 medium and incubated again for 30 min at 37 ◦ C and washed with PBS buffer. After treatment with Hg2+ ions the fluorescence emission gradually decreased in blue channel (Fig. 9). These results suggest that receptor 4 is an effective intracellular Hg2+ imaging agent with the ratiometric change
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in fluorescence emission, attributed to the working of the FRET phenomenon within the cellular systems. Moreover, we have calculated the LOD value for detection of Hg2+ ions in MCF-7 cell lines and it was observed that the lowest concentration of Hg2+ ions that can be image using derivative 4 was found to be around 1.5 ± 0.5 M. This study indicates that derivative 4 is a very good candidate for the detection of Hg2+ ions in submillimolar concentration (Fig. S25 and S26 in the Supplementary Information). 4. Conclusion In conclusion, we designed and synthesized AIEE active fluorescent probe 4 which shows fluorescence resonance energy transfer (FRET) in presence of Hg2+ ions in mixed aqueous media. The aggregates of derivative 4 can also be used as a ratiometric fluorescent probe for cell imaging of Hg2+ ions in MCF-7 cell lines. Acknowledgments M.K. and V.B. are thankful to UGC [ref. no. 42-282/2013(SR)] and Science and Engineering Research Board (SERB), New Delhi (ref no. SR/S1/OC-69/2012). Grants under the University with Potential for Excellence (UPE) scheme of the University Grants Commission (UGC) is acknowledged for providing infrastructural facilities to perform the research work. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.snb.2017.04.074. References [1] D.W. Boening, Ecological effects, transport, and fate of mercury: a general review, Chemosphere 40 (2000) 1335–1351. [2] A. Renzoni, F. Zino, E. Franchi, Mercury levels along the food chain and risk for exposed populations, Environ. Res. 77 (1998) 68–72. [3] I. Hoyle, R.D. Handy, Dose-dependent inorganic mercury absorption by isolated perfused intestine of rainbow trout, Oncorhynchus mykiss, involves both amiloride-sensitive and energy-dependent pathways, Aquat. Toxicol. 72 (2005) 147–159. [4] G. Shanker, L.A. Mutkus, S.J. Walker, M. Aschner, Methylmercury enhances arachidonic acid release and cytosolic phospholipase A2 expression in primary cultures of neonatal astrocytes, Mol. Brain Res. 106 (2002) 1–11. [5] J.-D. Park, W. Zheng, Human exposure and health effects of inorganic and elemental mercury, J. Prev. Med. Pub. Health 45 (2002) 344–352. [6] N.K. Mottet, M.E. Vahter, J.S. Charleston, L.T. Friberg, Metabolism of methylmercury in the brain and its toxicological significance, Met. Ions Biol. Syst. 34 (1997) 371–403. [7] W.D. Atchison, M.F. Hare, Mechanisms of methylmercury-induced neurotoxicity, FASEB J. 8 (1994) 622–629. [8] G. Chen, Z. Guo, G. Zeng, L. Tanga, Fluorescent and colorimetric sensors for environmental mercury detection, Analyst 140 (2015) 5400–5443. [9] H.N. Kim, W.X. Ren, J.S. Kim, J. Yoon, Fluorescent and colorimetric sensors for detection of lead cadmium, and mercury ions, Chem. Soc. Rev. 41 (2012) 3210–3244. [10] T.R. Pavase, H. Lin, Q. Shaikh, Z. Li, Rapid detection methodology for inorganic mercury (Hg2+ ) in seafoodsamples using conjugated polymer(1,4-bis-(8-(4-phenylthiazole-2-thiol)-octyloxy)-benzene) (PPT) by colorimetric and fluorescence spectroscopy, Sens. Actuators B 220 (2015) 406–413. [11] R.R. Koner, S. Sinha, S. Kumar, C.K. Nandi, S. Ghosh, 2-Aminopyridine derivative as fluorescence ‘On?Off’ molecular switch for selective detection of Fe3+ /Hg2+ , Tetrahedron Lett. 53 (2012) 2302–2307. [12] M. Kumar, A. Dhir, V. Bhalla, R. Sharma, R.K. Puri, R.K. Mahajan, Highly effective chemosensor for mercury ions based on bispyrenyl derivative, Analyst 135 (2010) 1600–1605. [13] Z.-Q. Hu, C.-S. Lin, X.-M. Wang, L. Ding, C.-L. Cui, S.-F. Liu, H.Y. Lu, Highly sensitive and selective turn-on fluorescent chemosensor for Pb2+ and Hg2+ based on a rhodamine–phenylurea conjugate, Chem. Commun. 46 (2010) 3765–3767. [14] X. Zhang, Y. Xiao, X. Qian, A ratiometric fluorescent probe based on FRET for imaging Hg2+ ions in living cells, Angew. Chem. Int. Ed. 47 (2008) 8025–8029.
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Biographies Gurpreet Singh is working at Department of Chemistry, Guru Nanak Dev University as a PhD student. His research interests include the design, synthesis and evaluation of AIEE active fluorescent probes for detection and imaging of various toxic metal ions.
Shahi Imam Reja received his PhD from the Department of Chemistry, Guru Nanak Dev University in 2017. His research interests include studies in the synthesis and evaluation of the fluorescent probes as chemosensors and imaging agents. He has published 14 research papers in international journals. Vandana Bhalla received her PhD from the Department of Chemistry, Guru Nanak Dev University in 1998. She is presently working as assistant professor at Department of Chemistry, Guru Nanak Dev University. Her current research interests are supramolecular host–guest chemistry and catalysis. Davinder Kaur is working as PhD student in the Department of Botanical and Environmental Sciences, Guru Nanak Dev University. Her research interest is in bioactivity of natural compounds. Pardeep Kaur is working as PhD student in the Department of Botanical and Environmental Sciences, Guru Nanak Dev University. Her research interest is in bioactivity of natural compounds. Saroj Arora is working as Professor at Department of Botanical and Environmental Sciences, Guru Nanak Dev University. Her research interests include Genetic Toxicology, Animal Tissue Culture, and Bioactivity of Natural Compounds. She has published more than 120 research papers in national/international journals. Manoj Kumar is working as professor at the Department of Chemistry, Guru Nanak Dev University, Amritsar. He was conferred with Bronze medal by Chemical Research Society of India, Bangalore (CRSI) in 2011. His researchinterest includes supramolecular host–guest chemistry of cyclic and acyclic receptors, fluorescent probes and their bio imaging applications. He has published more than 164 research papers in international journals.