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DOI: 10.1039/C5AN00273G
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An ESIPT-based fluorescent probe for highly selective and ratiometric detection of mercury (II) in solution and in cells Biao Gua, Liyan Huanga, Naxiu Mia, Peng Yina, Youyu Zhanga*, Xinman Tub, Xubiao Luob, Shenlian Luob, Shouzhuo Yaoa 5
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Key Laboratory of Chemical Biology and Traditional Chinese Medicine Research (Ministry of Education), College of Chemistry and Chemical Engineering, Hunan Normal University, Changsha 410081, PR China b Key Laboratory of Jiangxi Province for Ecological Diagnosis-Remediation and Pollution Control, Nanchang, 330063, PR China 10
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A novel ratiometric fluorescent Hg2+ detecting system was rationally developed based on the typical excited state intramolecular proton transfer (ESIPT) characteristic of the latent fluorophore, 2-(1-(ptolyl)-1H-phenanthro[9,10-d]imidazol-2-yl)phenol (Pol) and the Hg2+-mediated the cleavage of vinyl group. The probe responds selectively to Hg2+ over various other metal ions with a larger bathochromic shift (~100 nm). The sensing mechanism was investigated in detail by fluorescence spectroscopy, NMR spectra and mass spectrometry. Taking the advantage of the enhancement effect of dichloromethane to the ESIPT efficiency, a facile dichloromethane extraction was introduced in process of the detection of Hg2+, which affords a high sensitivity for the probe with a detection limit of 7.8 × 10-9 M for Hg2+. By using the new strategy, the novel probe can be used for the detection of Hg2+ in practical water samples with good recovery. Moreover, the probe was successfully applied to the fluorescence image of Hg2+ in living cells. These results indicated that the probe and the proposed method have useful applications for Hg2+ sensing in biological and environmental sciences.
1. Introduction
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Mercury ion (Hg2+) is considered as one of the most toxic cation which puts human health and the environment at risks. Even at low concentrations, mercury and its compounds are known to be a very dangerous neurotoxin for living organisms. Due to their durability, easy transference and high biological accumulation,1 exposure to mercury and its compounds can cause a series of serious health problems, including DNA damage, mitosis impairment, and permanent damage to the central nervous system even in low concentration.2, 3 The maximum limit of Hg2+ in drinking water is 2 ppb in the standard of United States Environmental Protection Agency (1 × 10-8 M).4 Therefore, the development of reliable and convenient analytical methods for the determination of Hg2+ is of great importance and highly desirable.5, 6 Many analytical techniques such as neutron activation analysis,7 anodic stripping voltammeter,8 inductively coupled plasma mass spectrometer,9 optical probes,10 and so on, have been developed for Hg2+ detection. The methods based on fluorescent probes are more desirable due to its high sensitivity, low detection limit, operational simplicity11, 12 Although a number of fluorescent probes have been reported for detecting Hg2+ in the past few decades,13-19 the detection of Hg2+ were mainly based only on the fluorescent emission intensity changes of the probes, which might be interfered by variables such as probe concentrations, environmental factors, instrumental efficiency, etc. By contrast, the ratiometric fluorescent probes can This journal is © The Royal Society of Chemistry [year]
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provide a built-in correction that eliminates most of the aforementioned interferences by employing the ratio of two emissions at different wavelengths.20-22 However, so far, the ratiometric fluorescent probes for Hg2+ are still very scarce, and the reported ratiometric probes for Hg2+ showed limitations of low selectivity, relatively low sensitivity and/or not easy preparation.23-26 Therefore, it is eagerly demanded to develop more sensitive, more selective and conveniently prepared ratiometric fluorescent probes for Hg2+ detection. The excited state intramolecular proton transfer (ESIPT) strategy has drawn much attention in the design of ratiometric fluorescent sensors, because of predictable efficiency of the ESIPT process.27, 28 Moreover, ESIPT dyes generally possess a large Stokes shift, which offers an advantage that minimizes the self-absorption and eliminates the interference from auto-fluorescence for bioimaging applications.29-32 For some of ESIPT based fluorescent probes, the desired ratiometric fluorescence response was obtained.12, 33-36 Nevertheless, the fluorescence signal of the latent fluorophores possibly becomes weakened in aqueous or protonic solution due to the intermolecular H-bonding,37 which results in the reduction of sensing performance. Therefore, the development of ratiometric fluorescence probes with a facile and efficient methodology to detect Hg2+ are desired, which requires the integration of the unique reactivity of Hg2+ and feature of fluorophores. Recently, Skonieczny et al. reported the synthesis and optical properties of 2-(2′-hydroxyphenyl)-9,10-phenanthroimidazole [journal], [year], [vol], 00–00 | 1
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(HPI) derivatives and their analogues.38 These new interesting fluorescent dyes possess excellent photophysical and photochemical properties, such as typical ESIPT characteristic, high absorption coefficient and quantum yields, as well as a tunability of both absorption and emission bands, which allow them to be applied to the design of fluorescent sensors. However, few HPI-based probes have been studied for detection of analytes. It is well known that the derivatives of phenol are frequently protected as the corresponding vinyl ether, which can be rapidly deprotected by Hg2+ at room temperature.39-41 Thus, this reaction could be considered as the selective reaction toward Hg2+. Based on above consideration, it’s expected that a novel vinyl-ester of HPI can be developed as an Hg2+ probe through the selective deprotection process. The vinyl-ester of HPI can react with Hg2+ to release the ESIPT dye to generate dual emission signals, thereby making the ratiometric fluorescence detection possible. Motivated by this concept, a new ratiometric fluorescence probe ， 1-(p-tolyl)-2-(2-(vinyloxy)phenyl)-1H-phenanthro[9,10d]imidazole (Pvi) (Scheme 1), was rationally designed and synthesized. The probe is composed of a 2-(1-(p-tolyl)-1Hphenanthro[9,10-d]imidazol-2-yl)phenol (Pol) dye as the fluorophore and a vinyl group as the recognition unit. The choice of the fluorophore is based on the consideration that the extended delocalization and the steric restriction of rotational deactivation reduce the nonradiative deactivation.42 In addition, the emission of this dye exhibits pronounced red shift due to the effective ESIPT process. In probe Pvi, the protection of the OH group with a vinyl group will turn off ESIPT, thus the probe mainly displays the normal emission. However, upon removal of the vinyl group, effective ESIPT should follow, which leads to a red shift tautomer emission. When Hg2+ coexists with probe Pvi under appropriate conditions, the removal of the vinyl group from Pvi will occur, resulting in a substantial ratiometric response. Indeed, probe Pvi as a new probe for Hg2+ has many advantages including easy preparation, ratiometric detection of Hg2+ with high sensitivity and selectivity, and large emission spectral shift. In addition, the detection limit of probe Pvi in buffer solutions is significantly decreased by equivoluminal dichloromethane extraction. Moreover, practical application and cell imaging of the probe suggested that it can be potentially employed to sense Hg2+ in the environment and living systems.
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Scheme 1. The detection mechanism of probe Pvi. 85
2. Experimental 2.1. Materials and Instruments Salicylaldehyde, p-toluidine, potassium rert-butoxide, phenanthrenequinone, 3-mercaptopropyltrimethoxysilane, 1,2-
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The synthetic strategy for probe Pvi is shown in Scheme 2. Pvi was characterized by the standard NMR spectroscopy and mass spectrometry (see Figs. S6-S8).
Scheme 2. Synthetic route to Pvi.
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dibromoethane were purchased from Sigma-Aldrich company. Dimethyl sulfoxide (DMSO) and ethyl acetate were obtained from Sinopharm chemical reagent company. All other chemicals used in this work were of analytical grade and used without further purification. Milli-Q ultrapure water (Millipore, ≥ 18 Mῼ cm-1) was used throughout all experiments. Silica gel 300-400 mesh (37-54 µm) was used for column chromatography. The thinlayer chromatography (TLC) was carried out on silica gel plates (60F-254) using UV-light to monitor the reaction. 1H and 13C nuclear magnetic resonance (NMR) spectra were measured on a Bruker AVB-500 spectrometer using TMS as an internal standard. Electrospray mass spectrometry (ESI-MS) spectra were acquired on ZQ2000 mass spectrometer (Manchester, UK) and 6530 Accurate-Mass QTOF spectrometer coupled to an Agilent HPLC 1200 series (Agilent Technologies). UV-vis spectra were measured on a UV2450 spectrophotometer (Shimadzu). Fluorescence spectra were recorded at room temperature by the F-4500 fluorescence spectrophoto-meter (Hitachi Co., Japan) with the excitation and emission slit widths at 5 nm and 10 nm, respectively.
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Synthesis of 2-(2-bromoethoxy)benzaldehyde (M1) The Intermediate M1 was synthesized by following similar procedures.43 Freshly distilled salicylaldehyde (18.3 g, 150 mmol) and 1,2-dibromoethane (56.4 g, 300 mmol) were added into a 250 mL round-bottomed flask under a nitrogen atmosphere. Next, a solution of sodium hydroxide (7.0 g, 175 mmol) in water (53 ml) was added, and the resultant mixture was heated to 120 °C for 24 h. The reaction mixture was cooled to room temperature, and extracted by ethyl acetate. The organic layer was washed twice with brine, dried with anhydrous Na2SO4, filtered and concentrated. Distillation at 110 °C under reduced pressure afforded the M1 (17.2 g). Yield: 50%. 1H NMR (500 MHz, CDCl3): δ 10.54 (d, J = 0.6 Hz, 1H), 7.85 (dd, J = 7.7, 1.8 Hz, 1H), 7.55 (ddd, J = 8.5, 7.4, 1.8 Hz, 1H), 7.07 (t, J = 7.5 Hz, 1H), 6.96 (d, J = 8.4 Hz, 1H), 4.41 (d, J = 6.0 Hz, 2H), 3.71 (t, J = 6.0 Hz, 2H).13C NMR (126 MHz, CDCl3): δ 189.53, 160.37, 135.84, 128.50, 125.26, 121.51, 112.67, 68.24, 28.67.
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Analyst Accepted Manuscript
DOI: 10.1039/C5AN00273G
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DOI: 10.1039/C5AN00273G
Synthesis of 2-(vinyloxy)benzaldehyde (M2)
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2.4. Cell cultures and fluorescence imaging
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Synthesis of probe Pvi
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The synthesis of 3-mercaptopropylsilica (MPS) was performed according to the literature.44 2-(ethenyloxy)benzaldehyde (148.2 mg, 1.0 mmol), phenanthrenequinone (208.2 mg ,1.0 mmol), ptoluidine (107.2 mg, 1.0 mmol) and ammonium acetate (154.2 mg, 2.0 mmol) were dissolved in 10 mL 60% methanol, and 8.0 mg catalyst MPS was then added into the solution. After stirring at 50 °C for 12 h, the reaction mixture was poured into water (40 mL), and then extracted with ethyl acetate. The combined organic layers were washed with brine, dried with anhydrous Na2SO4, filtered, and concentrated. Purification by silica gel column chromatography (petroleum ether/ethyl acetate, 50:1 to 10:1, v/v) afforded probe Pvi as a yellow solid (332.7 mg). Yield: 78%. 1H NMR (500 MHz, CDCl3): δ 8.82 (dd, 7.9 Hz, 1H), 8.71 (d, J = 8.3 Hz, 2H), 7.72 (t, J = 7.4 Hz, 1H), 7.65 (dd, J = 11.2, 4.2 Hz, 1H), 7.61–7.48 (m, 2H), 7.42–7.19 (m,7H), 7.11 (td, J = 7.5, 0.8 Hz, 1H), 6.87 (d, J = 8.3 Hz, 1H), 6.27 (dd, J = 13.7, 6.1 Hz, 1H), 4.64 (dd, J = 13.7, 1.7 Hz, 1H), 4.36 (dd, J = 6.0, 1.6 Hz, 1H), 2.43 (s, 3H). 13C NMR (126 MHz, CDCl3): δ 155.10, 147.31, 132.86, 129.83, 129.63, 128.46, 128.19, 127.28, 126.20, 124.02, 123.07, 122.77, 121.02, 115.61, 96.63, 29.69. MS (EI) m/z: 426.57 (M+). HR-MS (ESI) calculated for C30H23N2O+ (M + H+): 427.1805, found 427.1802.
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3. Results and discussion 3.1. Spectral characteristics of probe Pvi
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The optical properties of Pvi were characterized using UV-Vis absorbance and fluorescence spectroscopy. As shown in Fig. 1, the probe exhibited a maximum absorption peak at 260 nm, corresponding to the π-π* transition of the π-conjugated core. When excited at 270 nm, probe Pvi displayed a maximum emission peak at 380 nm, which can be attributed to the emission from normal-type Pvi.45 0.7
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2.3. General Procedure for the Spectra Measurement The stock solutions of probe Pvi (1.0 mM) were prepared in CH3CN. Stock solutions of metal ions (20.0 mM) were prepared from HgCl2, LiCl, MgCl2.6H2O, CaCl2.2H2O, SrCl2.6H2O, BaCl2.2H2O, MnCl2.4H2O, CoCl2.6H2O, NiCl2.6H2O, ZnCl2, CdCl2.2.5H2O, Pb(NO3)2, FeCl2.4H2O, CuCl2.2H2O, AgNO3, FeCl3.6H2O in double-distilled water. Test solutions were prepared by dissolving 20 µL of Pvi stock solutions and the appropriate amount of analyte stock solution into a phosphate buffer (10 mM, pH 7.4). The mixture (the final volume is 2 mL containing 1% v/v CH3CN as a co-solvent) was incubated at room temperature for 80 min, followed by extraction with equivoluminal dichloromethane (DCM). Then, the fluorescence emission spectra of the resultant solutions were recorded at excitation wavelength of 334 nm (unless otherwise noted, all
The HeLa cells were incubated on 96-well plate in the culture medium and allowed to adhere for 24 h at 37 °C. Immediately prior to the imaging experiments, the cells were washed with phosphate buffered saline (PBS), incubated with 10 µM Pvi (in the culture medium containing 1% CH3CN) for 1 h at 37 °C, then washed with PBS for three times, and imaged. After incubating with 5 µM HgCl2 for another 1 h at 37 °C, the HeLa cells were washed with PBS three times and imaged again. Fluorescence imaging of intracellular Hg2+ in HeLa cells was recorded on an inverted fluorescence microscopy (Nikon, Eclipse Ti-S) with a 40x objective lens. Excitation wavelength of the laser was 380 nm.
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Fig. 1. Absorption spectra and fluorescence spectra of Pvi. Spectra were recorded after incubation in 10 mM PBS buffer (1% CH3CN, pH 7.4) and then addition of equivoluminal DCM.
3.2. Mechanism of the detection of Hg2+ 95
The new fluorescent probe Pvi, contains a 2-(1-(p-tolyl)-1Hphenanthro[9,10-d]imidazol-2-yl) phenol moiety as signal unit and a vinyl group as the recognition unit, In Pvi, the protection of the OH group with a vinyl group blocks ESIPT and results in short wavelength. Whereas, after the deprotection reaction
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The Intermediate compound M2 was synthesized according to the literature 43 Potassium rert-butoxide (5.3 g, 46.8 mmol) in 20 mL of DMSO was slowly added to the solution of 2-(2bromoethoxy)benzaldehyde (10.5 g, 45.8 mmol) dissolved in 20 mL of DMSO. After stirring overnight at room temperature, the reaction mixture was poured into ice and water (100 mL), and extracted by ethyl acetate. The extracts were dried with anhydrous Na2SO4, filtered and finally concentrated. The crude product was purified by distillation at 145 °C under reduced pressure to give the M2 (3.5 g). Yield: 52%. 1H NMR (500 MHz, CDCl3): δ 10.42 (d, J = 0.7 Hz, 1H), 7.85 (dd, J = 7.7, 1.8 Hz, 1H), 7.55 (ddd, J = 8.3, 7.4, 1.8 Hz, 1H), 7.15 (t, J = 7.5 Hz, 1H), 7.05 (d, J = 8.3 Hz, 1H), 6.68 (dd, J = 13.7, 6.0 Hz, 1H), 4.84 (dd, J = 13.7, 2.0 Hz, 1H), 4.58 (dd, J = 6.0, 2.0 Hz, 1H).13C NMR (126 MHz, CDCl3): δ 189.00, 158.86, 147.60, 135.76, 128.40, 126.04, 123.40, 116.94, 97.24.
spectral data were measured according to this method). The environmental water samples from the tap water and Xiangjiang River in Changsha city were passed through a microfiltration membrane before use. The pH values of the water samples were adjusted using a sodium phosphate buffer (10 mM, pH 7.4), and the water samples were then spiked with different concentrations of Hg2+ (0, 0.25 µM, 0.5 µM) that had been accurately prepared. The resulting samples were further treated with probe Pvi in 10 mM PBS buffer (1% CH3CN, pH 7.4) to give the final mixtures (2.0 mL) containing Pvi (final concentration 10 µM) and Hg2+ (final concentration 0.25 or 0.5 µM). The results were summarized in Table 1.
Absorbance
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Fig. 2. Fluorescence spectra of Pvi (10 µM) in the absence and presence of Hg2+. Spectra were recorded after incubation with Hg2+ in 10 mM PBS buffer (1% CH3CN) for 80 min and then addition of equivoluminal DCM. λex = 334
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nm. The inset shows the visual color change of Pvi with the addition of 0.4 equivalents of Hg2+ under a UV-lamp (365 nm). 25
To further confirm the above interaction mechanism, the product of probe Pvi + Hg2+ was isolated by a silica gel column and then was subjected to 1H NMR analysis. As displayed in Fig. 3, Pvi showed the characteristic alkenyl proton Ha from 4.3 to 6.5 ppm.
However, the peak of vinyl group in the product disappeared, at the same time, the hydroxyl proton signal Hb appeared at ~14 ppm, which indicated that Pvi undergo the oxymercuration followed by hydrolysis to generate fluorophore Pol. Moreover, the reaction mechanism was also confirmed by mass analysis, and a peak at m/z 400.65 corresponding to Pol was obtained (Fig. S13). Therefore, 1H NMR experiments and mass spectrometry results suggested the validity of the detection mechanism.
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In order to obtain a high-sensitive response for Hg2+, the effects of the experimental parameters, such as solvents, incubation time and pH were investigated. Since solvents play a key role in efficiency of the ESIPT,38 it is expected that an appropriate solvent can maximally recover the reduced ESIPT efficiency of fluorophore and improve the sensitivity of the probe. Thus, the fluorescence spectra of the product of Pvi + Hg2+ in various solvents were measured. The fluorescence product Pol was isolated by column chromatography after chemical preparation and its structure was conformed by 1H NMR, 13C NMR and MS (Figs. S9-S12). As shown in Fig. 4A, Pol exhibited two weak emission peaks at 380 nm and 477 nm in protic or polar solvents, which can be attributed to the enol form (E*) and the keto form (K*) of Pol, respectively. In low polar solvents, only one strong peak emerged at 477 nm that corresponded to the keto form (K*). Moreover, the fluorescence intensity of Pol in DCM was far higher than that in protic or polar solvent under the same condition. This is in agreement with the fact that the intramolecular H-bonding between the phenolic hydroxyl proton and the aromatic nitrogen favors ESIPT in aprotic solvents (DCM). In protic solvents (CH3OH), intermolecular H-bonding between Pol and the solvent competes with the intramolecular Hbonding, which limits the ESIPT process. These results indicated that DCM not only played an important role in recovering the reduced ESIPT efficiency of fluorophore but also in extracting fluorophore from buffer solution. Hence, in the following assay, the spectra of the resultant solutions were recorded when the reaction of Pvi with Hg2+ was completed in 10 mM PBS buffer (1% CH3CN, pH 7.4), and then extracted with equivoluminal DCM. Fig. 4B shows the time dependent fluorescent response in the presence of Hg2+. The fluorescent intensity at 477 nm reached a constant value when the incubation time reached to 80 min, suggesting that the reaction between Pvi and Hg2+ was completed within 80 min. Therefore, the incubation time was controlled at 80 min. Fig. 4C illustrates the effect of pH on the fluorescence response of Pvi to Hg2+. It was obvious that the fluorescent intensity at 477 nm increased with pH value and then remained unchanged at neutral to pH 10. These results indicated that the alkaline conditions promoted the hydrolysis reaction. Hence, physiological pH (pH 7.4) could be selected as an appropriate working pH in the following experiments.
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Fig. 3. 1 H NMR (500 MHz) spectra of Pvi (A) and the isolated product of Pvi + Hg2+ (B) in CDCl3.
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promoted by Hg2+, the OH group in probe will be released, enabling the ESIPT process to shift the fluorescence signal to a longer wavelength. The fluorescence of Pvi in the absence and presence of Hg2+ was investigated. As shown in Fig. 2, free probe exhibited an original emission peak at 380 nm, and then this peak decreased after Pvi was treated with Hg2+. Meanwhile, a new peak at 477 nm appeared, which can be attributed to the fluorescence emission of the reaction product. Thus, these results indicated that the probe reacted with Hg2+ at room temperature to generate fluorophore Pol. The strong emission of Pol at longer wavelength is resulted from excited state intramolecular proton transfer (ESIPT). The red shift (~100 nm) in maximum emission supports this mechanism, which is the typical characterization of ESIPT (Scheme 1). In addition, the color of the Pvi solution upon interaction with Hg2+ changed from blue to green under UV light at 365 nm (see Fig. 2 inset), which would allow the fluorescence naked eye detection of Hg2+.
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potentially competing species is a necessity for a new fluorescent probe with potential application in complex biological and environmental sample. Therefore, the selectivity experiments of probe Pvi were investigated. As shown in Fig 6, there was no significant fluorescence changes in the presence of the common cations, such as Li+, Mg2+, Ca2, Sr2+, Ba2, Mn2+, Co2+, Ni2+, Zn2+, Cd2+, Pb2+, Cu2+, Ag+, Fe3+ (200 µM for each). However, a much larger fluorescence enhancement and a red shift of about 100 nm were observed for Pvi upon addition of Hg2+ (5 µM). These results clearly demonstrated that Pvi was highly selective fluorescent probe for Hg2+, and also verified that the hydrolysis of aryl vinyl ether was specific toward Hg2+.
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Fig. 4. (A) Fluorescence spectra of Pol (10 µM) in DCM, EA, CH3CN, CH3OH and 10 mM PBS buffer (1% CH3CN, pH 7.4). Effects of incubation time (B) and pH (C) on the fluorescence responses of Pvi for Hg2+ detection. 5
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In order to validate its practicality in environmental science, we employed probe Pvi in a standard addition method to determine Hg2+ concentrations in water samples from tap water and Xiangjiang River in Changsha city. The detection results were summarized in Table 1. Satisfactory values between 96% and 102% were obtained for the recovery experiments, and it can meet the requirement of detection of Hg2+ in water sample. The result indicated that the proposed probe could be an excellent tool to detect Hg2+. Table 1. The measurement results of mercury in water samples recovery (%)
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Fig. 5. (A) Fluorescence spectra of Pvi (10 µM) in the presence of various concentrations of Hg2+ (0, 0.01, 0.03, 0.06, 0.1, 0.2, 0.4, 0.6, 1.0, 2.0, 3.0, 4.0, 5.0 µM). (B) Linear relationship between the ratio of emission intensities (I477nm/I380nm) and the concentration of Hg2+. Spectra were recorded after 80 min of incubation with Hg2+ in 10 mM PBS buffer (1% CH3CN, pH 7.4) and
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Under the optimal conditions, the analytical performance of probe Pvi was studied by fluorescence response towards various concentrations of Hg2+. As shown in Fig. 5A, the fluorescence intensity at 380 nm decreased accompanying the increase in emission wavelength at 477 nm. The emission ratio (I477nm/I380nm) was linearly proportional to the concentration of Hg2+ in the range of 0.01 to 3.0 µM (Fig. 5B). And, a linear regression equation for Hg2+, F = 0.0535 + 0.5713x (R2 = 0.9921) was obtained, where F refers to the ratio of emission intensities and x refers to the concentration of Hg2+. The limit of detection (LOD) (S/N=3, the concentration necessary to yield a net signal equal to three times the standard deviation of the background) was calculated to be 7.8 × 10-9 M, which is lower than the maximum permissible level of Hg2+ in drinking water (1 × 10-8 M). These results revealed that the probe could monitor Hg2+ levels both qualitatively and quantitatively.
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A highly selective response to the target species over other
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3.6. Detection of Hg2+ in living cells To further investigate the membrane permeability of probe Pvi and its ability to sense Hg2+ in living cells, the cell fluorescence imaging experiment was carried out. The fluorescence microscopic images acquired at 380 nm excitation displayed
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enhanced green fluorescence for the incubated HeLa cells with Pvi + Hg2+ in which the relative emission intensity was more intense compared to HeLa cells with probe Pvi (Figs. 7B and D). These results established that the probe was cell-permeable and capable of imaging of Hg2+ in living cells. The bright-field images (Figs. 7A and C) confirmed that the cells were viable throughout the imaging experiments.
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† Electronic Supplementary Information (ESI) available: [details of any supplementary information available should be included here]. See DOI: 10.1039/b000000x/ 45
References
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Fig. 7. (A) Bright-field image of HeLa cells incubated with Pvi (10 µM) for 1 h. (B) fluorescence image of (A). (C) Bright-field image of HeLa cells incubated with Pvi (10 µM) for 1 h and then treated with Hg2+ (5 µM) for
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another 1 h. (D) fluorescence image of (C). 15
4. Conclusion
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In summary, a new ratiometric fluorescence probe Pvi by combining 2-(1-(p-tolyl)-1H-phenanthro[9,10-d]imidazol-2-yl) phenol as the fluorophore and a vinyl group as a high selective recognition unit was successfully designed and synthesized. Dichloromethane can be used to extract the producted fluorophore Pol and recover its ESIPT efficiency for enhancing the sensitivity of Hg2+ detection. Furthermore, probe Pvi can successfully be applied to Hg2+ determinations in water samples and fluorescence imaging of Hg2+ in HeLa cells. This research demonstrated potential of the probe for environmental and biological applications.
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This work was supported by the National Natural Science Foundation of China (21375037, 21275051, 21475043), Scientific Research Fund of Hunan Provincial Science and Technology Department and Education Department (13JJ2020, 12A084), and Doctoral Fund of Ministry of Education of China (NO: 20134306110006).
Notes and references Key Laboratory of Chemical Biology and Traditional Chinese Medicine Research (Ministry of Education),College of Chemistry and Chemical Engineering, Hunan Normal University, Changsha 410081, PR China Fax: +86 73188872531; Tel: +86 73188865515;
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An ESIPT-based fluorescent probe for highly selective and ratiometric detection of mercury (II) in solution and in cells Biao Gua, Liyan Huanga, Naxiu Mia, Peng Yina, Youyu Zhanga*, Xinman Tub, Xubiao Luob, Shenlian Luob, Shouzhuo Yaoa 5
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Key Laboratory of Chemical Biology and Traditional Chinese Medicine Research (Ministry of Education), College of Chemistry and Chemical Engineering, Hunan Normal University, Changsha 410081, PR China b Key Laboratory of Jiangxi Province for Ecological Diagnosis-Remediation and Pollution Control, Nanchang, 330063, PR China 10
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A novel ratiometric fluorescent Hg2+ detecting system was rationally developed based on the typical excited state intramolecular proton transfer (ESIPT) characteristic of the latent fluorophore, 2-(1-(ptolyl)-1H-phenanthro[9,10-d]imidazol-2-yl)phenol (Pol) and the Hg2+-mediated the cleavage of vinyl group. The probe responds selectively to Hg2+ over various other metal ions with a larger bathochromic shift (~100 nm). The sensing mechanism was investigated in detail by fluorescence spectroscopy, NMR spectra and mass spectrometry. Taking the advantage of the enhancement effect of dichloromethane to the ESIPT efficiency, a facile dichloromethane extraction was introduced in process of the detection of Hg2+, which affords a high sensitivity for the probe with a detection limit of 7.8 × 10-9 M for Hg2+. By using the new strategy, the novel probe can be used for the detection of Hg2+ in practical water samples with good recovery. Moreover, the probe was successfully applied to the fluorescence image of Hg2+ in living cells. These results indicated that the probe and the proposed method have useful applications for Hg2+ sensing in biological and environmental sciences.
1. Introduction
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Mercury ion (Hg2+) is considered as one of the most toxic cation which puts human health and the environment at risks. Even at low concentrations, mercury and its compounds are known to be a very dangerous neurotoxin for living organisms. Due to their durability, easy transference and high biological accumulation,1 exposure to mercury and its compounds can cause a series of serious health problems, including DNA damage, mitosis impairment, and permanent damage to the central nervous system even in low concentration.2, 3 The maximum limit of Hg2+ in drinking water is 2 ppb in the standard of United States Environmental Protection Agency (1 × 10-8 M).4 Therefore, the development of reliable and convenient analytical methods for the determination of Hg2+ is of great importance and highly desirable.5, 6 Many analytical techniques such as neutron activation analysis,7 anodic stripping voltammeter,8 inductively coupled plasma mass spectrometer,9 optical probes,10 and so on, have been developed for Hg2+ detection. The methods based on fluorescent probes are more desirable due to its high sensitivity, low detection limit, operational simplicity11, 12 Although a number of fluorescent probes have been reported for detecting Hg2+ in the past few decades,13-19 the detection of Hg2+ were mainly based only on the fluorescent emission intensity changes of the probes, which might be interfered by variables such as probe concentrations, environmental factors, instrumental efficiency, etc. By contrast, the ratiometric fluorescent probes can This journal is © The Royal Society of Chemistry [year]
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provide a built-in correction that eliminates most of the aforementioned interferences by employing the ratio of two emissions at different wavelengths.20-22 However, so far, the ratiometric fluorescent probes for Hg2+ are still very scarce, and the reported ratiometric probes for Hg2+ showed limitations of low selectivity, relatively low sensitivity and/or not easy preparation.23-26 Therefore, it is eagerly demanded to develop more sensitive, more selective and conveniently prepared ratiometric fluorescent probes for Hg2+ detection. The excited state intramolecular proton transfer (ESIPT) strategy has drawn much attention in the design of ratiometric fluorescent sensors, because of predictable efficiency of the ESIPT process.27, 28 Moreover, ESIPT dyes generally possess a large Stokes shift, which offers an advantage that minimizes the self-absorption and eliminates the interference from auto-fluorescence for bioimaging applications.29-32 For some of ESIPT based fluorescent probes, the desired ratiometric fluorescence response was obtained.12, 33-36 Nevertheless, the fluorescence signal of the latent fluorophores possibly becomes weakened in aqueous or protonic solution due to the intermolecular H-bonding,37 which results in the reduction of sensing performance. Therefore, the development of ratiometric fluorescence probes with a facile and efficient methodology to detect Hg2+ are desired, which requires the integration of the unique reactivity of Hg2+ and feature of fluorophores. Recently, Skonieczny et al. reported the synthesis and optical properties of 2-(2′-hydroxyphenyl)-9,10-phenanthroimidazole [journal], [year], [vol], 00–00 | 1
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(HPI) derivatives and their analogues.38 These new interesting fluorescent dyes possess excellent photophysical and photochemical properties, such as typical ESIPT characteristic, high absorption coefficient and quantum yields, as well as a tunability of both absorption and emission bands, which allow them to be applied to the design of fluorescent sensors. However, few HPI-based probes have been studied for detection of analytes. It is well known that the derivatives of phenol are frequently protected as the corresponding vinyl ether, which can be rapidly deprotected by Hg2+ at room temperature.39-41 Thus, this reaction could be considered as the selective reaction toward Hg2+. Based on above consideration, it’s expected that a novel vinyl-ester of HPI can be developed as an Hg2+ probe through the selective deprotection process. The vinyl-ester of HPI can react with Hg2+ to release the ESIPT dye to generate dual emission signals, thereby making the ratiometric fluorescence detection possible. Motivated by this concept, a new ratiometric fluorescence probe ， 1-(p-tolyl)-2-(2-(vinyloxy)phenyl)-1H-phenanthro[9,10d]imidazole (Pvi) (Scheme 1), was rationally designed and synthesized. The probe is composed of a 2-(1-(p-tolyl)-1Hphenanthro[9,10-d]imidazol-2-yl)phenol (Pol) dye as the fluorophore and a vinyl group as the recognition unit. The choice of the fluorophore is based on the consideration that the extended delocalization and the steric restriction of rotational deactivation reduce the nonradiative deactivation.42 In addition, the emission of this dye exhibits pronounced red shift due to the effective ESIPT process. In probe Pvi, the protection of the OH group with a vinyl group will turn off ESIPT, thus the probe mainly displays the normal emission. However, upon removal of the vinyl group, effective ESIPT should follow, which leads to a red shift tautomer emission. When Hg2+ coexists with probe Pvi under appropriate conditions, the removal of the vinyl group from Pvi will occur, resulting in a substantial ratiometric response. Indeed, probe Pvi as a new probe for Hg2+ has many advantages including easy preparation, ratiometric detection of Hg2+ with high sensitivity and selectivity, and large emission spectral shift. In addition, the detection limit of probe Pvi in buffer solutions is significantly decreased by equivoluminal dichloromethane extraction. Moreover, practical application and cell imaging of the probe suggested that it can be potentially employed to sense Hg2+ in the environment and living systems.
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2.2. Synthesis of intermediates and probe
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Scheme 1. The detection mechanism of probe Pvi. 85
2. Experimental 2.1. Materials and Instruments Salicylaldehyde, p-toluidine, potassium rert-butoxide, phenanthrenequinone, 3-mercaptopropyltrimethoxysilane, 1,2-
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The synthetic strategy for probe Pvi is shown in Scheme 2. Pvi was characterized by the standard NMR spectroscopy and mass spectrometry (see Figs. S6-S8).
Scheme 2. Synthetic route to Pvi.
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dibromoethane were purchased from Sigma-Aldrich company. Dimethyl sulfoxide (DMSO) and ethyl acetate were obtained from Sinopharm chemical reagent company. All other chemicals used in this work were of analytical grade and used without further purification. Milli-Q ultrapure water (Millipore, ≥ 18 Mῼ cm-1) was used throughout all experiments. Silica gel 300-400 mesh (37-54 µm) was used for column chromatography. The thinlayer chromatography (TLC) was carried out on silica gel plates (60F-254) using UV-light to monitor the reaction. 1H and 13C nuclear magnetic resonance (NMR) spectra were measured on a Bruker AVB-500 spectrometer using TMS as an internal standard. Electrospray mass spectrometry (ESI-MS) spectra were acquired on ZQ2000 mass spectrometer (Manchester, UK) and 6530 Accurate-Mass QTOF spectrometer coupled to an Agilent HPLC 1200 series (Agilent Technologies). UV-vis spectra were measured on a UV2450 spectrophotometer (Shimadzu). Fluorescence spectra were recorded at room temperature by the F-4500 fluorescence spectrophoto-meter (Hitachi Co., Japan) with the excitation and emission slit widths at 5 nm and 10 nm, respectively.
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Synthesis of 2-(2-bromoethoxy)benzaldehyde (M1) The Intermediate M1 was synthesized by following similar procedures.43 Freshly distilled salicylaldehyde (18.3 g, 150 mmol) and 1,2-dibromoethane (56.4 g, 300 mmol) were added into a 250 mL round-bottomed flask under a nitrogen atmosphere. Next, a solution of sodium hydroxide (7.0 g, 175 mmol) in water (53 ml) was added, and the resultant mixture was heated to 120 °C for 24 h. The reaction mixture was cooled to room temperature, and extracted by ethyl acetate. The organic layer was washed twice with brine, dried with anhydrous Na2SO4, filtered and concentrated. Distillation at 110 °C under reduced pressure afforded the M1 (17.2 g). Yield: 50%. 1H NMR (500 MHz, CDCl3): δ 10.54 (d, J = 0.6 Hz, 1H), 7.85 (dd, J = 7.7, 1.8 Hz, 1H), 7.55 (ddd, J = 8.5, 7.4, 1.8 Hz, 1H), 7.07 (t, J = 7.5 Hz, 1H), 6.96 (d, J = 8.4 Hz, 1H), 4.41 (d, J = 6.0 Hz, 2H), 3.71 (t, J = 6.0 Hz, 2H).13C NMR (126 MHz, CDCl3): δ 189.53, 160.37, 135.84, 128.50, 125.26, 121.51, 112.67, 68.24, 28.67.
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Synthesis of 2-(vinyloxy)benzaldehyde (M2)
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Synthesis of probe Pvi
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The synthesis of 3-mercaptopropylsilica (MPS) was performed according to the literature.44 2-(ethenyloxy)benzaldehyde (148.2 mg, 1.0 mmol), phenanthrenequinone (208.2 mg ,1.0 mmol), ptoluidine (107.2 mg, 1.0 mmol) and ammonium acetate (154.2 mg, 2.0 mmol) were dissolved in 10 mL 60% methanol, and 8.0 mg catalyst MPS was then added into the solution. After stirring at 50 °C for 12 h, the reaction mixture was poured into water (40 mL), and then extracted with ethyl acetate. The combined organic layers were washed with brine, dried with anhydrous Na2SO4, filtered, and concentrated. Purification by silica gel column chromatography (petroleum ether/ethyl acetate, 50:1 to 10:1, v/v) afforded probe Pvi as a yellow solid (332.7 mg). Yield: 78%. 1H NMR (500 MHz, CDCl3): δ 8.82 (dd, 7.9 Hz, 1H), 8.71 (d, J = 8.3 Hz, 2H), 7.72 (t, J = 7.4 Hz, 1H), 7.65 (dd, J = 11.2, 4.2 Hz, 1H), 7.61–7.48 (m, 2H), 7.42–7.19 (m,7H), 7.11 (td, J = 7.5, 0.8 Hz, 1H), 6.87 (d, J = 8.3 Hz, 1H), 6.27 (dd, J = 13.7, 6.1 Hz, 1H), 4.64 (dd, J = 13.7, 1.7 Hz, 1H), 4.36 (dd, J = 6.0, 1.6 Hz, 1H), 2.43 (s, 3H). 13C NMR (126 MHz, CDCl3): δ 155.10, 147.31, 132.86, 129.83, 129.63, 128.46, 128.19, 127.28, 126.20, 124.02, 123.07, 122.77, 121.02, 115.61, 96.63, 29.69. MS (EI) m/z: 426.57 (M+). HR-MS (ESI) calculated for C30H23N2O+ (M + H+): 427.1805, found 427.1802.
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3. Results and discussion 3.1. Spectral characteristics of probe Pvi
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The optical properties of Pvi were characterized using UV-Vis absorbance and fluorescence spectroscopy. As shown in Fig. 1, the probe exhibited a maximum absorption peak at 260 nm, corresponding to the π-π* transition of the π-conjugated core. When excited at 270 nm, probe Pvi displayed a maximum emission peak at 380 nm, which can be attributed to the emission from normal-type Pvi.45 0.7
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2.3. General Procedure for the Spectra Measurement The stock solutions of probe Pvi (1.0 mM) were prepared in CH3CN. Stock solutions of metal ions (20.0 mM) were prepared from HgCl2, LiCl, MgCl2.6H2O, CaCl2.2H2O, SrCl2.6H2O, BaCl2.2H2O, MnCl2.4H2O, CoCl2.6H2O, NiCl2.6H2O, ZnCl2, CdCl2.2.5H2O, Pb(NO3)2, FeCl2.4H2O, CuCl2.2H2O, AgNO3, FeCl3.6H2O in double-distilled water. Test solutions were prepared by dissolving 20 µL of Pvi stock solutions and the appropriate amount of analyte stock solution into a phosphate buffer (10 mM, pH 7.4). The mixture (the final volume is 2 mL containing 1% v/v CH3CN as a co-solvent) was incubated at room temperature for 80 min, followed by extraction with equivoluminal dichloromethane (DCM). Then, the fluorescence emission spectra of the resultant solutions were recorded at excitation wavelength of 334 nm (unless otherwise noted, all
The HeLa cells were incubated on 96-well plate in the culture medium and allowed to adhere for 24 h at 37 °C. Immediately prior to the imaging experiments, the cells were washed with phosphate buffered saline (PBS), incubated with 10 µM Pvi (in the culture medium containing 1% CH3CN) for 1 h at 37 °C, then washed with PBS for three times, and imaged. After incubating with 5 µM HgCl2 for another 1 h at 37 °C, the HeLa cells were washed with PBS three times and imaged again. Fluorescence imaging of intracellular Hg2+ in HeLa cells was recorded on an inverted fluorescence microscopy (Nikon, Eclipse Ti-S) with a 40x objective lens. Excitation wavelength of the laser was 380 nm.
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Fig. 1. Absorption spectra and fluorescence spectra of Pvi. Spectra were recorded after incubation in 10 mM PBS buffer (1% CH3CN, pH 7.4) and then addition of equivoluminal DCM.
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The new fluorescent probe Pvi, contains a 2-(1-(p-tolyl)-1Hphenanthro[9,10-d]imidazol-2-yl) phenol moiety as signal unit and a vinyl group as the recognition unit, In Pvi, the protection of the OH group with a vinyl group blocks ESIPT and results in short wavelength. Whereas, after the deprotection reaction
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The Intermediate compound M2 was synthesized according to the literature 43 Potassium rert-butoxide (5.3 g, 46.8 mmol) in 20 mL of DMSO was slowly added to the solution of 2-(2bromoethoxy)benzaldehyde (10.5 g, 45.8 mmol) dissolved in 20 mL of DMSO. After stirring overnight at room temperature, the reaction mixture was poured into ice and water (100 mL), and extracted by ethyl acetate. The extracts were dried with anhydrous Na2SO4, filtered and finally concentrated. The crude product was purified by distillation at 145 °C under reduced pressure to give the M2 (3.5 g). Yield: 52%. 1H NMR (500 MHz, CDCl3): δ 10.42 (d, J = 0.7 Hz, 1H), 7.85 (dd, J = 7.7, 1.8 Hz, 1H), 7.55 (ddd, J = 8.3, 7.4, 1.8 Hz, 1H), 7.15 (t, J = 7.5 Hz, 1H), 7.05 (d, J = 8.3 Hz, 1H), 6.68 (dd, J = 13.7, 6.0 Hz, 1H), 4.84 (dd, J = 13.7, 2.0 Hz, 1H), 4.58 (dd, J = 6.0, 2.0 Hz, 1H).13C NMR (126 MHz, CDCl3): δ 189.00, 158.86, 147.60, 135.76, 128.40, 126.04, 123.40, 116.94, 97.24.
spectral data were measured according to this method). The environmental water samples from the tap water and Xiangjiang River in Changsha city were passed through a microfiltration membrane before use. The pH values of the water samples were adjusted using a sodium phosphate buffer (10 mM, pH 7.4), and the water samples were then spiked with different concentrations of Hg2+ (0, 0.25 µM, 0.5 µM) that had been accurately prepared. The resulting samples were further treated with probe Pvi in 10 mM PBS buffer (1% CH3CN, pH 7.4) to give the final mixtures (2.0 mL) containing Pvi (final concentration 10 µM) and Hg2+ (final concentration 0.25 or 0.5 µM). The results were summarized in Table 1.
Absorbance
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3.3. Optimization of the detection conditions
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+ 4.0 µM Hg
1.0 0.8 0.6
2+
55
+ 0.8 µM Hg
0.4 0.2
only Pvi
0.0 350
20
400 450 500 550 Wavelength (nm)
600
60
Fig. 2. Fluorescence spectra of Pvi (10 µM) in the absence and presence of Hg2+. Spectra were recorded after incubation with Hg2+ in 10 mM PBS buffer (1% CH3CN) for 80 min and then addition of equivoluminal DCM. λex = 334
65
nm. The inset shows the visual color change of Pvi with the addition of 0.4 equivalents of Hg2+ under a UV-lamp (365 nm). 25
To further confirm the above interaction mechanism, the product of probe Pvi + Hg2+ was isolated by a silica gel column and then was subjected to 1H NMR analysis. As displayed in Fig. 3, Pvi showed the characteristic alkenyl proton Ha from 4.3 to 6.5 ppm.
However, the peak of vinyl group in the product disappeared, at the same time, the hydroxyl proton signal Hb appeared at ~14 ppm, which indicated that Pvi undergo the oxymercuration followed by hydrolysis to generate fluorophore Pol. Moreover, the reaction mechanism was also confirmed by mass analysis, and a peak at m/z 400.65 corresponding to Pol was obtained (Fig. S13). Therefore, 1H NMR experiments and mass spectrometry results suggested the validity of the detection mechanism.
70
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In order to obtain a high-sensitive response for Hg2+, the effects of the experimental parameters, such as solvents, incubation time and pH were investigated. Since solvents play a key role in efficiency of the ESIPT,38 it is expected that an appropriate solvent can maximally recover the reduced ESIPT efficiency of fluorophore and improve the sensitivity of the probe. Thus, the fluorescence spectra of the product of Pvi + Hg2+ in various solvents were measured. The fluorescence product Pol was isolated by column chromatography after chemical preparation and its structure was conformed by 1H NMR, 13C NMR and MS (Figs. S9-S12). As shown in Fig. 4A, Pol exhibited two weak emission peaks at 380 nm and 477 nm in protic or polar solvents, which can be attributed to the enol form (E*) and the keto form (K*) of Pol, respectively. In low polar solvents, only one strong peak emerged at 477 nm that corresponded to the keto form (K*). Moreover, the fluorescence intensity of Pol in DCM was far higher than that in protic or polar solvent under the same condition. This is in agreement with the fact that the intramolecular H-bonding between the phenolic hydroxyl proton and the aromatic nitrogen favors ESIPT in aprotic solvents (DCM). In protic solvents (CH3OH), intermolecular H-bonding between Pol and the solvent competes with the intramolecular Hbonding, which limits the ESIPT process. These results indicated that DCM not only played an important role in recovering the reduced ESIPT efficiency of fluorophore but also in extracting fluorophore from buffer solution. Hence, in the following assay, the spectra of the resultant solutions were recorded when the reaction of Pvi with Hg2+ was completed in 10 mM PBS buffer (1% CH3CN, pH 7.4), and then extracted with equivoluminal DCM. Fig. 4B shows the time dependent fluorescent response in the presence of Hg2+. The fluorescent intensity at 477 nm reached a constant value when the incubation time reached to 80 min, suggesting that the reaction between Pvi and Hg2+ was completed within 80 min. Therefore, the incubation time was controlled at 80 min. Fig. 4C illustrates the effect of pH on the fluorescence response of Pvi to Hg2+. It was obvious that the fluorescent intensity at 477 nm increased with pH value and then remained unchanged at neutral to pH 10. These results indicated that the alkaline conditions promoted the hydrolysis reaction. Hence, physiological pH (pH 7.4) could be selected as an appropriate working pH in the following experiments.
30
Fig. 3. 1 H NMR (500 MHz) spectra of Pvi (A) and the isolated product of Pvi + Hg2+ (B) in CDCl3.
4 | Journal Name, [year], [vol], 00–00
This journal is © The Royal Society of Chemistry [year]
Analyst Accepted Manuscript
5
promoted by Hg2+, the OH group in probe will be released, enabling the ESIPT process to shift the fluorescence signal to a longer wavelength. The fluorescence of Pvi in the absence and presence of Hg2+ was investigated. As shown in Fig. 2, free probe exhibited an original emission peak at 380 nm, and then this peak decreased after Pvi was treated with Hg2+. Meanwhile, a new peak at 477 nm appeared, which can be attributed to the fluorescence emission of the reaction product. Thus, these results indicated that the probe reacted with Hg2+ at room temperature to generate fluorophore Pol. The strong emission of Pol at longer wavelength is resulted from excited state intramolecular proton transfer (ESIPT). The red shift (~100 nm) in maximum emission supports this mechanism, which is the typical characterization of ESIPT (Scheme 1). In addition, the color of the Pvi solution upon interaction with Hg2+ changed from blue to green under UV light at 365 nm (see Fig. 2 inset), which would allow the fluorescence naked eye detection of Hg2+.
Normalized Intensity
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DOI: 10.1039/C5AN00273G
1.2
(A)
MeCN:PBS=1:99 MeOH MeCN EA DCM
1.0 0.8 0.6 0.4 0.2
(B) Normalized Intensity
Normalized Intensity
1.2
350
1.0 0.8
35
0.6 0.4 0.2
400
450 500 550 Wavelength (nm)
0
600
20
40 60 Time (min)
80
100 40
I477
(C)
1.0 0.8
potentially competing species is a necessity for a new fluorescent probe with potential application in complex biological and environmental sample. Therefore, the selectivity experiments of probe Pvi were investigated. As shown in Fig 6, there was no significant fluorescence changes in the presence of the common cations, such as Li+, Mg2+, Ca2, Sr2+, Ba2, Mn2+, Co2+, Ni2+, Zn2+, Cd2+, Pb2+, Cu2+, Ag+, Fe3+ (200 µM for each). However, a much larger fluorescence enhancement and a red shift of about 100 nm were observed for Pvi upon addition of Hg2+ (5 µM). These results clearly demonstrated that Pvi was highly selective fluorescent probe for Hg2+, and also verified that the hydrolysis of aryl vinyl ether was specific toward Hg2+.
0.6 0.4
1.2
0.0 4
6
8
10
pH
Fig. 4. (A) Fluorescence spectra of Pol (10 µM) in DCM, EA, CH3CN, CH3OH and 10 mM PBS buffer (1% CH3CN, pH 7.4). Effects of incubation time (B) and pH (C) on the fluorescence responses of Pvi for Hg2+ detection. 5
10
15
20
45
2.5
(A)
1.0
2+
[Hg ]
F477 nm/F380 nm
0.6
0 µM
0.4 0.2
0.2
400 450 500 550 Wavelength (nm)
600
Hg2+, 200 µM for other metal ions). Spectra were recorded after 80 min of and then addition of with equivoluminal DCM. λex = 334 nm.
3.5. Detection of Hg2+ in water samples 50
55
In order to validate its practicality in environmental science, we employed probe Pvi in a standard addition method to determine Hg2+ concentrations in water samples from tap water and Xiangjiang River in Changsha city. The detection results were summarized in Table 1. Satisfactory values between 96% and 102% were obtained for the recovery experiments, and it can meet the requirement of detection of Hg2+ in water sample. The result indicated that the proposed probe could be an excellent tool to detect Hg2+. Table 1. The measurement results of mercury in water samples recovery (%)
spiked (µΜ)
recovered (µΜ)
0
not detected
0.25
0.253±0.013
101
0.5
0.49±0.041
98
0
not detected
0.25
0.256±0.016
102
0.5
0.48±0.142
96
(B)
1.5
Tap water
1.0
0.0 350
400
450
500
550
Wavelength (nm)
600
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 2+ Hg (µM)
Xiangjiang River water
Fig. 5. (A) Fluorescence spectra of Pvi (10 µM) in the presence of various concentrations of Hg2+ (0, 0.01, 0.03, 0.06, 0.1, 0.2, 0.4, 0.6, 1.0, 2.0, 3.0, 4.0, 5.0 µM). (B) Linear relationship between the ratio of emission intensities (I477nm/I380nm) and the concentration of Hg2+. Spectra were recorded after 80 min of incubation with Hg2+ in 10 mM PBS buffer (1% CH3CN, pH 7.4) and
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0.4
0.5
0.0
25
other ions
incubation with different analytes in 10 mM PBS buffer (1% CH3CN, pH 7.4)
2.0
5 µM
0.8
0.6
Fig. 6. Fluorescence responses of Pvi (10 µM) to various metal ions (5 µM for
sample 1.2
0.8
350
334 nm.
Under the optimal conditions, the analytical performance of probe Pvi was studied by fluorescence response towards various concentrations of Hg2+. As shown in Fig. 5A, the fluorescence intensity at 380 nm decreased accompanying the increase in emission wavelength at 477 nm. The emission ratio (I477nm/I380nm) was linearly proportional to the concentration of Hg2+ in the range of 0.01 to 3.0 µM (Fig. 5B). And, a linear regression equation for Hg2+, F = 0.0535 + 0.5713x (R2 = 0.9921) was obtained, where F refers to the ratio of emission intensities and x refers to the concentration of Hg2+. The limit of detection (LOD) (S/N=3, the concentration necessary to yield a net signal equal to three times the standard deviation of the background) was calculated to be 7.8 × 10-9 M, which is lower than the maximum permissible level of Hg2+ in drinking water (1 × 10-8 M). These results revealed that the probe could monitor Hg2+ levels both qualitatively and quantitatively.
2+
probe M
0.0
The concentration of Pvi and Hg2+ was 10 µM and 5 µM, respectively. λex =
3.4. The response performances of probe Pvi to Hg2+
Hg
1.0
then addition of equivoluminal DCM. λex = 334 nm.
A highly selective response to the target species over other
This journal is © The Royal Society of Chemistry [year]
60
3.6. Detection of Hg2+ in living cells To further investigate the membrane permeability of probe Pvi and its ability to sense Hg2+ in living cells, the cell fluorescence imaging experiment was carried out. The fluorescence microscopic images acquired at 380 nm excitation displayed
Journal Name, [year], [vol], 00–00 | 5
Analyst Accepted Manuscript
0.2
Normalized Intensity
Normalized Internsity
1.2
I477
0.0
0.0
Normalized Intensity
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DOI: 10.1039/C5AN00273G
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enhanced green fluorescence for the incubated HeLa cells with Pvi + Hg2+ in which the relative emission intensity was more intense compared to HeLa cells with probe Pvi (Figs. 7B and D). These results established that the probe was cell-permeable and capable of imaging of Hg2+ in living cells. The bright-field images (Figs. 7A and C) confirmed that the cells were viable throughout the imaging experiments.
40
† Electronic Supplementary Information (ESI) available: [details of any supplementary information available should be included here]. See DOI: 10.1039/b000000x/ 45
References
50
55
60
10
Fig. 7. (A) Bright-field image of HeLa cells incubated with Pvi (10 µM) for 1 h. (B) fluorescence image of (A). (C) Bright-field image of HeLa cells incubated with Pvi (10 µM) for 1 h and then treated with Hg2+ (5 µM) for
65
another 1 h. (D) fluorescence image of (C). 15
4. Conclusion
20
25
In summary, a new ratiometric fluorescence probe Pvi by combining 2-(1-(p-tolyl)-1H-phenanthro[9,10-d]imidazol-2-yl) phenol as the fluorophore and a vinyl group as a high selective recognition unit was successfully designed and synthesized. Dichloromethane can be used to extract the producted fluorophore Pol and recover its ESIPT efficiency for enhancing the sensitivity of Hg2+ detection. Furthermore, probe Pvi can successfully be applied to Hg2+ determinations in water samples and fluorescence imaging of Hg2+ in HeLa cells. This research demonstrated potential of the probe for environmental and biological applications.
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Acknowledgments 30
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This work was supported by the National Natural Science Foundation of China (21375037, 21275051, 21475043), Scientific Research Fund of Hunan Provincial Science and Technology Department and Education Department (13JJ2020, 12A084), and Doctoral Fund of Ministry of Education of China (NO: 20134306110006).
Notes and references Key Laboratory of Chemical Biology and Traditional Chinese Medicine Research (Ministry of Education),College of Chemistry and Chemical Engineering, Hunan Normal University, Changsha 410081, PR China Fax: +86 73188872531; Tel: +86 73188865515;
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