Development of a Rhodamine–Rhodanine-Based Fluorescent

Feb 6, 2014 - Chemical Sciences Division, Saha Institute of Nuclear Physics, 1/AF Bidhannagar, Kolkata 700064, India ... We introduce a new rhodamineâ...
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Development of a Rhodamine−Rhodanine-Based Fluorescent Mercury Sensor and Its Use to Monitor Real-Time Uptake and Distribution of Inorganic Mercury in Live Zebrafish Larvae Kallol Bera,† Anand Kant Das,§ Moupriya Nag,† and Soumen Basak*,† †

Chemical Sciences Division, Saha Institute of Nuclear Physics, 1/AF Bidhannagar, Kolkata 700064, India Department of Chemical Sciences, Tata Institute of Fundamental Research, Colaba, Mumbai 400005, India

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S Supporting Information *

ABSTRACT: We introduce a new rhodamine−rhodanine-based “turn-on” fluorescent sensor (RR1) and describe its application for detection of mercury, including in solution, in live cells, and in a living vertebrate organism. The sensor RR1, which is a onepot synthesis from rhodamine B, undergoes a rapid and irreversible 1:1 stoichiometric reaction with Hg2+ in aqueous medium. Using fluorescence correlation spectroscopy (FCS), RR1 was shown to detect the presence of as low as a 0.5 pM concentration of Hg2+. It may also lend itself to tagging with biomolecules and nanoparticles, leading to the possibility of organelle-specific Hg detection. Results of experiments with mammalian cells and zebrafish show that RR1 is cell and organism permeable and that it responds selectively to mercury ions over other metal ions. In addition, real-time monitoring of inorganic mercury ion uptake by cells and live zebrafish using this chemosensor shows that saturation of mercury ion uptake occurs within 20−30 min in cells and organisms. We also demonstrate the acquisition of high-resolution real-time distribution maps of inorganic mercury (Hg2+) in the zebrafish brain by using a simple fluorescence confocal imaging technique. rhodamine−rhodanine-based, water compatible, “turn-on” fluorescent sensor that can be prepared by a single-pot synthesis from rhodamine B in about 8 h and can respond to the presence of mercury with high selectivity and sensitivity. The sensor (RR1) was synthesized by treating rhodamine B with POCl3 followed by 3-aminorhodanine. After column chromatography using DCM/MeOH (100:1 v/v) as eluent, RR1 was obtained at 52% yield and was quite stable over 5 months, even when kept at room temperature. We speculated that introduction of the 3-aminorhodanine receptor to a rhodamine-based probe would (1) increase its affinity for Hg2+ in competitive aqueous media through anchoring of the thiosulphamide bond with Hg2+, (2) lead to its quick fluorescence and color responses (i.e., facilitate realtime detection) through subsequent coordination of the

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ercury is a highly reactive agent1−6 that causes grisly immunotoxic,7 genotoxic, and neurototoxic8 effects, including damages to the central nervous system,9 endocrine system,8 kidney,1 and other organs.4,6,8 Mercury poisoning, through food-chain accumulation or high dose exposure, can result in several diseases, including acrodynia, Hunter−Russell syndrome, and Minamata disease.5,9,10 Studies have shown higher mercury concentrations in brains of deceased and in blood of living patients with Alzheimer’s disease.9 Detection and quantification of mercury is thus of great interest for monitoring and preventing its contamination of the environment and the living world.1−5,7−20 A vast array of sensor systems, based on chromophores and fluorophores, polymers, DNAzymes,13 oligonucleotides,13,19,20 nanosystems,13,15 proteins,2,10 and so forth have been developed for detection of Hg2+, one of the most stable and lethal inorganic forms of mercury.15,16,19 However, the need still remains for simply prepared, selective sensors with fast response that work in an aqueous environment2,8,13 and can detect very low levels of mercury. Here, we introduce a novel © 2014 American Chemical Society

Received: December 21, 2013 Accepted: February 6, 2014 Published: February 6, 2014 2740

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spirolactum oxygen with Hg2+, (3) improve selectivity, and (4) couple Hg2+ irreversibly. The proposed sensing mechanism of the chemosensor RR1 is shown in Scheme 1. RR1 reacts with Hg2+ through (1) Scheme 1. Proposed Hg2+-Sensing Mechanism of RR1

Figure 1. Fluorescence titration of RR1 (10 μM) with Hg2+ (0−12 μM) in the water−ACN (60/40 v/v) mixture. Excitation: 520 nm.

mercury-promoted spirolactam ring-opening followed by cyclizations, (2) insertion of mercury to form a new seven member ring, which is cleaved, and (3) formation of a watersoluble mercury complex by solvent water. The rhodamine derivative RR1 is ideal for this application, because conversion of the rhodamine moiety from its benzenoid to quininoid form, due to breaking the conjugation-preventing C−N bond by reaction with Hg2+, restores the strongly fluorescent character of rhodamine that was suppressed in RR1 and gives it the character of a “turn-on” sensor. Formation of the 1,3,4-oxadiazole ring ensures irreversibility of the reaction, preventing transformation of the quinoid form of rhodamine back to the benzenoid form and giving permanence to its fluorescence enhancement.

Figure 2. Relative fluorescence response (excitation at 520 nm) of RR1 (10 μM) in the water−ACN (60/40 v/v) mixture in the presence of 1 equiv of Hg2+ (1), Cu2+ (2), Zn2+ (3), Ag+ (4), Pb2+ (5), Cd2+ (6), Ni2+ (7), Co2+ (8), Fe2+ (9), Mn2+ (10), Mg2+ (11), Ca2+ (12), Ba2+ (13), Li+ (14), K+ (15), Na+ (16), Cr2+ (17), and RR1 (18).

In pure acetonitrile medium, the sensor acts drastically differently and behaves as a reversible zinc ion sensor. Due to the absence of hydrogen bonding in acetonitrile medium, the sensor attacks the zinc ion through O−O end produced by rotation through the N−N single bond (see Supporting Information, Scheme S3). Fluorescence correlation spectroscopy (FCS) is a powerful single molecule spectroscopic tool to study the sensing capability of a chemosensor. The autocorrelation function of fluorescence intensity fluctuations, G(0), yields the diffusion time of the probe across the illuminated sample volume (typically a few femtoliters). The number of fluorescent particles in the sample volume is given by N = 1/G(0). Figure 3 shows the correlation functions obtained by varying the concentration of Hg2+ in a sample with a fixed concentration of RR1. With increasing [Hg2+], the value of G(0) clearly decreases, demonstrating the ability of this sensor to detect even a subnanomolar concentration of mercury (in our case, ∼0.5 pM) through the use of FCS. To the best of our knowledge, this is the first demonstration of the use of FCS in mercury sensing.15 In Vivo Detection of Mercury Ions. Owing to its chemical and spectroscopic properties, RR1 should be ideally suited to monitor mercury ions in different kinds of cells and in live organisms. To test this hypothesis, in vivo detection of mercury ions in HeLa, HEK293T (somatic cell), RN46A (serotonergic neuronal cell line), midbrain-derived MN9D (dopaminergic neuronal cell line), and rat primary cortical neuronal cultures

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RESULTS AND DISCUSSION Chemical and Spectroscopic Properties of RR1. RR1 forms a colorless solution in the water−acetonitrile (60/40 v/ v) mixture, indicating that it exists predominantly in the spirocyclic form. Addition of mercuric ion to RR1 in solutions causes instantaneous development of a pink color and a strong yellow fluorescence. This observation shows that the mercuryinduced ring-opening reaction takes place rapidly at room temperature. Figure 1 shows the result of fluorescence titration of RR1 (10 μM solution in the water−ACN mixture, 60/40 v/ v) with Hg2+. Upon addition of 1 equiv of Hg2+, the fluorescence emission intensity of RR1 underwent a 26-fold increase, and the emission maximum shifted from 579 to 583 nm. The reaction responsible for these changes reached completion well within the time frame (3 min) of these measurements. The irreversibility of the reaction was checked by EDTA titration (Figure S6). The fluorescence response of RR1 toward various other metal ions (Pb2+, Zn2+, Ca2+, Cd2+, Cr2+, Mg2+, Fe2+, Ag+, Cu2+, Mn2+, Ni2+, Ba2+, Co2+, Pb2+) was also examined under identical conditions; the maximum fluorescence enhancement occurred in the presence of Hg2+ (Figure 2). Counter anions of mercury salts had a negligible effect on the sensing process, as Hg(ClO4)2 and HgCl2 gave similar results. 2741

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Figure 3. Fluorescence autocorrelation curves obtained for RR1 (20 pM) in the water−ACN medium (60:40 v/v) containing (in order of decreasing maxima of G(0)) 0, 1, 5, 10, and 20 pM of Hg2+ (inset: 1/ G(0) vs [Hg2+] plot).

were evaluated. The different types of cultured cells and differentiated neuron cells were exposed to 5 μM of HgCl2 for 10−20 min at 37 °C, washed with PBS to remove the remaining mercury ions, and incubated with 5 μM of RR1 for 20 min. In a reverse experiment, the cells were first treated with 5 μM of RR1 for 20 min and then incubated with 5 μM of HgCl2 for 10 min at 37 °C after removal of the remaining chemosensor. Fluorescence microscopic images of the cells subjected to the two procedures show that RR1 enters cell membranes and reacts with mercury ions to form the fluorescent product (Figure 4). In addition, the detection limit of mercury ions in the in vivo system was also examined by measuring the fluorescence intensity of the treated HEK 293T cells with a fluorescence confocal imaging technique. It was found that mercury ions in HEK cells treated with more than 5 nM of Hg2+ were monitored by this method. The ability of a chemosensor to selectively monitor metal ions in cells is an important requirement for biological applications. As described above, RR1 promotes a mercuryion-selective fluorescence response in vitro. To examine whether this specificity is preserved in vivo, mammalian RN46A cells were treated with 5 μM of RR1 for 20 min, washed with PBS to remove the remaining chemosensor, and individually exposed to 100 μM of various metal ions including Mg2+, Zn2+, Fe2+, Cu2+, Cd2+, Mn2+, Pb2+, and Hg2+ for 20 min. Fluorescence analysis of these cells reveals that RR1 exhibits a greater than 22-fold selective response for mercury ions over other biologically relevant ions, such as Mg2+, Zn2+, Cu2+, and Fe2+ (Figure 5). To test the toxic effect of RR1 toward a cell, rat primary cortical neuronal cultures plated in 96-well plates were treated with high concentrations of the sensor. From the toxicity assay, it is clear that even a high concentration (100 μM) of RR1 is not toxic toward cells compared with 50 μM of H2O2 as a positive control (Figure 6). Importantly, RR1 shows a 24-fold greater response to mercury ions than to Cd2+ and Pb2+, prevalent toxic metals in the environment. The success encountered in the cell experiments encouraged an exploratory effort to determine if the chemosensor RR1 could be used to detect mercury ions in living organisms. Newly hatched zebrafish are relatively underdeveloped. Their eyes have yet to properly develop, and they still have an attached yolk sac. They are, therefore, often used as

Figure 4. Fluorescence confocal imaging of Hg2+ in live somatic, cortical culture, and neuronal cells. (1) Fluorescence confocal image of (a) HeLa, (b) cortical culture, (c) RN46A [serotonergic cell line], (d) HEK293T, and (e) MN9D [dopaminergic cell line] cells incubated with RR1 for 30 min, washed three times, and further incubated with 5 μM of Hg2+ for 30 min. (2) Bright field transmission image of (a) HeLa, (b) cortical culture, (c) RN46A, (d) HEK293T, and (e) MN9D cells cell incubated with RR1 (5 μM). (3) Fluorescence image of (a) HeLa, (b) cortical culture, (c) RN46A, (d) HEK293T, and (e) MN9D cells cell incubated with RR1 (5 μM). Excitation was at 543 nm using a Carl Zeiss LSM710 confocal system 543 nm laser and 40× objective lens. All scale bars = 10 μm.

a model organism for the study of vertebrate embryonic development and toxicology.6 To test the applicability of RR1 in a real living system, 5-dayold zebrafish larvae were treated with 50 μM of HgCl2 in E3 embryo media for 10 min at 28 °C, washed with PBS to remove the remaining mercury ions, and incubated in a solution containing 50 μM of RR1 for 20 min. In a reverse experiment, the zebrafish was first incubated with 50 μM of RR1 for 20 min at 28 °C and then exposed to 50 μM of HgCl2 for 10 min after removal of the remaining chemosensor. The results of fluorescence microscope analysis of these specimens show that mercury ions in zebrafish are fluorescently detected by RR1 (Figure 7). The facts suggest that RR1 can penetrate the tissue of a live zebrafish with ease. Despite global concerns, little is known about the mechanisms underlying transport and toxicity of different 2742

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Five-day-old live zebrafish were incubated with 50 μM of RR1 in E3 embryo media for 30 min at 28 °C, washed with E3 media to remove the remaining chemosensor, and anesthetized with tricaine (ethyl 3-aminobenzoate methanesulfonate salt). Immediately after addition of different concentrations of HgCl2 to the zebrafish in E3 media, the zebrafish were examined continuously under a fluorescence confocal imaging microscope for 1 h. At the end of the imaging, all larvae were alive, although mostly moribund. Strong accumulation of mercury ion (Hg2+) was observed in the rapidly dividing layer of the lens epithelium, visible as rings at the periphery of the eye lenses (Video S2). A relatively lower level of mercury was observed in the brain as fluorescence intensity is relatively lower with respect to various other organs (Figure 8).

Figure 5. Relative fluorescence response (excitation at 543 nm) of RR1 (5 μM) in RN46A cells in the presence of 1 equiv of Hg2+, Cu2+, Fe2+, Mg2+, Zn2+, Pb2+, Cd2+, and vehicle (veh).

Figure 6. Toxicity assay of RR1 toward cortical culture.

Figure 7. Bright field (a,c) and confocal (b,d) images of a 5-day-old zebrafish treated with RR1 (50 μM) in the absence (a,b) and presence of Hg2+ (50 μM). All scale bars = 200 μm.

Figure 8. Inorganic mercury (Hg2+) accumulation and distribution in 5 dpf zebrafish brain. Images of a 5-day-old zebrafish treated with both Hg2+ (20 μM) and RR1 (20 μM) (a) Upper portion, (b) bright field [scale bar 200 μm], and (c) high-resolution elemental distributions of Hg2+ zebrafish (zoom). Quantities of the different elements are plotted with a 40× water immersion objective. Scale bar = 50 μm.

mercury species.6 To investigate the impact of inorganic mercury (Hg 2+ ) on vertebrate development, we have successfully combined the zebrafish, a well-established developmental biology model system, with confocal fluorescence imaging. Tricaine-anesthetized 5-day-post-fertilization (dpf) whole zebrafish larvae were examined using fluorescence confocal imaging to obtain the time evolution of distribution maps of inorganic mercury within the live fish (see Supporting Information Video S1).

As the brain is the most delicate part of a living system and mercury instantly destroys the central nervous system, the ability to observe inside the live brain without brain sectioning is an extremely important aspect of the study of the mercury toxicity mechanism. Our experiments on the inorganic mercury-exposed zebrafish larvae showed specific mercury accumulation in the ventricular region of their brain (Figure 8a, Figure S14, Video S3). The distribution pattern in brain cells showed mercury to be confined to the cytoplasm but 2743

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excluded from the nucleus (Figure 8c). Using a synchrotronbased X-ray fluorescence imaging methodology, Korbas et al. have shown a mercury ion (inorganic and organic) distribution map inside the zebrafish organism.6 We have demonstrated here the acquisition of a high-resolution real-time distribution map of inorganic mercury inside the live zebrafish brain using a simple fluorescence confocal imaging technique (Figure 8c). The present images (Figure 8) are similar to those obtained by Korbas et al. The effectiveness of a chemosensor lies in its ability to penetrate the tissue it monitors. In this context, RR1 is capable of penetrating the blood brain barrier and spreads uniformly over all regions of tissues. To observe inorganic mercury distribution at different depths of the zebrafish brain, we have collected z-stack records in confocal imaging at a step size of 2 μm (Figure S15, Videos S4a−c). The fluorescence intensity distribution was found to vary with depth inside the brain. We also recorded high-resolution z-stack images of regions of the brain to obtain mercury ion distribution maps at the cellular level (Video S5). This simple approach demonstrates the power of fluorescence confocal imaging as a tool for investigating molecular toxicology of heavy metals (such as Hg2+) in such appropriate model systems as live zebrafish.

Scheme 2. Synthesis of RR1

dissolved in acetonitrile (10 mL), to which a solution of 3amino rhodanine (0.15 g, 0.1 mol) and triethylamine (5 mL) in acetonitrile (20 mL) was added dropwise over 30 min. After refluxing for 6 h, the solvent was removed under reduced pressure to give a reddish-violet solid. Water was then added to the mixture, and the aqueous part was extracted with dichloromethane (15 mL × 3). The organic layer was washed with water, dried over anhydrous Na2SO4, and filtered. Purification by column chromatography on silica gel (CH2Cl2) gave 0.25 g of yellow solid at 52% yield. The proposed molecular structure and its purity were confirmed by various spectroscopic analyses. 1 H NMR (CDCl3, 400 MHz): δ = 1.15 (t, J = 6.6 Hz, 12H, CH3), 3.30−3.37 (m, 8H, CH2), 3.734 (S, 2H, methylene CH2), 6.324−6.271 (m, 2H, ArH), 6.59 (d, 2H, J = 9.2, ArH), 6.54 (d, 2H, J = 9.2, ArH), 7.36 (d, 1H, J = 7.6 Hz, ArH), 7.68 (t, 1H, J = 7.4 Hz, ArH), 7.61 (t, 1H, J = 7.6 Hz, ArH), 8.03 (d, 1H, J = 7.6 Hz, ArH). 13C NMR (CDCl3, 400 MHz): δ = 194.3, 167.8, 163.6, (154.9, 154.8), (149.2, 148.9), 133.6, 131.0, 130.6, 129.8, 128.8, 124.8, 123.9, (107.8, 107.6), 104.8, (97.5, 97.0), 68.39, (44.45, 44.37), 32.4, (12.5, 12.4). HRMS (ESI+) m/z: [M + H]+ calcd for C31H32N4O3S2, 573.20; found, 573.00. Evidence of Formation of 4A. RR1 (50 mg, 0.18 mmol) was dissolved in acetonitrile (1.0 mL), to which 0.5 mL of an aqueous solution of mercury(II) chloride (100 mg, 0.18 mmol) was added dropwise. The mixture was stirred at 37 °C for 50 min. After evaporating the solvent by lyophilization, the crude product was subjected to column chromatography on silica gel (CH2Cl2/MeOH = 5:1) to give 91 mg of 4 (90% yield). HRMS (MALDI-TOF) m/z: [M + NaCl] calcd for C29H31ClN4NaO2S+, 557.17; found, 557.676 [Figure S13]. UV−Vis and Fluorescence Spectroscopy. Stock solutions of RR1 and biologically relevant analytes, including Na(I), K(I), Zn(II), Mg(II), Fe(II), Cu(II), Ca(II), Hg(II), Cd(II), Ni(II), Co(II), Mn(II), and Ba(II), were prepared in triple-distilled water. Absorption spectra were recorded on a Jasco V650 (Scinco) spectrophotometer, and fluorescence spectra were recorded using a Fluoromax equipped with a xenon lamp. Samples for absorption and emission measurements were contained in quartz cuvettes (1 mL volume). Excitation was provided at 520 nm with excitation and emission slit widths at 3 nm. All spectroscopic measurements were performed under physiological conditions (at 37 °C in PBS buffer, pH 7.4). Effect of pH. We also studied the stability of RR1 in acidic or basic pH solutions. From the experiment (as shown in Figure S9), strong fluorescence enhancement was observed at pH 4 which indicates spirocyclic ring-opening. These results clearly explain that this probe can be used in a broad range of pH (5− 10). HeLa, HEK293T, MN9D, and RN46A Cell Culture and Rat Primary Neuronal Cultures. Low-passage HeLa, HEK293T, and MN9D cells were cultured in DMEM media supplemented with 10% FBS, 50 units/ml penicillin, and 50 μg/mL streptomycin at 37 °C under humidified air containing

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EXPERIMENTAL SECTION Synthetic Materials and Methods. All solvents were of analytical grade and used without further purification. Disodium hydrogen orthophosphate dehydrate (Na2HPO4) and potassium dihydrogen orthophosphate (KH2PO4) were purchased from SD Fine Chem Ltd. (Mumbai, India). Sodium chloride (NaCl) was obtained from Fisher Scientific (Mumbai, India). Potassium chloride (KCl) and calcium dihydrate (CaCl2.2H2O) were obtained from SRL (Mumbai, India). Magnesium Sulfate was purchased from Anala R, Glaxo Laboratories (Mumbai India). Propidium iodide, poly-L-lysine, and Dulbecco’s Modified Eagle’s Medium (DMEM) were purchased from Sigma Aldrich, Inc. (St. Louis, MO). Penicillin/ streptomycin, fetal bovine serum (FBS), DMEM-F12 media, and trypsin were purchased from Gibco (Grand Island, NY). Hoechst 33342 was purchased from Molecular Probes (Eugene, OR). L-Glutamine, B-27 supplement, hepes, and neurobasal medium were purchased from Invitrogen. Petri dishes and 96well plates were purchased from Tarsons (Mumbai, India). 1H NMR and 13C NMR spectra were collected on a Bruker-400 spectrometer with chemical shifts reported as ppm (in CDCl3, TMS as internal standard). MALDI-MS mass spectra were measured on a Bruker Ultraflextreme MALDI-TOF instrument. Measurements of pH were made with a Sartorius PB 20 pH meter. Absorption spectra were collected on a Jasco V650 UV− vis spectrophotometer (Jasco, Japan). Fluorescence spectra were collected on a Spex Fluoromax 3 spectrofluorometer (Horiba Jovin Yvon, U.S.A.). CD spectra were recorded using a Jasco J720 spectropolarimeter (Jasco, Japan). Melting points were determined by using a digital auto melting point apparatus (Scientific International, India). Fluorescence correlation spectroscopy was carried out on a setup assembled in our laboratory. Confocal imaging experiments were carried out using a Carl Zeiss LSM 710 confocal system with 543 nm laser and 40× water immersion objective (Scheme 2. To a stirred solution of rhodamine B (0.4 g, 0.84 mmol) in 1, 2-dichloroethane (10 mL), 1 mL of phosphorus oxychloride was added. The solution was refluxed for 6 h and concentrated by lyophilization. The obtained crude acid chloride was 2744

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μM of RR1 in E3 media for 30 min at 28 °C. After washing with E3 media, various concentrations (0−100 μM) of HgCl2 were added to the zebrafish in E3 media, and fluorescence intensity was determined by a fluorescence confocal imaging microscope. Toxicity Assay. Rat primary cortical neuronal cultures plated in 96-well plates were treated with high concentrations of the sensor. The cells were assessed after 60 h for the extent of cell death. The cells were treated with 0.01 mg/mL concentrations of Hoechst 33342 (a DNA-intercalating dye that permeates membranes and hence labels all the cells present) and propidium iodide (PI, another DNA-intercalating dye that does not permeate live membranes and hence labels only the dead cells with damaged membranes) in TB for 10 min followed by washing with TB. The cells were imaged for Hoechst 33342 and PI fluorescence in a confocal microscope setup (LSM-710, Zeiss, Germany) using a 20× objective. A 690 nm pulsed light from a mode-locked Ti−sapphire laser (MaiTai, Spectra Physics, CA) was used for the two-photon excitation of Hoechst 33342. The fluorescence was separated from the excitation using a 690 nm dichroic mirror and detected using a photomultiplier tube (385−535 nm). PI was excited using a 543 nm laser (He−Ne, Zeiss), and the fluorescence was separated using a dichroic mirror and detected between 565 and 720 nm. Images were analyzed for the total number of cells (Hoechst 33342 fluorescent spots) and the number of dead cells (PI fluorescent spots) using an automated particle counter in Image J. The ratio of cells which are alive (PI negative) to the number of total cells was reported as the % viability.

5% CO2 in T-25 canted-neck flasks. For RN46A serotonergic cells, DMEM-F12 (1:1) was used instead of regular DMEM. For rat primary cortical neuronal cultures, pregnant female Wistar rats were obtained from the animal facility at the Tata Institute of Fundamental Research (TIFR). All animal handling procedures were approved by the Animal Ethics Committee of TIFR. Briefly, neurons were isolated from the cortex of 17-dayold embryos. The cortex was removed under sterile conditions in Hank’s Balanced Salt Solution (HBSS) containing hepes, 50 units/ml penicillin, and 50 μg/mL streptomycin, followed by a trypsin digestion (0.25%) solution for 15 min at 37 °C, trituration, and plating on poly-L-lysine-coated coverslipbottomed Petri dishes. Cells were grown in Neurobasal media supplemented with 2% b-27 supplement, 0.5% penicillin/streptomycin, and 0.25% L-glutamine. For toxicity experiments, rat cortical neurons were plated in 96-well plates. Medium was changed every 48 h. Imaging Studies of Cells. For imaging studies, all cell types were cultured in poly-L-lysine-coated (0.1 mg/mL) homemade coverslip-bottomed Petri dishes. For rat primary cortical cultures, imaging was completed after 4 days. Cell imaging was performed using a confocal microscope (LSM 710, Carl Zeiss, Germany) with a 40× water immersion objective. Fluorescence images were recorded at an interval of 0.5 μm in the longitudinal (Z) direction. Different cell types were incubated with 5 μM of RR1 in Thomson’s buffer (TB, 146 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 0.8 mM MgSO4, 0.4 mM KH2PO4, 0.3 mM Na2HPO4, 5 mM dextrose, and 20 mM Na-HEPES, pH adjusted to 7.4) for 20 min. Subsequent washes were given with TB to remove the remaining sensor. The cells were then exposed to 5 μM of HgCl2 for 10 min. The cells incubated under both conditions were imaged (excitation wavelength, 543 nm; emission wavelength, 557 nm). Imaging of Zebra Fish Incubated with Mercury Ions and RR1. Zebrafish was kept at 28 °C and maintained at optimal breeding conditions. For mating, male and female zebrafish were maintained in one tank at 28 °C on a 12 h light/ 12 h dark cycle, and then the spawning of eggs was triggered by giving light stimulation in the morning. Almost all eggs were fertilized immediately. The 5-day-old zebrafish was maintained in E3 embryo media (15 mM NaCl, 0.5 mM KCl, 1 Mm MgSO4, 1 mM CaCl2, 0.15 mM KH2PO4, 0.05 mM Na2HPO4, 0.7 mM NaHCO3, 10−5% methylene blue, pH 7.5). The 5-dayold zebrafish was incubated with 50 μM of HgCl2 in E3 media for 10 min at 28 °C. After washing with PBS to remove the remaining mercury ions, the zebrafish was further incubated with 50 μM of RR1 for 20 min at 28 °C. Alternatively, the 5day-old zebrafish was incubated with 50 μM of RR1 in E3 media for 20 min at 28 °C. After washing with PBS to remove the remaining sensor, the zebrafish was further incubated with 50 μM of HgCl2 for 10 min at 28 °C. The zebrafish was imaged using a confocal microscope. Real-Time Uptake of Mercury Ions by MN9D cells. MN9D cells were seeded in a glass-bottom Petri dish at a density of 103 cells per well in culture media. After 24 h, the culture media were replaced with fresh media, and the cells were incubated with 20 μM of RR1 in culture media for 30 min. After washing with Thomson’s buffer to remove the remaining sensor, various concentrations (0−20 μM) of HgCl2 were added to the cells in culture media, and fluorescence intensity was determined using a confocal microscope. Real-Time Uptake of Mercury Ions by 5-Day-Old Zebrafish. The 5-day-old zebrafish was incubated with 100

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CONCLUSION In conclusion, we have developed a new fluorescent sensor RR1 based on the fluorophore rhodamine B. The distinct color and fluorescence changes due to the spirolactam ring-opening makes this derivative extremely useful for sensing ions through fluorescence or naked-eye detection in aqueous solution. The irreversibility of RR1−mercury interaction ensures the desired response even in biological media containing other competitive mercury chelators (such as cysteine, GSH), which distinguishes it from sensors based on reversible mercury binding. Moreover, its fluorescence emission intensity increases in linear fashion with concentration of Hg2+ and over a wide pH span (∼5−10), which makes it potentially useful for quantification of Hg2+ ions in aqueous solution for practical analysis. This probe can also be used in fluorescence imaging assays for in vivo monitoring of mercury in various cells, including mammalian cell lines, primary and differentiated cells, and in vertebrate organisms. In particular, it can enable convenient acquisition of real-time distribution maps of Hg2+ accumulation in vertebrate tissues (including the brain) for understanding mechanisms of mercury toxicity. The diagram below shows that there exists a possibility for tagging the sensor with biomolecules (like proteins,16,17 DNA,4,18−20, etc.) and various types of systems (like nanoparticles6,14,16,20,21). The rhodanine moiety has two active methylene protons that can take part in a condensation type of reaction to generate a model system RR2 (Scheme 3 and Supporting Information, Scheme S4), which can be tagged with the cysteine residue of a protein.17 This methodology can be used to resolve the problem related to the low solubility of small organic molecules in biological solutions21 and to develop a protein, DNA,16 or liposome-based mercury detection and 2745

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Analytical Chemistry

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Scheme 3. Possible Uses of a Proposed Sensor (RR2) for (a) Organelle-Specific Detection and (b) Tagging of Biomolecules and Nanoparticles for Development as Mercury Sensors

detoxification system8 as well as an organelle-specific mercury sensor to study different types of biophysical processes related to mercury poisoning.4,16,18−20,22−25

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ASSOCIATED CONTENT

S Supporting Information *

Synthesis, characterization, and additional spectroscopic data. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We sincerely thank Dr. S. Maiti (TIFR) for assistance in setting up the FCS apparatus in our laboratory and allowing use of his cell culture facility and Dr. M. Sonawane (TIFR) for allowing us to use his fish culture facility. We also thank Bidyut Sarkar and Raja Kumar Rit for their constant support and help. We acknowledge financial support from CSIR, Government of India, and the BARD project at the Saha Institute.

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REFERENCES

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dx.doi.org/10.1021/ac404160v | Anal. Chem. 2014, 86, 2740−2746