A sensitive water-soluble fluorescent probe based on umpolung and

Subscriber access provided by TULANE UNIVERSITY

New Analytical Methods

A sensitive water-soluble fluorescent probe based on umpolung and aggregation-induced emission strategies for selective detection of Hg2+ in living cells and zebrafish Tang Gao, Xueyan Huang, Shuai Huang, Jie Dong, Kai Yuan, Xueping Feng, Tingting Liu, Kunqian Yu, and Wenbin Zeng J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b06895 • Publication Date (Web): 01 Feb 2019 Downloaded from http://pubs.acs.org on February 4, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 32

Journal of Agricultural and Food Chemistry

A sensitive water-soluble fluorescent probe based on umpolung and aggregation-induced emission strategies for selective detection of Hg2+ in living cells and zebrafish Tang Gao1, Xueyan Huang1, Shuai Huang1, Jie Dong2, Kai Yuan3, Xueping Feng3, Tingting Liu4, Kunqian Yu4, Wenbin Zeng1,* 1

Xiangya School of Pharmaceutical Sciences, Central South University,

Changsha 410013, China. 2

Central South University of Forestry and Technology, Changsha 410004,

China. 3

4

Xiangya Hospital, Central South University, Changsha 410078, China. State key Laboratory of Drug Research, Shanghai Institute of Materia

Medica, Chinese Academy of Sciences, Shanghai 201203, China. * Corresponding author: Wenbin Zeng, Tel: 86-731-82650459; E-mail: [email protected] (W. Zeng).

ACS Paragon Plus Environment


Abstract: Using Hg2+-induced umpolung reaction and aggregation induced emission (AIE), we have rationally developed a water soluble fluorescent probe 2,2'-(((4-(4,5-bis(4-methoxyphenyl)-1-phenyl-1H-imidazol-2-yl)phenyl)methylene)bi s(sulfanediyl))diethanol (MPIPBS) for Hg2+ detection. MPIPBS was found to have high selectivity and sensitivity toward Hg2+ detection. The mechanism of MPIPBS response to Hg2+ was verified by 1H-NMR titration, HPLC and HRMS spectroscopy. The detection limit was examined to be 1.45 nM, which is lower than most reported probes for Hg2+. Taking advantage of excellent optical properties of AIEgen, a paper based sensor for Hg2+ detection was fabricated by immobilizing the MPIPBS on Waterman test paper. Meanwhile, MPIPBS showed satisfactory analytical performance in real water and urine samples. Further, thanks to the high water solubility, cell membrane permeability and low cytotoxicity, MPIPBS was further used to detect Hg2+ both in living cells and zebrafish. We anticipate that the prepared probe was available to detect Hg2+ in environment and biosamples. Keywords ： Umpolung; Aggregation-induced emission; Fluorescent probe; Detection of Hg2+; Cell imaging; Zebrafish imaging.


Page 2 of 32

Page 3 of 32


Introduction As a extremely toxic heavy metal cation, Hg2+ is usually accumulated in fish and bacteria, when it enters the environment. In the bacteria and living system, Hg2+ is easily converted into neurotoxic methylmercury.1 Due to the high biological accumulation and easily passing through biological membranes and gastrointestinal tissues, Hg2+ accumulated in the food chain is harmful to DNA and central nervous system.2,3 In additon, the US Environmental Protection Agency (USEPA) limits the mercury ion content in drinking water to no more than 10 nM (2 ppb). 4 Hence, the development of effective, facile, and reliable methods to detect Hg2+ in environmental and/or biological samples are of great significance. Currently, several sensitive detection methods for detecting Hg2+, including atomic absorption spectroscopy (AAS),5 atomic fluorescence spectrometry (AFS),6 liquid chromatography (LC),7 and inductively coupled plasma-mass spectrometry (ICP-MS) 8, have been developed. Although these methods can sensitively detect Hg2+ with a range from submicrogram to nanogram per liter,9 these techniques have some certain disadvantages. For example, the cost of the ICPMS instrument is high, and the AAS is oftern affected by the nonlinearity of the calibration curve, especially in the higher absorbance range. Complicated pre-treatment procedures are usually needed before the sample analysis. In contrast, fluorescent probes are considered to be an effective means of detecting heavy metal ions and biomolecules due to their high sensitivity, selectivity and ease of operation.10-13



Recently, using the strategy of coordination or irreversible reactions, many fluorescent probes for Hg2+ have been construscted.14-19 However, fluorescent probes designed based on ligand complexation often show low selectivity due to interference from other metal cations (Ag+, Cu2+, Pb2+, Fe2+ and Fe3+)20-25 or anions (I, CN and PO4).24-26 In contrast, probes utilizing specific chemical reactions induced by Hg2+ have superior selectivity.27-29 On the other hand, most reported reaction-based probes detect Hg2+ in organic or aqueous organic solvent mixtures.30-35 In addition, the hydrophobic fluorophore of probes tend to aggregate in aqueous solution and quench the fluorescence,

35-37

which may result in low detection sensitivity in aqueous

solution.23,38 Furthermore, most of fluorescent probe for Hg2+ show the limit of detection (LOD) higher than the USEPA’s limit (2 ppb, 10 nM), which seriously restrict its application for real water samples, let alone in complex biosystems such as urine, plasma, living cells or bacteria.14,33,39,40 Thus, exploring novel fluorescent probe for Hg2+ with good water solubility and outstanding sensing properties is highly demanded. In 2001, Tang et al first proposed the concept of aggregation-induced emission.37 In contrast to aggregation-induced quenching (ACQ) fluorophores (fluorescein, rhodamine, and cyanine),41 the fluorogens with character of aggregation-induced emission have high emission efficiency, strong photostability, and large Stokes’ shift, which benifits for sensing applications with high sensitivity. In this context, the development of AIE probes and exploration of their biomedical applications have


Page 4 of 32

Page 5 of 32


attracted intense research interest.42-45 One of the working mechanisms of AIE-based sensors was turning ‘‘free’’ AIE molecules with intramolecular motion.46 42 Although many excellent AIE-based probes have been reported to detect mercury ions (Table S1), there are only few reported water-soluble AIE-based probes for detecting mercury ions in living cells and organism.47,48 Taking above in mind, we intent to develope water-soluble AIE-based fluorescent probe for selective detection of Hg2+. Our group successfully synthesized a fluorophore TIB with AIE properties.49 To enhance the intramolecular electron transfer and red shift the emission wavelength, We introduce push-pull electrons into the fluorophore. Literature studies have shown that Hg(II) induced dethioacetalization can be used to detect Hg2+.18,36,39 To improve the water-solubility, 2-mercaptoethanol was introduced in our design strategy. As expected, in the presence of Hg2+, the probe MPIPBS could be cleaved readily due to the Hg2+-induced umpolung reaction on the polyaryl-substituted imidazoles-based acyclic thioacetals, and subsequentently, compound MPIB was released with a free aldehyde group in aqueous solution. This chemical transformation from thioacetal to aldehyde modulated the water solubility and ICT efficiency of the probe, and resulted in the turn-on fluorescence emission (Scheme 1). Fluorescent probe MPIPBS was able to detect low concentrations of Hg2+ (limit of detection = 1.45 nM). Portable paper-based sensors have been designed for detection of Hg2+ by loading MPIPBS on Waterman test paper. In addition, MPIPBS was also used to detect Hg2+ in real water



samples and urine samples. More importantly, due to its good biocompatibility, MPIPBS was further used to detect Hg2+ in living cells and zebrafish. Experimental Section Materials and apparatus. Vitamin B1, 4-methoxybenzaldehyde, aniline and terephthaloaldehyde were purchased from Energy Chemical Co., Ltd (China). Ammonium acetate was purchased from Sinopharm Chemical Reagent Co., Ltd (China). 2-mercaptoethanol, and p-toluenesulfonic acid were obtained from Aladdin company (Shanghai, China). Other chemical reagents and solvents were used without further puriﬁcation. All inorganic salts LiCl, NaCl, Mg(NO3)2, CuCl2, CaCl2, Pb(NO3)2, CoCl2, NiCl2, KCl, ZnCl2, BaCl2, FeCl2, Al(NO3)3·9H2O, CrCl2, HgCl2, AgNO3, Cd(NO3)2·4H2O, NaOAc, NaBr, NaCl, Na2CO3 and Na3PO4 were obtained from Sinopharm Chemical Reagent Co., Ltd (China). Cysteine (Cys), homocysteine (Hcys) and glutathione (GSH) were purchased from Sigma-Aldrich. Ultrapure water was prepared by Millipore water purification system. UV-2550 scanning spectrophotometer (Shimadzu) was uesed to perforem UV-vis absorption spectra. Fluorescent spectra were collected on Shimadzu RF-5301. Particle size tests were conducted on Zestier Nano ZS (Malvern Instruments Ltd, Uk) at 25 oC. 1H NMR and 13C

NMR were measured in CD3OD, CDCl3 or DMSO-d6 (TMS as the internal

standard) on a Bruker AVII- 500 MHz and 125 MHz spectrometer, respectively. HRMS was conducted on an Orbitrap Velos Pro LC-MS spectrometer (Thermo Scientific).


Page 6 of 32

Page 7 of 32


Fluorescence spectroscopic studies. The stock solutions of Li+, Mg2+, Na+, Cu2+, Ca2+, Pb2+, Co2+, Ni2+, K+, Zn2+, Ba2+, Fe2+, Al3+, Cr2+, Hg2+, Ag+, Cd2+, AcO−, Br−, Cl−, CO32−, PO43− and biothiols were prepared in deionized water. O2•− , The stock solutions of hydrogen peroxide (H2O2), hydroxyl radical (•OH), nitroxyl (HNO) and nitric oxide (NO) were prepared according the literature.50-52 The stock solution of probe MPIPBS (1 mM) was dissolved in DMSO, and then diluted to 10 μM with PBS (10 mM, pH 7.4). A volume of 10 μL of MPIPBS solution (1 mM) was added to 990 μL of PBS buffer containg different concentrations of Hg2+ solutions. The solution was mixed by stirring and incubated for 30 minutes at room temperature. The test sample for the selective experiment was carried out by adding a suitable volume of metal ion mother liquor to a solution containing MPIPBS (10 μM) and making the final solution volume to 1 mL. To investigate the practicality of MPIPBS towards Hg2+, the detection was performed in real river water samples and dilutted urine through the standard addition method,53 respectively. Water samples were taken from Xianjiang River (Changsha, Hunan) and filtered througth a 0.22 μm membrane. After that, different concentration of Hg2+ was addded into the water samples. Then, 10 μL of MPIPBS solution (1 mM) was added to 990 μL of water samples. Urine samples were obtained from healthy volunteers and filtered througth 0.22 μm membrane before used. Collected the filtrate and centrifuged for 30 minutes at 15000 rpm. The supernatant was diluted by 50 times using PBS buffer. Finally, 10 μL of MPIPBS solution (1 mM) was added to urine samples(990 μL) with different contents of Hg2+.



All the samples were incubated for 30 min and then tested by fluorescence spectrometers. 1HNMR

titrations of MPIPBS with Hg2+. MPIPBS (26.7 mM) was dissolved in

500 μL of DMSO-d6 solution, followed by the addition of 0.5 and 1 equivalent of D2O dissolved mercury ions. After shaking the NMR tube for 30 minutes, its 1H NMR spectrum was measured at room temperature. Cell culture and confocal imaging. MDA-MB-231cells, were cultured in DMEM medium (Sigma-Aldrich) at 37 oC and 5% CO2. The medium contained 10% fetal bovine serum (Gibco), 100 units/mL penicillin (Gibco), and 50 μg/mL streptomycin (Gibco). Incubated the cells (0.6 × 106/mL)in a 6-well plate with a coverslip on the bottom for 24 hours. Cells were incubated with HgCl2 (5 μM or 10 μM) in DMEM for 1 h at 37 °C, and then washed twice with PBS to remove the remaining Hg2 + ions. Then the MDA-MB-231cells were incubated with MPIPBS (10 μM) for 1 hour. Then the residual probe from medium was washed with PBS. Removed the coverslip and added appropriate amount of DAPI staining 15 min, and then washed several times with PBS. A drop of 50% glycerol (glycerol:PBS = 1:1) was added to the slide and observed by confocal fluorescence microscopy. Cell viability assay. The cytotoxicity of MPIPBS was studied by CCK-8 assay. Briefly, 100 μL of cell suspension was placed in a 96-well plate. The plates were pre-incubated in an incubator for 24 hours (37 oC, 5% CO2). 10 μL of different concentrations of MPIPBS were added to the plates. The plates were incubated for 48


Page 8 of 32

Page 9 of 32


hours in an incubator. 10 μL of CCK8 solution was added to each well. The plates were incubated for 1 hour in an incubator. The absorbance at 450 nm was measured with a microplate reader. to reflect the viable cell viability of each well.54 Zebrafish imaging. The zebrafish eggs in the sedimentation tank are collected and immediately fertilized. The embryos were then transferred to distilled water and incubated at 28 °C for 3 days in an incubator. The 3 days old zebrafish larvae were incubated with different concentrations of mercury ions (0 μM, 5 μM, 10 μM) at 28 °C for 2 hours and then washed three times with distilled water. Then, it was incubated with 10 μM MPIPBS for 1 hour and washed twice with distilled water to remove the residual probe. Finally, zebrafish larvae were fixed with 1.5% agarose and imaged by laser confocal scanning microscopy (ZEISS LSM 880). Results and discussion Synthesis of MPIPBS. Compound The synthetic route of MPIPBS was shown in Scheme S1. The synthesis of compound MPIB was based on the method we reported previously.48 In the presence of p-toluene sulfonic acid, MPIB reacted with 2-mercaptoethanolto afford MPIPBS with yield 62%.The structure of MPIPBS was characterized with 1HNMR, 13CNMR and mass spectrometry. Spectroscopic characterization of MPIB and MPIPBS. Firstly, we investigated the optical properties of MPIB and MPIPBS. As shown in Figure S1, the luminescent color of the MPIB solution was turned from blue (455 nm) to yellow (550 nm) as the solvent polarity was gradually increased. Furthermore, as the polarity of the solvent



increased, the emission intensity of the MPIB solution decreased, which was consistent with twisted intramolecular charge transfer (TICT) effect.55 However, due to the weak ICT effect, the fluorescence intensity of MPIPBS was weakly affected by the solvent (Figure S2). To further examine the character of TICT, the electron cloud distributions of MPIB and MPIPBS were calculated through density functional theory ((B3LYP level of theory, 6-31G (d3, 3p) basis set). The transition of the fluorophore from the highest occupied molecular orbital (HOMO) to the lowest unoccupied orbit (LUMO and LUMO+1) corresponds to the transition from S0 to S1.56 For fluorophores with TICT properties, the electron cloud of the HOMO orbital is usually distributed on the electron donating group, while the electron cloud of the LUMO and LUMO + 1 orbitals is distributed on the electron withdrawing group.57 As illustrated in Figure 1, the electron cloud of the HOMO orbital of MPIB was mainly distributed on the electron donating group, and the electron cloud of the LUMO and LUMO+ 1orbitals was distributed on the electron acceptor. This electron cloud distribution in MPIB is similar to the fluorophore with TICT properties as reported.52 Compared with MPIB, the electron distribution in the LUMO of MPIPBS is located on the whole molecule, which indicates that MPIPBS has a weak TICT effect. To explore the AIE characteristics of MPIB, we investigated the fluorescence emission spectra of MPIB in a mixed solvent of water and DMSO. As shown in Figure 2a, when the water content increased from 0 to 20%, the fluorescence emission intensity of MPIB decreased with a slight red shift. This change is due to an


Page 10 of 32

Page 11 of 32


increase in the water content resulting in an increase in the polarity of the surrounding environment. When the water content was in the range of 20% to 80% (Figure 2b), the fluorescence emission of MPIB was quenched due to the effect of the TICT. Conversely, as the water content increased from 80% to 99%, the fluorescence emission intensity of MPIB increased by 11 times and the emission wavelength shifted from 530 nm to 495 nm. This phenomenon is due to the fact that MPIB forms aggregates in high water content solutions and limits intramolecular rotation to ultimately activate AIE. The above results indicate that MPIB has TICT and AIE properties. Fluorescence response to Hg2+. Since MPIPBS has high water solubility, all experiments with MPIPBS were conducted in PBS buffer (10 mM, pH = 7.4, containing 1% DMSO). The absorption spectrum of the probe MPIPBS has a typical tail absorption peak of Mie scattering (Figure 3a). The aggregates of MPIPBS have average size of 96 nm in PBS (Figure S3). Upon addition of 302.4 nM Hg2+, the maximum absorption peak was red-shift, and the absorption peak at 380 nm was increased (Figure 3a). Moreover, after the addition of Hg2+ the average particle size increased to 178.7 nm (Figure S4). MPIPBS exhibited weak emission at 490 nm in PBS (Figure 3b). In the presence of Hg2+, the fluorescence emission at 490 nm was significantly enhanced (Figure 3b). The enhancement of fluorescence emission is due to the formation of aggregates upon addition of Hg2+.



In order to optimize the detection conditions, the experimental parameters including incubation time and pH effects were investigated. The detection kinetics of MPIPBS was carried out (Figure S5). The result showed that the response of MPIPBS to Hg2+ was almost completed within 30 min. Figure S6 illustrated the effect of pH on the fluorescence response of MPIPBS to Hg2+. MPIPBS (10 μΜ) was not affected by the pH from 4.10–13.09. As the pH increased from 4.10 to 7.40, the fluorescence intensity at 490 nm increased in the presence of 302.4 nM Hg2+. The results showed that alkaline conditions could promote the Hg2+-induced umpolung reaction. The above results indicated that MPIPBS is suitable for detecting Hg2+ in the physiological pH range. Hence, physiological pH (pH 7.4) was selected as the working pH in the following experiments. We used fluorescence titration to study the sensitivity of MPIPBS for Hg2+ detection. At 380 nm excitation, MPIPBS showed weak emission at 490 nm in PBS solution, and intensity gradually increased after adding different concentrations of. Hg2+ (Figure 3c). As shown in Figure 3d, the fluorescence intensity at 490 nm showed a good linear relationship (R2 = 0.99407) with Hg2+ in the range of 0–302.4 nM. The limit of detection (3σ/slope) of MPIPBS for Hg2+ was determined to be 1.45 nM (0.29 ppb), lower the maximum allowable level of Hg2+ in drinking water by the USEPA. Furthermore, we compared this probe to other reported AIE-based probes. (Table S1). These results indicated that MPIPBS has a high sensitivity.


Page 12 of 32

Page 13 of 32


Mechanism of MPIPBS to detect Hg2+. To explore the detection mechanism of MPIPBS, we performed a 1HNMR titration of MPIPBS in the absence and presence of 0.5 or 1.5 equiv. of Hg2+. As indicated in Figure 4, upon addition 1.5 equiv. Hg2+ to MPIPBS in DMSO-d6-D2O (9/1, v/v), the signal of the thioacetal-based proton at 5.21ppm disappeared. A new signal of the −CHO at 10.03 ppm appeared. At the same time, the proton signals in the benzene ring were shifted to the downfield, and the spectral similarity to that of MPIB. Furthermore, the reaction of MPIPBS with Hg2+ was verified by HPLC and HRMS. As shown in Figure S7a, the retention time of MPIPBS was 11.5 min. The peak of MPIPBS disappeared after the addition of 10 μM Hg2+, and a new peak was produced at 19.2 minutes. The retention time of this chromatographic peak was basically the same as that of MPIB (Figures S7b and S7c). In addition, the reaction product of MPIPBS and Hg2+ was further verified by HRMS. As shown in Figure S8, a peak at m/z 461.1852 was observed. These results provided the solid proof for the proposed response mechanism (Scheme S2), in which Hg2+ induced MPIPBS hydrolysis to MPIB in situ. The aggregation of MPIB in aqueous solution resulted in enhanced fluorescence emission. The selectivity of MPIPBS. A highly selectiveness is important for fluorescent probe with potential application in environmental samples and biological evaluation. Therefore, the selectivity experiments of MPIPBS were investigated. The selectivity of MPIPBS toward other species, including Li+, Na+, Ag+, Mg2+, Cu2+, Ca2+, Pb2+, Co2+, Ni2+, K+, Zn2+, Ba2+, Fe2+, Al3+, Cr2+, Cd2+, AcO−, Br−, Cl−, CO32−, PO43−, O2•− ,



H2O2, •OH, HNO, NO and biothiol was investigated. As shown in Figure S9 , there were not obvious fluorescence enhanced in the presence of other species, such as Li+, Na+, Mg2+, Cu2+, Ca2+, Pb2+, Co2+, Ni2+, K+, Zn2+, Ba2+, Fe2+, Al3+, Cr2+, Ag+, Cd2+, AcO−, Br−, Cl−, CO32−, PO43− biothiols, O2•− , H2O2, •OH, HNO and NO (10 μM for each). However, upon addition of Hg2+ (302.4 nM), an obvious fluorescence enhancement at 490 nm was observed for MPIPBS. These results clearly demonstrated that MPIPBS could selectively detect Hg2+. Detection of Hg2+ by paper test strips. Based on the positive results of our probe for Hg2+ detection, we further fabricated a simple paper strip to detect Hg2+. A paper test strip system was developed by dipping a neutral filter paper into a solution of MPIPBS (0.67 mM) in THF, and then dried in air. The different concentrations of Hg2+ was sprayed on the test paper. As shown in Figure S10, the fluorescence of test paper loaded with MPIPBS was blue. After the addition of Hg2+, the fluorescence of the test paper was observed to change from blue to green. With this method, the lowest detectable Hg2+ concentration was 0.30μM. In short, the result indicates that MPIPBS can be developed to the test strip for detection of Hg2+. Hg2+ detection in real samples. Due to the high sensitivity and selectivity of MPIPBS, we evaluate the practicality of MPIPBS for detecting Hg2+ in river water and urine samples. Figure S11a shows the fluorescence emission spectra of different Hg2+ concentrations in river water samples. With the quantitative standard curve behind in hand (Figure S11b), three samples added with Hg2+ were tested. Each


Page 14 of 32

Page 15 of 32


sample was measured independently three times. The recovery rate of Hg2+ in water samples was 95.2-103.2%, and RSD was less than 5.0% (Table 1). In addition, three urine samples with different Hg2+ concentrations were tested using the detection curve in diluted urine samples (Figure S12). The results in Table 1 indicated satisfactory recoveries. The above results prove that the probe MPIPBS can effectively detect Hg2+ in complex samples. Detection of Hg2+ in living cells. The excellent Hg2+ sensing performance of MPIPBS inspired us to evaluate its ability to detect Hg2+ in living cells. As shown in Figure 5b, MDA-MB-231cells stained by 10 μM MPIPBS alone showed weak fluorescence in the green channel. Upon addition of Hg2+ (5 μM or 10 μM), the fluorescence intensity in the green channel was increased. The overlay image revealed that the green fluorescence was distributed throughout the cytoplasm. This phenomenon was proved the good cell-membrane permeability of MPIPBS. Interesting, the fluorescent images shown in Figure 5e and Figure 5h clearly indicated that the intracellular fluorescence intensity exhibited an uneven distribution, which may be related to the distribution of Hg2+ in the cells.58 These results indicated MPIPBS is able to penetrate into MDA-MB-231 cells and could be used for the detection of Hg2+ in living cells. In addition, the CCK-8 assay was carried out. As depicted in Figure S13, the cell viability of GES-1 cells remains around 95% upon addition of MPIPBS (10 μM). The above results indicate that MPIPBS has good biocapability at low concentration.



Detection of Hg2+ in zebrafish. Human consumption of mercury-contaminated fish is one of the sources of mercury poisoning.59,60 We investigated whether MPIPBS can be used to detect Hg2+ in zebrafish. As depicted in Figure 6, the 3 days old zebrafish treated with MPIPBS alone exhibited no fluorescent signal. However, the zebrafish treated with both Hg2+ (5 μM) and MPIPBS exhibited green fluorescence. Interestingly, confocal images of the larvae indicated fluorescence intensities were located in the yolk sak and the digestive organs of the larvae, such as the liver and the gut. After treatment of the larvae with 10 μM Hg2+, the fluorescence intensity of the larvae was significantly enhanced. The Hg2+ dose-dependent fluorescence intensity increase in zebrafish demonstrates that MPIPBS is capable to detect Hg2+ in living organisms. In summary, we have rationally developed a novel water soluble fluorescent probe MPIPBS for the detection of Hg2+, and the detection mechanism was proved by 1HNMR titration, HPLC and HRMS spectroscopy. It is worth noting that MPIPBS exhibits a high sensitivity and selective response to Hg2+ in the wide pH range of 4-10. In particular, due to the AIE effect the detection limit of MPIPBS is 1.45 nM, which is superior to most of the reported mercury ion probes. MPIPBS was developed into the paper-based devices for point-of-care detection of Hg2+ with naked eyes. Moreover, MPIPBS was successfully applied for the quantitative detection of Hg2+ in real water and urine samples with high sensitivity and the satisfying detection performance. Furthermore, owing to the


Page 16 of 32

Page 17 of 32


high water solubility, cell membrane permeability and low cytotoxicity, MPIPBS was used to visualize Hg2+ both in living cells and zebrafish. Hence, MPIPBS can be employed to detect Hg2+ in environmental and biosamples. Conflict of interest There are no financial conflicts Acknowledgements We are grateful for the financial supports from National Natural Science Foundation of China (81671756), Key Research Project of Science and Technology Foundation of Hunan Province (2017SK2093), the State Key Laboratory of Drug Research (SIMM1803KF-14) and the Fundamental Research Funds for the Central Universities of Central South University (2018zzts041). The authors also acknowledge the NMR measurements by the Modern Analysis and Testing Center of CSU. Supporting Information Synthetic scheme of probe MPIPBS; Fluorescence emission spectrum of MPIB and MPIPBS; DLS of MPIPBS; Fluorescence detection kinetics of MPIPBS against Hg2+; HPLC chromatograms of MPIPBS in the absence and presence of Hg2+; HRMS of reaction product of probe MPIPBS with Hg2+; Selectivity test of MPIPBS. Photographs of the fluorescence change of the paper test strips; Fluorescence spectra of MPIPBS in the presence of Hg2+; NMR spectrums of new compounds. This material is available free of charge via the Internet at http://pubs.acs.org. References



(1) Zhang, T.; Kim, B.; Levard, C. m.; Reinsch, B. C.; Lowry, G. V.; Deshusses, M. A.; Hsu-Kim, H. Methylation of mercury by bacteria exposed to dissolved, nanoparticulate, and microparticulate mercuric sulfides. Environ. Sci. Technol. 2012, 46, 6950-6958. (2) Grandjean, P.; Weihe, P.; White, R. F.; Debes, F. Cognitive performance of children prenatally exposed to “safe” levels of methylmercury. Environ. Res. 1998, 77, 165-172. (3) Takeuchi, T.; Morikawa, N.; Matsumoto, H.; Shiraishi, Y. A pathological study of Minamata disease in Japan. Acta Neuropathol.1962, 2, 40-57. (4) Mercury Update: Impact of Fish Advisories. EPA Fact Sheet EPA-823-F-01-011; EPA, Office of Water: Washington, DC, 2001. (5) Filippelli, M. Determination of trace amounts of organic and mercury in biological materials by graphite furnace atomic absorption spectrometry and organic mercury speciation by gas chromatography. Anal. Chem. 1987, 59, 116-118. (6) Labatzke, T.; Schlemmer, G. Ultratrace determination of mercury in water following EN and EPA standards using atomic fluorescence spectrometry. Anal. Bioanal. Chem. 2004, 378, 1075-1082. (7) Wang, M.; Feng, W.; Shi, J.; Zhang, F.; Wang, B.; Zhu, M.; Li, B.; Zhao, Y.; Chai, Z. Development of a mild mercaptoethanol extraction method for determination of mercury species in biological samples by HPLC–ICP-MS. Talanta 2007, 71, 2034-2039.


Page 18 of 32

Page 19 of 32


(8) Chen, H.; Chen, J.; Jin, X.; Wei, D. Determination of trace mercury species by high performance liquid chromatography–inductively coupled plasma mass spectrometry after cloud point extraction. J. Hazard Mater. 2009, 172, 1282-1287. (9) Anderson, K. A. Mercury analysis in environmental samples by cold vapor techniques. In Encyclopedia of Analytical Chemistry; Meyes, R. A., Ed.; Wiley: New York, 2006. (10) Tian, X.; Tong, X.; Li, Z.; Li, D.; Kong, Q.; Yang, X., In vivo fluoride ion detection and imaging in mice using a designed near-infrared ratiometric fluorescent probe based on IR-780. J. Agric. Food Chem. 2018, 66, 11486-11491. (11) Quang, D. T.; Kim, J. S. Fluoro-and chromogenic chemodosimeters for heavy metal ion detection in solution and biospecimens. Chem. Rev. 2010, 110, 6280-6301. (12) Hu, Q.; Li, W.; Qin, C.; Zeng, L.; Hou, J., Rapid and visual detection of benzoyl peroxide in food by a colorimetric and ratiometric fluorescent probe. J. Agric. Food Chem. 2018, 66, 10913-10920. (13) Zhang, P.; Tian, Y.; Liu, H.; Ren, J.; Wang, H.; Zeng, R.; Long, Y.; Chen, J., In vivo imaging of hepatocellular nitric oxide using a hepatocyte-targeting fluorescent sensor. Chem. Commun. 2018, 54, 7231-7234. (14) Feng, Y.; Kuai, Z.; Song, Y.; Guo, J.; Yang, Q.; Shan, Y.; Li, Y. A novel “turn-on” thiooxofluorescein-based colorimetric and fluorescent sensor for Hg2+ and its application in living cells. Talanta 2017, 170, 103-110.



(15)

Bhatta,

S.

R.;

Mondal,

B.;

Vijaykumar,

G.;

Page 20 of 32

Thakur,

A.

ICT–

isomerization-induced turn-on fluorescence probe with a large emission shift for mercury ion: application in combinational molecular logic. Inorg. Chem. 2017, 56, 11577-11590. (16) Chen, L.; Yang, L.; Li, H.; Gao, Y.; Deng, D.; Wu, Y.; Ma, L. Tridentate lysine-based fluorescent sensor for Hg (II) in aqueous solution. Inorg. Chem. 2011, 50, 10028-10032. (17) Song, C.; Yang, W.; Zhou, N.; Qian, R.; Zhang, Y.; Lou, K.; Wang, R.; Wang, W. Fluorescent theranostic agents for Hg2+ detection and detoxification treatment. Chem. Commun. 2015, 51, 4443-4446. (18) Yu, X.; Pan, S.; Li, K.; Li, L.; Li, M.; Shi, L.; Liu, Y. A reaction-based ratiometric fluorescent sensor for detection of Hg (II) ions both in cells and bacteria. Chem. Commun. 2018, 54, 4955-4958. (19) Chen, G.; Guo, Z.; Zeng, G.; Tang, L. Fluorescent and colorimetric sensors for environmental mercury detection. Analyst 2015, 140, 5400-5443. (20) Aliberti, A.; Vaiano, P.; Caporale, A.; Consales, M.; Ruvo, M.; Cusano, A. Fluorescent chemosensors for Hg2+ detection in aqueous environment. Sens. Actuators B Chem. 2017, 247, 727-735. (21) Zong, L.; Xie, Y.; Li, Q.; Li, Z. A new red fluorescent probe for Hg2+ based on naphthalene diimide and its application in living cells, reversibility on strip papers. Sens. Actuators B Chem. 2017, 238, 735-743.


Page 21 of 32


(22) Kim, I.; Lee, N.; Jeong, Y.; Chung, Y.; Cho, B.; Lee, E. Micellar and vesicular nanoassemblies of triazole-based amphiphilic probes triggered by mercury (II) ions in a 100% aqueous medium. Chem. Commun. 2014, 50, 14006-14009. (23) Fang, W.; Zhang, G.; Chen, J.; Kong, L.; Yang, L.; Bi, H.; Yang, J. An AIE active probe for specific sensing of Hg2+ based on linear conjugated bis-Schiff base. Sens. Actuators B Chem. 2016, 229, 338-346. (24) Zong, L.; Wang, C.; Song, Y.; Xie, Y.; Zhang, P.; Peng, Q.; Li, Q.; Li, Z. A dual-function probe based on naphthalene diimide for fluorescent recognition of Hg2+ and colorimetric detection of Cu2+. Sens. Actuators B Chem. 2017, 252, 1105-1111. (25) Zhang, S.; Niu, Q.; Lan, L.; Li, T. Novel oligothiophene-phenylamine based Schiff base as a fluorescent chemosensor for the dual-channel detection of Hg2+ and Cu2+ with high sensitivity and selectivity. Sens. Actuators B Chem. 2017, 240, 793-800. (26) Srivastava, P.; Razi, S. S.; Ali, R.; Gupta, R. C.; Yadav, S. S.; Narayan, G.; Misra, A. Selective naked-eye detection of Hg2+ through an efficient turn-on photoinduced electron transfer fluorescent probe and its real applications. Anal. Chem. 2014, 86, 8693-8699. (27) Zhang, X.; Xiao, Y.; Qian, X. A ratiometric fluorescent probe based on FRET for imaging Hg2+ ions in living cells. Angew. Chem. Int. Ed. 2008, 47, 8025-8029.



(28) Nolan, E. M.; Lippard, S. J. A “turn-on” fluorescent sensor for the selective detection of mercuric ion in aqueous media. J. Am. Chem. Soc. 2003, 125, 14270-14271. (29) Lee, M. H.; Lee, S. W.; Kim, S. H.; Kang, C.; Kim, J. S. Nanomolar Hg(II) detection using Nile blue chemodosimeter in biological media. Org. Lett. 2009, 11, 2101-2104. (30) Chen, Y.; Zhang, W.; Cai, Y.; Kwok, R. T.; Hu, Y.; Lam, J. W.; Gu, X.; He, Z.; Zhao, Z.; Zheng, X. AIEgens for dark through-bond energy transfer: design, synthesis, theoretical study and application in ratiometric Hg2+ sensing. Chem. Sci. 2017, 8, 2047-2055. (31) Zhou, Y.; He, X.; Chen, H.; Wang, Y.; Xiao, S.; Zhang, N.; Li, D.; Zheng, K. An ESIPT/ICT modulation based ratiometric fluorescent probe for sensitive and selective sensing Hg2+. Sens. Actuators B Chem. 2017, 247, 626-631. (32) Jiao, Y.; Liu, X.; Zhou, L.; He, H.; Zhou, P.; Duan, C. A schiff-base dual emission ratiometric fluorescent chemosensor for Hg2+ ions and its application in cellular imaging. Sens. Actuators B Chem. 2017, 247, 950-956. (33) Hu, J.; Hu, Z.; Liu, S.; Zhang, Q.; Gao, H.; Uvdal, K. A new ratiometric fluorescent chemodosimeter based on an ICT modulation for the detection of Hg2+. Sens. Actuators B Chem. 2016, 230, 639-644. (34) Chan, J.; Dodani, S. C.; Chang, C. J. Reaction-based small-molecule fluorescent probes for chemoselective bioimaging. Nat. Chem. 2012, 4, 973-984.


Page 22 of 32

Page 23 of 32


(35) Yang, Y.; Shen, R.; Wang, Y-Z.; Qiu, F.; Feng, Y.; Tang, X.; Bai, D.; Zhang, G.; Liu, W. A selective turn-on fluorescent sensor for Hg(II) in living cells and tissues. Sens. Actuators B Chem. 2018, 255, 3479-3487. (36) Gao, Y.; Ma, T.; Ou, Z.; Cai, W.; Yang, G.; Li, Y.; Xu, M.; Li, Q. Highly sensitive and selective turn-on fluorescent chemosensors for Hg2+ based on thioacetal modified pyrene. Talanta 2018, 178, 663-669. (37) Luo, J.; Xie, Z.; Lam, J. W.; Cheng, L.; Chen, H.; Qiu, C.; Kwok, H. S.; Zhan, X.; Liu, Y.; Zhu, D. Aggregation-induced emission of 1-methyl-1, 2, 3, 4, 5-pentaphenylsilole. Chem. Commun. 2001, 1740-1741. (38) Deng, M.; Wang, Y.; Men, G.; Shang, H.; Jiang, S. Reusable highly sensitive and selective fluorescent sensor for Hg2+ detection in water based on a thermoresponsive copolymer. Sens. Actuators B Chem. 2016, 234, 609-615. (39) Cheng, X.; Li, Q.; Qin, J.; Li, Z. A new approach to design ratiometric fluorescent probe for mercury (II) based on the Hg2+-promoted deprotection of thioacetals. ACS Appl. Mater. Interfaces 2010, 2, 1066-1072. (40) García-Calvo, J.; Vallejos, S.; García, F. C.; Rojo, J.; García, J. M.; Torroba, T. A smart material for the in situ detection of mercury in fish. Chem. Commun. 2016, 52, 11915-11918. (41) Johnson, I.; Spence, M. 2010. The Molecular Probes Handbook 11th edn (Carlsbad, CA: Life Technologies Corporation).



Page 24 of 32

(42) Gao, M.; Tang, B. Z. Fluorescent sensors based on aggregation-induced emission: recent advances and perspectives. ACS Sens. 2017, 2, 1382-1399. (43)

La,

D.

D.;

Bhosale,

S.

V.;

Jones,

L.

A.;

Bhosale,

S.

V.

Tetraphenylethylene-based AIE-active probes for sensing applications. ACS Appl. Mater. Interfaces 2018, 10, 12189-12216. (44) Mei, J.; Leung, N. L.; Kwok, R. T.; Lam, J. W.; Tang, B. Z. Aggregation-induced emission: together we shine, united we soar! Chem. Rev. 2015, 115, 11718-11940. (45) Huang, Y.; Zhang, P.; Gao, M.; Zeng, F.; Qin, A.; Wu, S.; Tang, B. Z., Ratiometric detection and imaging of endogenous hypochlorite in live cells and in vivo achieved by using an aggregation induced emission (AIE)-based nanoprobe. Chem. Commun. 2016, 52, 7288-7291. (46) Qian, J.; Tang, B. Z. AIE luminogens for bioimaging and theranostics: from organelles to animals. Chem. 2017, 3, 56-91. (47) Neupane, L. N.; Oh, E-T.; Park, H. J.; Lee, K-H. Selective and sensitive detection of heavy metal ions in 100% aqueous solution and cells with a fluorescence chemosensor based on peptide using aggregation-induced emission. Anal. Chem. 2016, 88, 3333-3340. (48) Neupane, L. N.; Hwang, G. W.; Lee, K-H. Tuning of the selectivity of fluorescent peptidyl bioprobe using aggregation induced emission for heavy metal


Page 25 of 32


ions by buffering agents in 100% aqueous solutions. Biosens. Bioelectron. 2017, 92, 179-185. (49) Gao, T.; Cao, X.; Ge, P.; Dong, J.; Yang, S.; Xu, H.; Wu, Y.; Gao, F.; Zeng, W. A self-assembled fluorescent organic nanoprobe and its application for sulfite detection in food samples and living systems. Org. Biomol. Chem. 2017, 15, 4375-4382. (50) Xiao, H.; Wu, C.; Li, P.; Tang, B. Simultaneous fluorescence visualization of endoplasmic reticulum superoxide anion and polarity in myocardial cells and tissue. Anal. Chem. 2018, 90, 6081-6088. (51) Zhou, Y.; Zhang, X.; Yang, S.; Li, Y.; Qing, Z.; Zheng, J.; Li, J.; Yang, R. Ratiometric visualization of NO/H2S cross-talk in living cells and tissues using a nitroxyl-responsive two-photon fluorescence probe. Anal. Chem. 2017, 89, 4587-4594. (52) Drago, R. S.; Paulik, F. The reaction of nitrogen (II) oxide with diethylamine. J. Am. Chem. Soc. 1960, 82, 96-98. (53) Zhao, J.; Huang, M.; Zhang, L.; Zou, M.; Chen, D.; Huang, Y.; Zhao, S. Unique approach to develop carbon dot-based nanohybrid near-infrared ratiometric fluorescent sensor for the detection of mercury ions. Anal. Chem. 2017, 89, 8044-8049.



(54) Gao, T.; Yang, S.; Cao, X.; Dong, J.; Zhao, N.; Ge, P.; Zeng, W.; Cheng, Z. Smart self-assembled organic nanoprobe for protein-specific detection: design, synthesis, application, and mechanism studies. Anal. Chem. 2017, 89, 10085-10093. (55) Li, K.; Liu, Y.; Li, Y.; Feng, Q.; Hou, H.; Tang, B. Z. 2, 5-bis (4-alkoxycarbonylphenyl)-1, 4-diaryl-1, 4-dihydropyrrolo [3, 2-b] pyrrole (AAPP) AIEgens: tunable RIR and TICT characteristics and their multifunctional applications. Chem. Sci. 2017, 8, 7258-7267. (56) Naito, H.; Nishino, K.; Morisaki, Y.; Tanaka, K.; Chujo, Y. Solid ‐ state emission of the anthracene-o-carborane dyad from the twisted-intramolecular charge transfer in the crystalline state. Angew. Chem. Int. Ed. 2017, 56, 254-259. (57) Yang, J.; Liau, K.; Li, C.; Chen, M. Meta conjugation effect on the torsional motion of aminostilbenes in the photoinduced intramolecular charge-transfer state. J. Am. Chem. Soc. 2007, 129, 13183-13192. (58) Agarwalla, H.; Mahajan, P. S.; Sahu, D.; Taye, N.; Ganguly, B.; Mhaske, S. B.; Chattopadhyay, S.; Das, A. A switch-on NIR probe for specific detection of Hg2+ ion in aqueous medium and in mitochondria. Inorg. Chem. 2016, 55, 12052-12060. (59) Wang, X.; Wu, F.; Wang, W. In vivo mercury demethylation in a marine fish (Acanthopagrus schlegeli). Environ. Sci. Technol. 2017, 51, 6441-6451. (60) Björnberg, K. A.; Vahter, M.; Englund, G. S. Methylmercury, amalgams, and children’s health. Environ. Health Persp. 2006, 114, A149-A150.


Page 26 of 32

Page 27 of 32


Figures and captions

Scheme 1. Chemical structures of probe MPIPBS and design strategy via the Hg2+-induced umpolung reaction and the AIE mechanism.

Figure 1. Molecular orbital plots of MPIPBS (top) and MPIB (down) in the ground states and excited states.



Figure 2. (a) Fluorescence emission spectra of MPIB in DMSO/water mixtures with different water fractions (fw). (b) Fluorescence intensity of MPIB (10 μM) at 495 nm versus the different water fractions. The excitation wavelength was 380 nm. Inset: photographs of MPIB in DMSO/water mixtures with 0 and 99% water content taken under a 365 nm hand-held UV lamp.

Figure 3. (a) UV-vis absorption spectra of MPIPBS (10 μM) in the absence and presence of Hg2+ (302.4 nM) in pH 7.4 PBS buffer (10 mM containing1% DMSO). (b) Fluorescence spectra of MPIPBS (10 μM) in the absence and presence of Hg2+ (302.4 nM) in PBS buffer (10 mM; pH 7.4; containing 1% DMSO). The excitation


Page 28 of 32

Page 29 of 32


wavelength was 380 nm. Inset picture: photograph of solution MPIPBS before and after addition of Hg2+. (c) Fluorescence titration of MPIPBS (10 μM) by Hg2+ in PBS buffer (10 mM; pH 7.4; containing1% DMSO). (d) Plot of fluorescence intensity of MPIPBS solution as a function of the Hg2+ concentration in the ranges of 0–302.4 nM. Error bars are ±SD, n=3.

Figure 4. (a) Proposed mechanism of dethiolation of Hg2+ for MPIPBS. (b) 1H NMR (500 MHz) spectra of MPIPBS (26.7 mM). (c) 1H NMR (500 MHz) spectra of MPIPBS (26.7 mM) upon addition of 0.5 equiv. of Hg2+ in DMSO-d6-D2O (9/1, v/v) (d) 1H NMR (500 MHz) spectra of MPIPBS (26.7 mM) upon addition of 1.5 equiv. of Hg2+ in DMSO-d6-D2O (9/1, v/v). (e) 1HNMR (500 MHz) spectra of MPIB.



Page 30 of 32

Table 1. Results for detection of Hg2+ in Xiangjiang River water and urine samples Samples

Added

Found

Recovery

RSD

(nmolL-1)

(nmolL-1)

(%)

(%, n=3)

Xiangjiang River

53.6

51.2

95.5

3.81

water

107.2

110.6

103.2

2.92

160.8

158.7

98.7

4.71

214.4

210.8

98.3

2.47

53.6

55.3

103.2

4.16

107.2

101.6

94.8

1.78

160.8

150.8

93.8

2.34

214.4

215.7

100.6

5.26

Urine samples

Figure 5. Fluorescence confocal microscopy images of MDA-MB-231cells stained with 10 μM MPIPBS for 40 min and DAPI (1.0 μM) in the absence and presence of various concentrations of Hg2+ (5 μM and 10 μM) at 37 oC. Blue channel 450-470 nm; Ex =358 nm, Green channel: 480–500 nm; Ex: 400nm. Scale bar: 7.5 μm.


Page 31 of 32


Figure 6. Confocal microscopy images of 3-day-old zebrafish; (a, d and g) Bright field of zebrafish larvae exposure to 0 μM, 5 μM and 10 μM Hg2+, respectively; (b, e and h) fluorescent images of zebrafish larvae exposure to 0 μM, 5 μM and 10 μM Hg2+ respectively; (c, f and i) Overlay image of (a and b), (d and e) and (g and h) respectively; Ex: 405 nm. Em: 450–520 nm.



Graphical Abstract


Page 32 of 32

A sensitive water-soluble fluorescent probe based on umpolung and

Recommend Documents