Aggregation-Induced Emission-Based Chemodosimeter Approach for

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An AIE-based Chemodosimeter Approach for Selective Sensing and Imaging of Hg(II) and Methylmercury Species Amrita Chatterjee, Mainak Banerjee, Dipratn G Khandare, Ram Gawas, Starlaine Chrizelle Mascarenhas, Anasuya Ganguly, Rishabh Gupta, and Hrishikesh Joshi Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b02663 • Publication Date (Web): 08 Nov 2017 Downloaded from http://pubs.acs.org on November 8, 2017

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Analytical Chemistry

An AIE-based Chemodosimeter Approach for Selective Sensing and Imaging of Hg(II) and Methylmercury Species Amrita Chatterjee,†* Mainak Banerjee,†* Dipratn G. Khandare,† Ram U. Gawas,† Starlaine C. Mascarenhas,‡ Anasuya Ganguly,‡ Rishabh Gupta,† Hrishikesh Joshi† *E-mail: [email protected], [email protected]; Tel: +91-832-2580-320 (A.C.); Tel: +91-832-2580-347 (M.B.) † Department of Chemistry, BITS, Pilani, Goa Campus, NH 17B Bypass Road, Zuarinagar, Goa 403726, INDIA. ‡ Department of Biological Sciences, BITS, Pilani, Goa Campus, NH 17B Bypass Road, Zuarinagar, Goa 403726, INDIA. ABSTRACT: Methylmercury (CH3Hg+) is the common form of organic mercury and is more toxic than its inorganic or elemental forms. Mercury is emanated in the course of various natural events and human activities and converts to methylmercury by anaerobic organisms. CH3Hg+ are ingested by fish and subsequently bioaccumulated in their tissue, and eventually, enter the human diet, causing serious health issues. Therefore, selective and sensitive detection of bioaccumulated CH3Hg+ in fish samples is essential. Herein, the development of a simple, highly sensitive and selective AIE-based turn-on probe for both inorganic mercury ions and organicmercury species is reported. The probe’s function is based on mercury ion-promoted transmetallation reaction of aryl boronic acid. The probe, a TPE-monoboronic acid (1), was successfully utilized for AIE-based fluorescence imaging study on methylmercury contaminated live cells and zebrafish for the first time. Both Hg(II) and CH3Hg+ ensued a fast transmetallation of TPE-boronic acid (1) causing drastic reduction in the solubility of the resulting product (TPE-HgCl/TPE-HgMe) in the working solvent system. At the dispersed phase, the aggregated form of TPE-mercury ions recovers planarity because of restricted rotational freedom promoting aggregation induced emission. Simple design, cost-effective synthesis, high selectivity, inexpensive instrumentation, fast signal transduction, low limit of detection (0.12 ppm) are some of the key merits of this analytical tool.

Mercury is considered as one of the most toxic and highly dangerous metal ions because of its diverse toxicological profile.1 Mercury is emanated in the course of various natural events and human activities and converts to its most common organic form, methylmercury (CH3HgX; X = halides) by anaerobic organisms.2,3 Methylmercury is even more toxic to living being than its inorganic forms (HgX2).4 Because of their lipid solubility, methylmercury species can readily cross biological membranes and act as powerful neurotoxin to many eukaryotes including fish, animals, and humans.2,3 Methylmercury intoxications are manifold which include prenatal brain damage, cognitive and motion disorders, vision and hearing loss etc.5 CH3Hg+ exposure to human is mostly due to marine sea food consumption with socio-economical costs estimated to be several billions of dollars per year worldwide.6 The unfortunate epidemics of Minamata Bay7 in Japan and in Iraq8 by methylmercury have proven the lethal intimidation of methylmercury species to human health. Owing to the severity and rising effects of mercury, Environmental Protection Agency (EPA) has set quite rigid thresholds for mercury species9,10 which demands the development of sensitive, selective and cost-effective probes for on-site detection of mercury species, especially methylmercury.10 In general, complex hyphenated techniques such as high performance liquid chromatography (HPLC),11 gas chromatog-

raphy (GC),12 atomic absorption spectroscopy (AAS),13 inductively coupled plasma mass spectroscopy (ICPMS),12 neutron activation analysis,14 and X-ray fluorescence spectrophotometry15 are some of the common techniques that used for detection of Hg(II) and methylmercury. The major limitations of these methods are requirement of costly and complicated instruments, longer data acquisition time, involvement of trained personnel etc. In recent years, to prevail these constraints as well as for easy and quick screening of a good number of mercury specifics, various sensor based approaches have been developed by utilizing polymeric materials,16 nucleic acids,17 metal nanoparticles,18 surface-enhanced Raman scattering (SERS) platforms19,20 and fluorescent organic molecules.21-26 Among them, fluorimetric chemosensors based on conventional dye molecules have drawn enormous attention because of simplicity and high selectivity with only limitation of aggregation caused quenching effect (ACQ) which mostly limits their use in vivo.27,28 Surprisingly, most of them could able to recognize only inorganic mercury22,23,29,30 and those which can detect organic mercury31-39 are much less in number. In the recent past, the concern of aggregation caused quenching (ACQ) among fluorimetric probes has been well-addressed by the introduction of aggregation induced emission (AIE) systems.40-48 Over last decade, AIE has emerged as most promising among fluoremetric sensors, in particular, for in vivo and in vitro applications restoring high selectivity and sensitivity.48

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Among various systems developed so far, tetraphnylethylene (TPE) derivatives have received highest attention because of their easy synthesis and functionalization.49-69 Till date, a couple of TPE-based chemosonsors have been reported for mercury ions.70,71 However, the probes introduced by Liu et al.70 and Neupane et al.71 use an intricate biomolecular recognition approach and strategically limited to recognition of inorganic Hg(II) form only. In continuation of our efforts towards development of various fluorescent probes for toxic analytes,6669,72,71 herein, we report, a TPE-boronic acid chemodosimeter (1) which selectively reacts with Hg2+ as well as methylmercurals in the presence of other metal ions by Hg-promoted transmetallation reaction.74,75 The probe is found equally effective in detecting inorganic mercury ions and methylmercury in aqueous solution as well as in living organisms. The probe was successfully utilized for AIE-based fluorescence imaging study on methylmercury affected live cells and zebrafish for the first time.

EXPERIMENTAL SECTION Chemicals and Reagents. 4-Bromobenzophenone and benzophenone were purchased from Sigma Aldrich (India). TiCl4 was purchased from Spectrochem Pvt. Ltd. Mumbai (India). All other common chemicals and solvents of AR grade were obtained from different commercial suppliers and were used without further purification. Ultrapure water was obtained from Millipore system and purged with N2 for 15 min prior to use. Instruments and Measurements. NMR spectra were recorded on Bruker Avance (300 MHz and 400 MHz) NMR spectrometer. Fluorescence spectra were taken on a JASCO FP6300 spectrofluorimeter. Transmission electron micrograph (TEM) and field emission scanning electron microscopy (FESEM) images were recorded on a Philips CM-200 transmission electron microscope and a Quanta 250 FEG (FEI make), respectively. Beckman Coulter DelsaTM Nano particle size analyzer was used for DLS study. IR spectra were recorded on IR Affinity-1 FTIR Spectrophotometer, Shimadzu. Elemental analysis was carried out on Vario elementar CHNS analyzer. Mass spectra were obtained from Agilent 6400B LCMS (ESI). General Procedure THF was dried over sodium and freshly distilled before use. The reactions were monitored by thin layer chromatography (TLC) carried out on 0.25 mm silica gel plates (60F-254) using UV light (254 or 365 nm) for visualization. Stock solution of compound 1 (1 mM) was prepared in THF. The solutions of mercuric chloride and methylmercuric chloride were prepared in deionized water (MilliQ, 18 MΩ) and THF, respectively. Deionized water and / or THF was used as per requirement for dilution purpose. For study of the effect of different metal ions stock solutions were prepared by dissolving respective chloride / nitrate salts in deionized water. All solutions were subjected to filtration through 0.22 µm syringe filter in order to avoid any interference by any particulate matter in fluorescence measurement. After addition of analyte each solution was incubated for 10 min before recording the corresponding

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fluorescence spectrum; the excitation wavelength was 345 nm and the emission was measured from 350 to 650 nm. All the experiments were performed at room temperature. Synthesis of 4-(1,2,2-Triphenylvinyl)phenylboronic Acid (1)76 1-(4-Bromophenyl)-1,2,2-triphenylethene (2) was prepared from an equimolar mixture of 4-bromobenzophenone and benzophenone using McMurry cross-coupling reaction according to the reported procedure.77 Next, compound 2 (0.410 g, 1 mmol) was dissolved in anhydrous THF (4 mL) in a 50 mL two-neck round bottom flask under nitrogen atmosphere. The mixture was cooled to −78 °C and n-BuLi (1.25 mL, 2 mmol) was added slowly. After the mixture was stirred for 3 h at the same temperature, triethyl borate (0.580 mL, 5 mmol) was injected. After 2 h, the mixture was slowly warmed to room temperature. HCl (3 M, 5 mL) solution was added slowly to the reaction mixture and it was stirred for another 3 h. The mixture was extracted with dichloromethane (3 x 20 mL) and the combined organic extracts were washed with water (1 x 10 mL) and brine solution (2 x 10 mL), and dried over anhydrous sodium sulfate. The crude product thus obtained was purified through a silica-gel column using a mixture of petroleum ether and ethyl acetate (5:1 by volume) as eluent. The probe 1 was obtained as white solid in 71% yield. 1H NMR (400 MHz, CDCl3, δ): 6.56 (d, 2H, J = 6.8 Hz), 6.87 (d, 2H, J = 6.4 Hz), 7.02−7.14 (m, 15H); 13C NMR (100 MHz, CDCl3, δ): 114.6, 126.2, 126.4, 127.6, 127.7, 131.35, 131.37, 131.40, 132.7, 136.0, 140.0, 140.6, 144.0, 144.1, 154.5; ESI-MS (+ve): m/z 377 [M+H]; Anal. Calc. for C26H21BO2: C, 83.00; H, 5.63; Found: C, 83.13; H, 5.54. Method for Cytotoxicity Study For cell culture and imaging HEK293 cells (Human Embryonic Kidney cells) maintained in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% (v/v) fetal bovine serum were used. The probe was dissolved in THF to make a 1 mM stock solution. 5 x 104 cells/well viable HEK cells were plated in a 24-well plate. After 24 h incubation at 37 oC in a humidified 5% CO2 incubator, the probe solution was added into the culture media present in the wells of the plate to achieve final concentrations of the probe in the media as 1 µM, 5 µM, 10 µM, 20 µM and 40 µM and they were incubated for further 24 h. Cytotoxicity was analysed by MTT assay. The cell viability (viable cells in a microscope field expressed as the percentage of total cells in that field) was calculated using Trypan Blue viability assay at every time and for every compound concentration. Each set of experiments was triplicated and the average results are presented. Method for Imaging of Methylmercurals in Live HEK Cells and Zebrafish For fluorescence imaging of methylmercurals accumulation in living cells and vertebrate organisms HEK cells and zebrafish (at different stages of life) were chosen.

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For study on zebrafish, one-day old, two-day old and threeday old zebrafish embryos were grown and subsequently treated with probe 1 and CH3HgCl. At a time, two of the embryos at their respective stages were taken in a 30 mm dish containing 2 mL of the embryo medium. To the medium, 10 µM of the probe was added and incubated for 30 min followed by washing with embryo medium. Then 1 µM of methylmercuric chloride in THF was added to the medium and incubated for another 30 min followed by washing with the embryo medium. One set of embryos at each stage were exposed only to the probe and another set only to methylmercury to serve as controls for validating the fluorimetric imaging studies on combined exposure to both (the probe and organomercury species). Untreated embryo controls (at each stage) were maintained as well. Fluorescence was monitored under a DAPI filter using the fluorescence microscope (Olympus IX51), captured and reported. The viability of the exposed embryo was monitored at regular intervals by checking the heartbeats using optical microscopy. Each experimental analysis was repeated thrice for better reproducibility of data.

To understand the sensing characteristic of probe 1 an equivalent study was conducted. It was seen that the fluorescence intensities of initially non-fluorescent solution of probe 1 (30 µM) in 90% water‒THF mixture intensified upon gradual addition of HgCl2 proving its effectiveness in the fast recognition of Hg(II) ions in solution (Figure 1). A separate time dependent study with equimolar mixture of probe 1 and HgCl2 (both 30 µM) revealed that 10 min of incubation time is good enough for obtaining maximum fluorescence response from the medium. Up to 9 fold increase of fluorescence intensity was observed upon addition of 1.0 equiv of Hg(II) to the probe solution.

RESULTS AND DISCUSSION Synthesis and Characterization of Probe 1. The probe, a TPE-monoboronic acid (1), is a literature known compound71 and was synthesized by following a two-step procedure in good overall yields (50% over two steps). At first, an equimolar mixture of 4-bromobenzophenone and benzophenone was converted to TPE-Br (2) by McMurry cross-coupling reaction in 71% yield (see SI for details).77 The spectral data matched well with the reported values. Next, compound 2 was reacted with n-BuLi at −78 °C followed by treatment with triethyl borate to get the intermediate TPE-borate ester which on acid hydrolysis in situ produces the desired product, 1. The formation of probe 1 was established by 1H NMR, 13C NMR, ESI-MS and CHN. The NMR data were in good agreement with the reported values.71 A broad singlet at δ 5.92 in 1H NMR indicates the presence of -B(OH)2 residue. The ESI-MS showed molecular ion peak at m/z 377 (M+H). Solvent Screening and Fluorimetric Response of Probe 1 towards Hg(II) Ions To find out the most suitable solvent system for fluorimetric studies, the proportion of water-THF was varied to identify the ratio of water-THF at which probe 1 starts aggregating leading to fluorescence emission by AIE effect. Probe 1 was readily soluble and non-fluorescent in pure THF and almost insoluble in water. Because of its poor solubility in water gradual addition of water in THF solution of probe 1, keeping probe concentration fixed at 30 µM, allows to retain its fluorescence response manifesting the AIE behavior. The fluorescence intensity of probe 1 upon photo-excitation at 345 nm was plotted with the water volume fractions (see SI, Figure S1). A sharp rise in fluorescence intensity was observed over 95% of water−THF which allowed setting the ideal working solvent system for fluorimetric studies as 90% of water−THF. As seen in figure S1, fluorescence response of probe 1 in the said solvent system is nominal.

Figure 1. (A) Fluorescence response of probe 1 (30 µM) upon addition of HgCl2 (0-2.0 equiv) in 90% water-THF mixture [λex 345 nm]; (B) Inset: plot of the increment in emission against no. of equiv of HgCl2; (C) An image showing the fluorescence change of 1 (30 µM) upon addition of 1.0 equiv of Hg2+ ions in 90% water–THF after 10 min.

Mechanistic Aspects of Hg2+ Sensing by Probe 1 and Spectral Features

Equivalent study showed that the saturation attains at 1.0 equiv of Hg(II) (30 µM) (inset of Figure 1). Further addition

Figure 2. A schematic representation of sensing process of probe 1 towards Hg2+ and CH3Hg+ by turn-on type AIE.

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of Hg(II) ions does not show any substantial changes in fluorescence intensity indicating complete utilization of the existing probe molecules by spontaneous transmetallation reaction (C-B to C-Hg).74,75 As expected, transmetallation of TPEboronic acid (1) ensued drastic reduction in the solubility of the resulting product (TPE-HgCl) in the working solvent system (90% water‒THF). As soon as the organomercury derivatives form by gradual addition of Hg(II) ions in the medium they go out of the working solvent system and form a dispersed phase. The aggregated form of TPE-mercury ions recovers planarity due to restriction on rotational freedom triggering aggregation induced emission. The sensing event is schematically represented in figure 2. In order to investigate Hg(II) triggered transmetallation of TPE boronic acid (1) transmission electronmicroscopy (TEM), field emission scanning electron microscopy (FESEM) and dynamic light scattering (DLS) were performed to identify the aggregates generated from 1 upon addition Hg(II) ions in the solution. In DLS study, the particle size of probe 1 in 90% water-THF was measured before and after the addition of equivalent amount of Hg(II) and the development of nanoaggregates was observed (Figure 3); the average particle size of probe 1 before addition of Hg(II) in working solution was about 36 nm which after addition of 30 µM (1 equiv) of Hg(II) in working solution became approximately 690 nm. TEM images revealed that while simple probe solution formed small particles (Figure 4A), larger aggregates were seen upon addition of 1.0 equiv of Hg2+ ions (Figure 4B) supporting the results obtained in DLS study. Similar results were obtained in

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FESEM analysis (see SI, Figure S2). These experimental results strongly support spontaneous formation of TPE based hydrophobic organomercurals, which aggregate and further show turn-on type fluorescence by AIE mechanism. Considering two possible modes of transmetallation of TPE-boronic acid (1) with Hg(II) ions (i.e. 1:1 or 1:2 ratio) a job-plot analysis was conducted to ascertain the actual binding stoichiometry of the probe-Hg2+ conjugate. The total concentration of probe and Hg2+ ions was kept constant (at 30 µM) and the mole fraction of Hg2+ was changed from 0 to 1. As shown in Job’s plot analysis, it can be observed that significant enhancement of fluorescence intensity of TPE-boronic acid (1) was resulted due to the reaction with Hg2+ (Figure 5). The fluorescence intensity reached to maximum at a mole fraction of about 0.5 of Hg2+ and then dropped down at higher mole fraction confirming that a 1:1 stoichiometry was mostly occurring for the transmetallation of Hg2+ with probe 1 suggesting TPE-HgCl is preferably formed. The formation of TPE-HgCl as the result of transmetallation reaction was indicated by IR (see SI, Figure S3) and further established by carrying out a CHN analysis. For this purpose, the same reaction was performed in larger quantity and the precipitate was collected. The presence of an additional band at 1450 cm-1 in the IR spectrum is a clear indication of existence of C-Hg bond in the resulting product (see SI for details, Figure S3).78 The CHN analysis of the solid product further indicated the formation of TPE-HgCl as the major product. Due to insolubility of the product in common organic solvents NMR or MS analysis could not be carried out. Presumably, second transmetallation does not take place because of considerable amount of steric bulk around Hg(II) ion due to the presence of two bulky TPE units.

Figure 3. Particle size analysis of probe 1 and the reaction mixture after addition of 1.0 equiv of Hg2+ in 90% water–THF.

Figure 5. Job's plot for the determination of the stoichiometry of probe 1 and Hg2+ ions (total concentration 30 µM; λex 345 nm).

Figure 4. TEM images of (A) probe 1 and (B) after addition of 1 equiv of Hg(II) in the same probe solution. Scale bar is 200 nm for both the images.

Selectivity Study of Probe 1 towards Hg(II) Ions. Next, the selectivity of the probe was investigated in the presence of various metal ions analogously under the identical condition. The fluorescence spectra were recorded after addition of 1.0 equiv of each metal ion including Al3+, Ba2+, Ca2+, Cd2+, Co2+, Fe2+, Fe3+, Mn2+, Ni2+, Pb2+, Sr3+, Zn2+ and Ag+ in

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the probe solution (30 µM). As depicted in figure 6, the probe showed hardly any response toward all other metal ions. The selectivity of probe 1 towards Hg2+ is so high that it remains unperturbed by the presence of many other challenging metal ions in a mixture. This was established by carrying out competition experiments in the presence of both mercury(II) and large excess (5 times of Hg2+) of respective metal ions (Figure 6). As expected, the Hg2+-induced fluorescence output of probe 1 remained indifferent in the presence or absence of other metal ions. It divulges that probe 1 preserves high specificity towards Hg2+ in the presence of various other competing metal ions. The specificity is an upshot of the fact that Hg2+ is only capable of fast transmetallation with the boronic acid residue

Figure 6. Maximum fluorescence response of probe 1 (30 µM) upon addition of different analytes in 90% water-THF mixture [λex 345 nm].

of the probe to form hydrophobic organomercury species, which in turn go to the disperse phase in the working solvent system due to decrease in solubility resulting in turn-on fluorescence response. Measurement of Detection Limit

To measure the limit of detection the response of probe 1 towards lower concentration range of HgCl2 was plotted (see SI, Figure S4). It was found that probe 1 responds to Hg(II) ions linearly at a wide concentration range (0.6 x 10-6 – 3.0 x 10-5 M) with R2 = 0.992 and from the linear plot the limit of detection of the probe was estimated to be 0.12 ppm of Hg(II) (Figure S4C). Fluorimetric Response of Probe 1 towards Methylmercurals The most interesting feature of the probe is its ability to detect methylmercurals in aqueous solutions with same efficiency as Hg(II) ions. For this purpose an equivalence study was conducted for probe 1 against gradual addition of CH3HgCl keeping all other parameters same as in the case of Hg(II) ions. As expected, the fluorescence intensities of initially non-

fluorescent solution of probe 1 (30 µM) in 90% water‒THF mixture intensified upon gradual addition of CH3HgCl and goes up to 8 fold of the baseline (Figure 7). In this case, the saturation attains at 1.0 equiv of CH3HgCl (30 µM) as well (Figure 7). It may be presumed that the chloride is getting replaced by TPE unit to form corresponding organic mercury species (Me-Hg-TPE) which because of its poor solubility forms a disperse phase and starts fluorescing by AIE mechanism.

Figure 7. (A) Fluorescence response of a probe 1 (30 µM) upon addition of CH3HgCl (0-2.0 equiv) in 90% water-THF mixture [λex 345 nm]; (B) Inset: plot of the increment in emission against no. of equiv of CH3HgCl.

Fluorescence Imaging in Live Cells and Zebrafish An efficient sensing tool for mercury is expected to be capable of detecting traces of organic mercury in living organisms. This opens up its real world applications including identification of mercury contaminated fish and other living organisms. The efficiency of probe 1 was assessed for fluorescence monitoring of methylmercurals accumulation in living cells as well as in vertebrate organisms. For this purpose CH3HgCl contaminated HEK cell-line and zebrafish at different stages of life were chosen. At first, the cytotoxicity of the probe at different concentrations to HEK cells was examined to ascertain cell sustainability at a desired probe concentration (see SI, Figure S5). Therefore, 70% confluent HEK cells were treated with five different concentrations of the probe, serially diluted in DMEM (i.e. 40 µM, 20 µM, 10 µM, 5 µM, and 1 µM) in a 24 well plate followed by 24 h of incubation. Untreated culture was also maintained. Cytotoxicity was analysed by standard MTT assay. The percentages of viable cells relative to untreated controls were estimated. As shown in figure S5, the cell viability is over 80% even at 40 µM of probe concentration after exposure for 24 h. It implies that moderately high concentration of the probe is non-toxic to living organisms and therefore, it can be used for the detection of methylmercurals in them.

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Since no cytotoxic effect was observed at various levels of probe 1 in the cell viability experiment, the median possible probe concentration i.e. 10 µM was chosen for cellular fluorescence imaging studies. The HEK cells were separately incubated with 10 µM of the probe and 1 µM of CH3HgCl for 30 min, respectively and their fluorescence images were recorded under fluorescence microscope (Figure 8). Another set of HEK cells were incubated with 1 µM of CH3HgCl for 30 min followed by PBS wash to remove non-accumulated methymercury. This was followed by incubation with 10 µM of probe for 30 min and their fluorescence images were recorded. As shown in figure 8d, an intense blue fluorescence was observed from the cells contaminated with CH3HgCl and subsequently treated with probe 1, which indicates that probe molecules can efficiently cross cell membranes and undergo transmetallation with intra-cellular organomercurals to convert them to AIE-active TPE derivative. However, the cells show no or very weak fluorescence in the absence of organomercurals (Figure 8b). The sensitivity of this probe was found to be extremely high since accumulation of methylmercury species in HEK cells at low concentration (1 µM) is good enough to express high fluorescence intensity from intra-cellular region. Same study was continued in vertebrates like zebrafish for invivo imaging and monitoring of methylmercury accumulation (Figure 8e-h and Figure S6 of SI). For this study, one-day old, two-day old and three-day old zebrafish embryos were exposed to 10 µM of probe 1 in the presence and absence of 1 µM CH3HgCl. Separate embryos at each stage were exposed only to the probe and another set only to methylmercury species to serve as controls for validating the fluorescence signals obtained on combined exposure to both (the probe and methylmercury species). At the same time, one set of untreated embryos was also maintained for each case. The images taken in fluorescence microscope (IX51) revealed that

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ability of TPE-boronic acid, 1 in identifying the accumulation of organomercury species in cells and living organisms.

CONCLUSION In summary, we have developed a simple and cost-effective AIE-active fluorescent chemodosimeter for organomercury and inorganic mercury ions. The probe, a TPE-monoboronic acid (1), efficiently detects Hg(II) ions or CH3Hg+ by mercury ion-promoted fast transmetallation reaction of aryl boronic acid. The poor solubility of the resulting product (TPEHgCl/TPE-HgMe) in the working solvent system forces them to form a dispersed phase and emit strong blue fluorescence. At the dispersed phase, the aggregated form of TPE-mercury ions recovers planarity due to restricted rotational freedom triggering aggregation induced emission. Simple design, easy synthesis, high selectivity, fast signal transduction, low limit of detection make probe 1 a viable alternative to the existing analytical tools for the detection of Hg(II) ions or CH3Hg+.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: Synthetic procedure for 2, solvent effect on the probe, FESEM images, plots for LOD, IR study, cytotoxicity study, detailed study of methylmercury accumulation on zebrafish, spectra of probe 1 (PDF).

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] (A.C.); [email protected] (M.B.); Tel: +91-832-2580-320 (A.C.), +91-8322580-347 (M.B.); Fax: +91-832-2557-033.

Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENT A.C. thanks UGC-DAE (CSR-KN/CRS-91/2016-17/1132) for financial support. D.G.K. and R.U.G. are thankful to BITS, Pilani for research fellowships. The authors thankfully acknowledge Prof. N. N. Ghosh of the same department for extending DLS facility. The authors also acknowledge SAIF-IITB, Mumbai, India for TEM images and the Central Research Facility, BITS Pilani KK Birla Goa Campus for FESEM images. Figure 8. Detection of CH3HgCl in HEK cells and three-day old zebrafish with probe 1. HEK cells and zebrafish were incubated with 10 µM of probe 1 and 1 µM of CH3HgCl in 90% water-THF mixture. Images of HEK cells in absence (a,b) and presence (c,d) of CH3HgCl; zebrafish in absence (e,f) and presence (g,h) of CH3HgCl. Scale bar for a, c and d is 200 µm.

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CH3HgCl was spread all across the body of zebrafish because of which the whole fish glow blue (image h of Figure 8). The preliminary in vitro and in vivo studies undoubtedly prove the

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Table of Contents (TOC).

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Figure 1. (A) Fluorescence response of probe 1 (30 µM) upon addition of HgCl2 (0-2.0 equiv) in 90% waterTHF mixture [λex 345 nm]; (B) Inset: plot of the increment in emission against no. of equiv of HgCl2; (C) A picture of fluorescence change of 1 (30 µM) upon addition of 1.0 equiv of Hg2+ ions in 90% water–THF after 10 min. 395x280mm (72 x 72 DPI)



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Figure 2. Sensing process of probe 1 based on AIE mechanism. 654x251mm (72 x 72 DPI)


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Figure 3. Particle size analysis of probe 1 and the reaction mixture after addition of 1.0 equiv of Hg2+ in 90% water–THF. 110x36mm (300 x 300 DPI)



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Figure 4. TEM images of (A) probe 1 and (B) after addition of 1 equiv of Hg(II) in the same probe solution. Scale bar is 200 nm for both the images. 805x291mm (72 x 72 DPI)


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Figure 5. Job's plot for determining the stoichiometry of probe 1 and Hg2+ ions (total concentration 30 µM; λex 345 nm). 104x74mm (300 x 300 DPI)



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Figure 6. Maximum fluorescence response of probe 1 (30 µM) upon addition of different analytes in 90% water-THF mixture [λex 345 nm]. 274x158mm (72 x 72 DPI)


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Figure 7. (A) Fluorescence response of a probe 1 (30 µM) upon addition of CH3HgCl (0-2.0 equiv) in 90% water-THF mixture [λex 345 nm]; (B) Inset: plot of the increment in emission against no. of equiv of CH3HgCl. 296x209mm (300 x 300 DPI)



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Figure 8. Detection of CH3HgCl in HEK cells and three-day old zebrafish with probe 1. HEK cells and zebrafish were incubated with 10 µM of probe 1 and 1 µM of CH3HgCl in 90% water-THF mixture. Images of HEK cells in absence (a,b) and presence (c,d) of CH3HgCl; zebrafish in absence (e,f) and presence (g,h) of CH3HgCl. Scale bar for a, c and d is 200 µm. 1104x446mm (72 x 72 DPI)


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346x137mm (72 x 72 DPI)


Aggregation-Induced Emission-Based Chemodosimeter Approach for

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