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A water-soluble, low-cytotoxic and sensitive fluorescent probe based on poly(ethylene glycol) for detecting sulfide anion in aqueous media and imaging inside live cells
Polymer xxx (2013) 1e7
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A water-soluble, low-cytotoxic and sensitive fluorescent probe based on poly(ethylene glycol) for detecting sulfide anion in aqueous media and imaging inside live cells Fangyuan Zheng a, Min Wen a, Fang Zeng a, *, Shuizhu Wu a, b, * a b
College of Materials Science & Engineering, South China University of Technology, Guangzhou 510640, PR China State Key Laboratory of Luminescent Materials & Devices, South China University of Technology, Guangzhou 510640, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 June 2013 Received in revised form 29 July 2013 Accepted 11 August 2013 Available online xxx
Sulfide anions are generated not only as a byproduct from industrial processes but also in biosystems. Hence, fluorescent probes for detecting sulfide anion which are water soluble, sensitive, selective and biocompatible are highly sought-after. In this study, we report a water-soluble, low-cytotoxic and sensitive fluorescent sensor for detecting sulfide anion. In this probe, the strong electron-withdrawing dinitrobenzenesulfonate ester group is incorporated onto fluorescein fluorophore, and correspondingly the fluorescence of fluorescein is efficiently quenched; while when the dinitrobenzenesulfonate ester is cleaved by the nucleophilic sulfide anion, the substantial fluorescence enhancement can be observed. Furthermore, poly(ethylene glycol) is coupled onto the fluorophore to impart the probe water-soluble and low cytotoxicity. The probe is capable of permeating the cell membrane and realizing sulfide anion monitoring and imaging in live cells and real sample. This technically-simple modification strategy may be suitable for fabricating some other fluorescent probes with enhanced biocompatibility and water solubility. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Fluorescence Sulfide Water soluble
1. Introduction To design and synthesize selective and sensitive sensing systems to detect various chemically and biologically pertinent species has attained a significant interest [1e3]. Among these species, sulfide anion is a very important member. Sulfide is a toxic anion, generated not only as a byproduct in industrial processes but also in biosystems due to microbial reduction of sulfate by anaerobic bacteria and sulfide generation from the sulfur-containing amino acids in meat proteins [4]. In addition, sulfide is employed in the production of sulfur and sulfuric acid, dyes and cosmetic manufacturing, production of wood pulp, etc. [5,6]. Most of the sulfide present in raw waters is derived from natural sources and industrial processes. It is particularly noticeable in some groundwaters, depending on source rock mineralogy and microorganisms present [7,8]. Exposure to a high level of sulfide can lead to
* Corresponding authors. College of Materials Science and Engineering, State Key Laboratory of Luminescent Materials & Devices, South China University of Technology, Guangzhou 510640, PR China. Tel.: þ86 20 22236262; fax: þ86 20 22236363. E-mail addresses: [email protected] (F. Zeng), [email protected] (S. Wu).
irritation in mucous membranes, unconsciousness, and respiratory paralysis. Once protonated, sulfide anion turns into HS (under acidic pH, HS converts to H2S) and becomes even more toxic and caustic [8]. Recent studies have shown that protonated sulfide is involved in multiple physiological processes in central nervous system, respiratory system, gastrointestinal system and endocrine system [9,10]; it is also related to diseases like Alzheimer’s disease, Down’s syndrome, diabetes, and liver cirrhosis [11,12], Therefore, the detection of sulfide anions is of high importance from industrial, environmental and biological points of view. Many approaches, such as colorimetric, electrochemical analysis and gas chromatography, have been employed to measure and trace sulfide anion [13e17]. However, relatively high cost, timeconsuming processes, destruction of tissues or cell lysates and lack of temporal and spatial resolution prohibit them from having applications in many biological studies [18,19]. On the other hand, fluorescent techniques are extremely attractive in this regard due to their simplicity, high sensitivity and real-time detection, as well as the capability for nondestructive detection of biological events in live cells or tissues [20e55]. Until now, several elegant fluorescent probes for sulfide anions have been reported [18,19,56e70]. In general, these probes were
0032-3861/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.polymer.2013.08.021
Please cite this article in press as: Zheng F, et al., A water-soluble, low-cytotoxic and sensitive fluorescent probe based on poly(ethylene glycol) for detecting sulfide anion in aqueous media and imaging inside live cells, Polymer (2013), http://dx.doi.org/10.1016/j.polymer.2013.08.021
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designed based on: (a) Reduction reaction between an azide group and S2 [19,56e59]; (b) Formation of a copper-centered coordination complex in which Cu2þ can be released by binding with S2 [18,60e67]; (c) The nucleophilic nature of S2 [68e70]. However, much improvement is still needed for S2 probes for possible environmental and bio-related applications in terms of water solubility, low toxicity and usability in biological matrixes. On the other hand, previous researches demonstrated that, electron-withdrawing groups such as dinitrophenyl ether or dinitrobenzenesulfonyl, when coupled onto fluorophores, can efficiently quench the fluorescence of the fluorophores [71e73]; while the cleavage of these groups will restore the fluorescence. By using this protocol, a couple of elegant thiol probes have been designed based on the thiol-mediated cleavage of dinitrophenyl ether or dinitrobenzenesulfonyl group due to the nucleophilicity of thiols [71e73]. However, these probes are not water soluble and only usable in water/solvent mixture (e.g. ethanol or dimethylformamide); in addition, the response time is usually relatively long (dozens of minutes). Compared to thiols, sulfide anion is a much more effective nucleophile and reacts readily with sulfonate esters. This fact along with the above research results concerning thiol probes provided us with a clue for the design of novel fluorescent probes for sulfide anion. We thus anticipated that, incorporating the strong electron-withdrawing group dinitrobenzenesulfonate ester onto fluorescein fluorophore could significantly diminish the fluorescence; while when the dinitrobenzenesulfonate ester is cleaved by the more nucleophilic sulfide anion, we should observe a substantial fluorescence turnon response. Furthermore, if the well-known hydrophilic polymer poly(ethylene oxide) (PEO) [29], which has been used for a wide range of biomedical applications because of its biocompatible, non-toxic, non-antigenic and non-immunogenic properties, is coupled onto the fluorophore, we could ensure water solubility, cell membrane permeability and low-cytotoxicity for the probe as well. With these ideas in mind, in this study, we demonstrate a fluorescent turn-on probe for S2, which is based on the nucleophilicity of sulfide anion and features good water solubility, low cytotoxicity and high sensitivity. The schematic illustration for the selective sensing for sulfide anion by the probe is shown in Fig. 1. The salient features of this probe include: First, the preparation of the probe is technically simple, and it can be easily obtained through a two-step-reaction synthetic route. In addition, the probe is able to selectively detect sulfide anion in totally aqueous media (100% water); and it is also capable of permeating the cell membrane and tracing sulfide anion in live cells. Moreover, with a relatively short response time, this probe is also very sensitive with a low detection limit of 150 nM.
Fig. 1. Schematic illustration for the structure of the probe and its selective detection of sulfide anion. (The probe: 102 mg/mL in pH 7.4 HEPES buffered water, sulfide anion: 8 105 M).
2.2. Synthesis of the probe The probe was synthesized through a two-step reaction. First, methoxypolyethylene glycol amine (average molecular weight 2000) (0.4 g, 0.2 mmol) and fluorescein isothiocyanate (79 mg, 0.2 mmol) were dissolved in 5 mL DMF in a flask. The reaction mixture was stirred under N2 at room temperature overnight, afterwards the solvent was removed under reduced pressure. The residue was dissolved in 3 mL CH2Cl2, and then 30 mL diethyl ether was added to precipitate the product. The mixture solution was centrifuged (10,000 r/s, 8 min). And the intermediate product 1 was obtained as a solid (442 mg, 92%). 1H NMR (CDCl3, 400 MHz) d ppm: 3.37 (methoxy protons of PEG), 3.63 (methylene protons of PEG backbone), 6.5e8.2 (fluorescein aromatic protons). Second, 1 (430 mg, 0.18 mmol) and N,N-diisopropylethylamine (65 mg, 0.5 mmol) were dissolved in 5 mL CH2Cl2 in a flask, the mixture was stirred under N2. Then 2,4-dinitrobenzenesulfonyl chloride (134 mg, 0.5 mmol) was added into the above solution dropwise. The solution was stirred at room temperature overnight. After that, the solution was filtered, 30 mL of CH2Cl2 was added into the filtrate, and the filtrate was then washed with deionized water. The combined organic phase was dried over anhydrous MgSO4. Then the solvent was evaporated under vacuum and the residue was dissolved in 3 mL CH2Cl2, and then 30 mL diethyl ether was added to precipitate the product. The mixture solution was centrifuged (10,000 r/s, 8 min). And the product (the probe) was obtained as a yellow solid (452 mg, 96%). 1H NMR (CDCl3, 400 MHz) d ppm: 3.37 (methoxy protons of PEG), 3.63 (methylene protons of PEG backbone), 6.5e8.2 (fluorescein aromatic protons), 8.3e8.7 (dinitrobenzene protons). 2.3. Cytotoxicity
2. Experimental
The cell line, L929 (murine aneuploid fibro-sarcoma cell) was incubated in RPMI1640 medium supplemented with 10% fetal bovine serum (FBS) at 37 C with 5% CO2. The cytotoxicity of the probe against L929 cells was assessed by MTT assay according to ISO 10993-5.
2.1. Materials and reagents
2.4. Cell incubation and imaging
Methoxypolyethylene glycol amine (average molecular weight around 2000, PEG2000), fluorescein isothiocyanate, N,N-diisopropylethylamine, 2,4-dinitrobenzenesulfonyl chloride, sodium salts of anion (S2, F, Cl, Br, I, CO2 3 ; 2 2 2 NO and 4(2-hydroxyethyl)-13 ; S2 O3 ; SO3 ; HSO3 ; SO4 ), piperazineethanesulfonic acid (HEPES) were purchased from Aldrich. The purified water used in this study was the tripledistilled water which was further treated by ion exchange columns and then by a Milli-Q water purification system. N, NDimethyl-formamide (DMF) was dried with CaH2 and vacuum distilled. Methanol and dichloromethane were analytically pure solvents and distilled before use.
Two cell lines, Hela (human cervical cancer cell) and L929 (murine aneuploid fibrosarcoma cell), were incubated in RPMI1640 medium supplemented with 10% Fetal Bovine Serum (FBS, Invitrogen). One day before imaging, cells were passed and plated on 30-mm glass culture dishes. For the experiments, cells were washed with RPMI1640, incubated in RPMI1640 medium containing N-methylmaleimide (which is a trapping reagent of thiol species) for 20 min and then the probe at 37 C under 5% CO2 for 2 h, and then treated with Na2S (2 mM and 8 mM respectively) for 30 min. After that, the cells were washed with PBS for three times and then imaged on an Olympus IX71 inverted fluorescence microscope equipped with a DP72 color CCD (blue light excitation).
Please cite this article in press as: Zheng F, et al., A water-soluble, low-cytotoxic and sensitive fluorescent probe based on poly(ethylene glycol) for detecting sulfide anion in aqueous media and imaging inside live cells, Polymer (2013), http://dx.doi.org/10.1016/j.polymer.2013.08.021
F. Zheng et al. / Polymer xxx (2013) 1e7
For the cells being treated with the probe only (without Na2S), one day before imaging, cells were passed and plated on 30-mm glass culture dishes and allowed to grow to 50e70% confluence. Afterwards, cells were washed with RPMI1640, incubated in RPMI1640 medium containing the probe at 37 C under 5% CO2 for 2 h, the cells were washed with PBS for three times and then imaged on an Olympus IX71 inverted fluorescence microscope. 2.5. Measurements 1
H NMR spectra were recorded on a Bruker Avance 400 MHz NMR spectrometer. Fluorescence spectra were recorded on a Hitachi F-4600 fluorescence spectrophotometer. Absorbance measurements were carried out using a Hitachi U-3010 UVevis spectrophotometer. MALDI-TOF mass spectra were measured by using UltrafleXtreme MALDI-TOF system. 3. Results and discussion 3.1. Synthesis of the probe The probe was readily synthesized through a two-step reaction. First, an amine-terminated PEG (methoxypolyethylene glycol amine, with the average molecular weight of 1780) was reacted with fluorescein isothiocyanate to produce an intermediate product, and the hydroxyl groups on fluorescein moiety were then reacted with 2,4-dinitrobenzenesulfonyl chloride to form the probe. The synthetic route is shown in Scheme 1. The intermediate product and the synthesized probe were characterized by 1H NMR and the matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (Figs. S1 and S2). Unlike a smallmolecular compound which has a definite molecular weight, a polymer sample is actually a mixture of polymer chains with different numbers of repeat units. The average molecular weight of the probe is determined to be 2430, and the polydispersity index is 1.1. In addition, due to the highly efficient reaction between isothiocyanate group and primary amine, as well as the highly efficient reaction between the hydroxyl groups on fluorescein moiety and 2,4-dinitrobenzenesulfonyl chloride, the synthetic reactions of the probe readily take place, and the probe can be easily reproducible. 3.2. Fluorescent turn-on sensing for sulfide anion in totally aqueous media (100% water) The fluorescence spectra of the probe in aqueous solution upon addition of varied amounts of sulfide anions are shown in Fig. 2A. A working curve is also established by plotting the increment of emission intensities at 518 nm vs. sulfide anion concentration, as
3
shown in Fig. 2B. It is clear that, the probe is essentially nonfluorescent in aqueous media (pH 7.4 HEPES buffered water), while the addition of increasing concentrations of sulfide anion elicits a dramatic change in the emission spectra, and an intense new peak around 518 nm can be observed; and at the sulfide anion concentration of 15 mM, the fluorescence enhancement at 518 nm is enhanced up to 340-fold, as shown in Fig. 2. When the sulfide amount is at low concentration range (<5 mM), the fluorescence intensity increases relatively slow. As the sulfide concentration is further increased, the fluorescence intensity increases steadily with the increasing concentration of the added sulfide anions; while when the sulfide concentration is higher than 35 mM, the fluorescence intensity increases slowly with the increasing concentration of sulfide, and finally the curve of fluorescence intensity vs. Csulfide levels off. Furthermore, the significant fluorescence amplification can also be observed visually by the emission of the probe solution from dark to bright green (in web version) upon introduction of sulfide anion as displayed in Fig. 1. Moreover, the detection limit toward sulfide anion is determined to be 150 nM (Fig. S3). To confirm that the fluorescence sensing response of the probe to sulfide anion is indeed due to the cleavage of the 2,4dinitrobenzenesulfonyl group, MALDI-TOF mass spectrometry was used to investigate the reaction of the probe with sulfide anion (since both the probe and the product resulted from the reaction of the probe and sulfide anion are polymer compounds, HPLC can’t be used to monitor the reaction). Into the probe (102 mg/mL) solution in pH 7.4 HEPES buffered water, Na2S (10 105 M) was added under stirring, 10 min later the water was evaporated under vacuum, and the solid residue (reaction product) was purified and then used for mass spectrometric measurement. The MALDI-TOF MS spectra for both the probe and the product are given in Fig. 3. It can be seen that, the molecular weight difference (w231) between the product and the corresponding probe equals the molecular weight of the 2,4dinitrobenzenesulfonyl group. This analysis strongly suggests that upon addition of Na2S, the 2,4-dinitrobenzenesulfonyl group is indeed cleaved from the probe and the fluorescein moiety is restored. For this sensing strategy, in the absence of the sulfide anion, the incorporation of 2,4-dinitrobenzenesulfonate ester group results in the quenching of fluorescence of the probe; while in the presence of sulfide anion, the cleavage of 2,4-dinitrobenzenesulfonyl will restore the fluorescein moiety, thereby leading to the fluorescence enhancement of the probe. 3.3. Selectivity and anti-interference performance of the sensing system To evaluate the selectivity of the probe towards sulfide anion, the fluorescence response of the probe in the presence of some
Scheme 1. Synthesis route for the probe.
Please cite this article in press as: Zheng F, et al., A water-soluble, low-cytotoxic and sensitive fluorescent probe based on poly(ethylene glycol) for detecting sulfide anion in aqueous media and imaging inside live cells, Polymer (2013), http://dx.doi.org/10.1016/j.polymer.2013.08.021
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Fig. 2. (A) Fluorescence spectra of the probe (102 mg/mL) in the presence of different amounts of sulfide anion in pH 7.4 HEPES buffered water. (B) Fluorescence intensity of the probe (102 mg/mL) at 518 nm as a function of sulfide anion concentration in pH 7.4 HEPES buffered water. (lexc ¼ 490 nm) The spectra were recorded 10 min after addition of sulfide anion.
2 2 anions including F, Cl, Br, I, CO2 3 ; NO3 ; S2 O3 ; SO3 ; 2 HSO ; SO were recorded. And the comparison of the fluorescence 3 4 intensities at 518 nm is shown in Fig. 4A. It is obvious that, only sulfide anion elicits a significant fluorescence intensity enhancement, whereas the addition of other anions leads to almost no fluorescence change, even though the concentration of these anions is much higher than that of S2. These results confirm that the probe shows an excellent selectivity toward S2 over other competitive anion. This is probably because, sulfide anion is a highly effective nucleophile and reacts readily with sulfonate esters. On the other hand, the interference of the above species was also tested. Fig. 4B shows the fluorescence response of the probe towards S2 in the presence of other competitive anions. It is obvious that, generally the co-existence of these species does not interfere with the reaction of sulfide anion with the probe as well as the subsequent fluorescence enhancement. These results suggest that the probe can function as a highly selective probe for S2. Moreover, we also compared the fluorescence response of the probe over time in the presence of S2, three thiols (glutathione, cysteine or homocysteine), as shown in Fig. S4. It is clear that, in the presence of sulfide anion, the fluorescence intensity of the probe solution quickly reaches a plateau in about 10 min; while with the respective addition of the three thiols, the fluorescence intensity of the probe solution increases much slower, and will only reach the plateau after several hours. The results indicate that, the reaction between S2 and the probe is complete in about 10 min, which is much faster than that with the three thiols. This is why we choose 10 min as the reaction length for all fluorescence measurements. Compared to thiols, sulfide anion is a much more effective
nucleophile and reacts readily with sulfonate esters. Hence, the biologically-relevant thiols like glutathione (GSH), cysteine (Cys) and homocysteine (Hcy) react with sulfonate ester more slowly than sulfide. It can be seen from this figure that, as for GSH, even at the high concentration of 1 mM (ten-fold of that of sulfide anion), GSH still couldn’t lead to the fluorescence enhancement to the extent as sulfide does. Furthermore, we also investigated possible interference by other biologically relevant substances, including various amino acids and ascorbic acid, as shown in Fig. S5. It is clear that these biologically relevant compounds do not lead to any fluorescence change, even though the concentration of these anions is much higher than that of S2. This ensures that, the probe has quite good selectivity for sulfide anion over abundant biologically-relevant thiols and other compounds. 3.4. Sulfide anion sensing in running water To evaluate the efficacy of this probe’s application in real sample, the probe was applied to monitoring sulfide anion in running water. As shown in Table 1, this probe works quite well in running water with quite good recovery. The results given in Table 1 also show that the detected concentrations of the sulfide anion are in good agreement with those added in the samples. In addition, the composition of the running water does not significantly interfere with the detection of sulfide anion, indicating the capability of this probe for being used in water quality monitoring. According to World Health Organization, the maximum recommended sulfide concentration in drinking water is less than 500 mg/L (about
Fig. 3. Mass spectra of the as-prepared probe (A) and the purified reaction product upon addition of sulfide anion (B).
Please cite this article in press as: Zheng F, et al., A water-soluble, low-cytotoxic and sensitive fluorescent probe based on poly(ethylene glycol) for detecting sulfide anion in aqueous media and imaging inside live cells, Polymer (2013), http://dx.doi.org/10.1016/j.polymer.2013.08.021
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Fig. 4. (A) Fluorescence intensities at 518 nm for the probe (102 mg/mL) in the presence of 15 mM S2 and 100 mM different anions respectively (lexc ¼ 490 nm). (B) Fluorescence intensities at 518 nm for the probe (102 mg/mL) in the presence of 15 mM of S2 and with the addition of 100 mM of different anions (lexc ¼ 490 nm). All fluorescence intensities were measured in pH 7.4 HEPES buffered water and 10 min after the addition of anions.
15 mM);7 hence with the detection limit of 0.15 mM, this probe can be used in the detection for sulfide anion in water quality monitoring. This probe is mainly intended to be used for detecting sulfide levels in running water, and the only possible interferent for the sensing e biothiols are rarely found in ground water; hence the sensor has the potential for being used in water quality monitoring. 3.5. Imaging of sulfide anion in live cells To investigate the biocompatibility of the probe, the cytotoxicity of the probe was evaluated using L929 cell line by MTT assay in compliance with ISO 10993-5. And the results are shown in Fig. 5. The probe shows a cell viability of 98% at the test concentration of 102 mg/mL. Even at the concentration of 5 102 mg/mL, the cell viability can still reach 94%. This indicates that the probe generally has no cytotoxicity, probably due to the incorporation of the biocompatible PEG. The feasibility of the probe for fluorescence imaging of sulfide anion in the living cells was also investigated. The favorable features of the probe include excitation and emission in the visible region, a significant fluorescence turn-on signal, high sensitivity, high selectivity, low cytotoxicity, and functioning well at physiological pH. These desirable characters are conducive to imaging S2 in living cells. Hence, we explored the capability of the probe to track sulfide level in two commonly-used cell lines e Hela (human cervical cancer cell) and L929 (murine aneuploid fibrosarcoma cell). To exclude the effects of any endogenous biothiols, N-methylmaleimide, a known trapping reagent of thiol species, was incubated with the cells before the cells were treated with the sensor. Fig. 6 and Fig. S6 display the fluorescent microscope images for the two kinds of cells incubated with the probe before and after being treated with different concentrations of S2. Fluorescence
microscope images of both Hela and L929 live cells loaded with the probe for 2 h at 37 C show no fluorescence intracellularly, as shown in Fig. 6A and D. The control experiment on cells without the probe gives no fluorescence either under the same exposure condition. By contrast, the intracellular fluorescence for the probestained cells exposed to 2 mM and 8 mM of S2 for 30 min at 37 C gives rise to bright green fluorescence, as shown in Fig. 6B, C, E and F (The concentrations (2 mM or 8 mM) were not the concentrations for the intracellular sulfide, but those for the sulfide donor in the culture media. Generally, only a small portion of sulfide donor can be internalized by the cells). These data establish that the probe is cell membrane permeable and is able to respond to sulfide anion inside living cells, as well as can be utilized to track sulfide level change inside live cells. 4. Conclusion In summary, we have successfully constructed a water-soluble and sensitive fluorescent turn-on sensing system for sulfide anion, which is usable in totally aqueous media. The sensing system features simplicity in synthesis, fast-responding, sensitive and selective detection of sulfide anion. Furthermore, the sensing system has good cell membrane permeability as well as very low cytotoxicity. The incorporation of 2,4-dinitrobenzenesulfonate ester group onto fluorescein can effectively quench the fluorescence of the fluorophore; while upon addition of sulfide anion, the sulfidemediated cleavage of 2,4-dinitrobenzenesulfonyl group occurs, resulting in the recovery of the strong fluorescence of the
Table 1 Determination of sulfide anion in running water. Samples
Amount of sulfide anion (mol L1)
Running water
0 1.00 1.50 2.00 2.50 3.00
Added
Recovery (%)
Found 105 106 105 105 105
e 1.02 1.55 2.03 2.57 3.11
105 105 105 105 105
e 102 103.3 101.5 102.8 103.7
In the test solutions, running water was 10-fold diluted. The measurements were conducted 10 min after the addition of sulfide.
Fig. 5. Cytotoxic effects against L929 cells upon 24 h of incubation. Control: L929 cells in the absence of the probe; others: L929 cells in the presence of the probe (102 mg/ mL, 2 102 mg/mL, 5 102 mg/mL respectively).
Please cite this article in press as: Zheng F, et al., A water-soluble, low-cytotoxic and sensitive fluorescent probe based on poly(ethylene glycol) for detecting sulfide anion in aqueous media and imaging inside live cells, Polymer (2013), http://dx.doi.org/10.1016/j.polymer.2013.08.021
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Fig. 6. Fluorescence imaging of HeLa and L929 cells incubated with the probe before (A and D) and after (B and E: 2 mM S2; C and F: 8 mM S2) being treated with sulfide.
fluorescein moiety. Moreover, the sensing system can be used for imaging sulfide anion inside live cells. And this probe exhibits a low detection limit of 0.15 mM. This approach offers some useful insights for realizing technically-simple fluorescent turn-on sensing in the detection assays for other analytes. Acknowledgment This work was supported by the National Key Basic Research Program of China (Project No. 2013CB834702) and NSFC (Project No. 21025415, 21174040). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.polymer.2013.08.021. References [1] de Silva AP, Gunaratne HQN, Gunnlaugsson T, Huxley AJM, McCoy CP, Rademacher JT, et al. Chem Rev 1997;97:1515e66. [2] Esteban-Gomez D, Platas-Iglesias C, de Blas A, Fabbrizzi L, Rodriguez-Blas T. Chem Eur J 2008;14:5829e38. [3] Martínez-Manez R, Sancenon F. Chem Rev 2003;103:4419e76. [4] Hydrogen sulfide (environmental health criteria, no. 19). Geneva: World Health Organization; 1981. [5] Gosselin RE, Smith RP, Hodge HC, Braddock J. In: Clinical toxicology of commercial products. 5th ed. Baltimore, MD: Williams and Wilkins; 1984III-198e 202. [6] Patwardhan SA, Abhyankar SM. Colourage 1988;35:15e8. [7] WHO. Guidelines for drinking-water quality. 2nd ed. Geneva: World Health Organization; 1996. [8] Meyer B, Ward K, Koshlap K, Peter L. Inorg Chem 1983;22:2345e50. [9] Li L, Rose P, Moore PK. Annu Rev Pharmacol Toxicol 2011;51:169e87. [10] Boehning D, Snyder SH. Annu Rev Neurosci 2003;26:105e31. [11] Eto K, Asada T, Arima K, Makifuchi T, Kimura H. Biochem Biophys Res Commun 2002;293:1485e8. [12] Kamoun P, Belardinelli MC, Chabli A, Lallouchi K, Chadefaux-Vekemans B. Am J Med Genet 2003;116:310e1. [13] Balasubramanian S, Pugalenthi V. Wat Res 2000;34:4201e6. [14] Bings NH, Bogaerts A, Broekaert JAC. Anal Chem 2008;80:4317e47.
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A water-soluble, low-cytotoxic and sensitive fluorescent probe based on poly(ethylene glycol) for detecting sulfide anion in aqueous media and imaging inside live cells Fangyuan Zheng a, Min Wen a, Fang Zeng a, *, Shuizhu Wu a, b, * a b
College of Materials Science & Engineering, South China University of Technology, Guangzhou 510640, PR China State Key Laboratory of Luminescent Materials & Devices, South China University of Technology, Guangzhou 510640, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 June 2013 Received in revised form 29 July 2013 Accepted 11 August 2013 Available online xxx
Sulfide anions are generated not only as a byproduct from industrial processes but also in biosystems. Hence, fluorescent probes for detecting sulfide anion which are water soluble, sensitive, selective and biocompatible are highly sought-after. In this study, we report a water-soluble, low-cytotoxic and sensitive fluorescent sensor for detecting sulfide anion. In this probe, the strong electron-withdrawing dinitrobenzenesulfonate ester group is incorporated onto fluorescein fluorophore, and correspondingly the fluorescence of fluorescein is efficiently quenched; while when the dinitrobenzenesulfonate ester is cleaved by the nucleophilic sulfide anion, the substantial fluorescence enhancement can be observed. Furthermore, poly(ethylene glycol) is coupled onto the fluorophore to impart the probe water-soluble and low cytotoxicity. The probe is capable of permeating the cell membrane and realizing sulfide anion monitoring and imaging in live cells and real sample. This technically-simple modification strategy may be suitable for fabricating some other fluorescent probes with enhanced biocompatibility and water solubility. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Fluorescence Sulfide Water soluble
1. Introduction To design and synthesize selective and sensitive sensing systems to detect various chemically and biologically pertinent species has attained a significant interest [1e3]. Among these species, sulfide anion is a very important member. Sulfide is a toxic anion, generated not only as a byproduct in industrial processes but also in biosystems due to microbial reduction of sulfate by anaerobic bacteria and sulfide generation from the sulfur-containing amino acids in meat proteins [4]. In addition, sulfide is employed in the production of sulfur and sulfuric acid, dyes and cosmetic manufacturing, production of wood pulp, etc. [5,6]. Most of the sulfide present in raw waters is derived from natural sources and industrial processes. It is particularly noticeable in some groundwaters, depending on source rock mineralogy and microorganisms present [7,8]. Exposure to a high level of sulfide can lead to
* Corresponding authors. College of Materials Science and Engineering, State Key Laboratory of Luminescent Materials & Devices, South China University of Technology, Guangzhou 510640, PR China. Tel.: þ86 20 22236262; fax: þ86 20 22236363. E-mail addresses: [email protected] (F. Zeng), [email protected] (S. Wu).
irritation in mucous membranes, unconsciousness, and respiratory paralysis. Once protonated, sulfide anion turns into HS (under acidic pH, HS converts to H2S) and becomes even more toxic and caustic [8]. Recent studies have shown that protonated sulfide is involved in multiple physiological processes in central nervous system, respiratory system, gastrointestinal system and endocrine system [9,10]; it is also related to diseases like Alzheimer’s disease, Down’s syndrome, diabetes, and liver cirrhosis [11,12], Therefore, the detection of sulfide anions is of high importance from industrial, environmental and biological points of view. Many approaches, such as colorimetric, electrochemical analysis and gas chromatography, have been employed to measure and trace sulfide anion [13e17]. However, relatively high cost, timeconsuming processes, destruction of tissues or cell lysates and lack of temporal and spatial resolution prohibit them from having applications in many biological studies [18,19]. On the other hand, fluorescent techniques are extremely attractive in this regard due to their simplicity, high sensitivity and real-time detection, as well as the capability for nondestructive detection of biological events in live cells or tissues [20e55]. Until now, several elegant fluorescent probes for sulfide anions have been reported [18,19,56e70]. In general, these probes were
0032-3861/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.polymer.2013.08.021
Please cite this article in press as: Zheng F, et al., A water-soluble, low-cytotoxic and sensitive fluorescent probe based on poly(ethylene glycol) for detecting sulfide anion in aqueous media and imaging inside live cells, Polymer (2013), http://dx.doi.org/10.1016/j.polymer.2013.08.021
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F. Zheng et al. / Polymer xxx (2013) 1e7
designed based on: (a) Reduction reaction between an azide group and S2 [19,56e59]; (b) Formation of a copper-centered coordination complex in which Cu2þ can be released by binding with S2 [18,60e67]; (c) The nucleophilic nature of S2 [68e70]. However, much improvement is still needed for S2 probes for possible environmental and bio-related applications in terms of water solubility, low toxicity and usability in biological matrixes. On the other hand, previous researches demonstrated that, electron-withdrawing groups such as dinitrophenyl ether or dinitrobenzenesulfonyl, when coupled onto fluorophores, can efficiently quench the fluorescence of the fluorophores [71e73]; while the cleavage of these groups will restore the fluorescence. By using this protocol, a couple of elegant thiol probes have been designed based on the thiol-mediated cleavage of dinitrophenyl ether or dinitrobenzenesulfonyl group due to the nucleophilicity of thiols [71e73]. However, these probes are not water soluble and only usable in water/solvent mixture (e.g. ethanol or dimethylformamide); in addition, the response time is usually relatively long (dozens of minutes). Compared to thiols, sulfide anion is a much more effective nucleophile and reacts readily with sulfonate esters. This fact along with the above research results concerning thiol probes provided us with a clue for the design of novel fluorescent probes for sulfide anion. We thus anticipated that, incorporating the strong electron-withdrawing group dinitrobenzenesulfonate ester onto fluorescein fluorophore could significantly diminish the fluorescence; while when the dinitrobenzenesulfonate ester is cleaved by the more nucleophilic sulfide anion, we should observe a substantial fluorescence turnon response. Furthermore, if the well-known hydrophilic polymer poly(ethylene oxide) (PEO) [29], which has been used for a wide range of biomedical applications because of its biocompatible, non-toxic, non-antigenic and non-immunogenic properties, is coupled onto the fluorophore, we could ensure water solubility, cell membrane permeability and low-cytotoxicity for the probe as well. With these ideas in mind, in this study, we demonstrate a fluorescent turn-on probe for S2, which is based on the nucleophilicity of sulfide anion and features good water solubility, low cytotoxicity and high sensitivity. The schematic illustration for the selective sensing for sulfide anion by the probe is shown in Fig. 1. The salient features of this probe include: First, the preparation of the probe is technically simple, and it can be easily obtained through a two-step-reaction synthetic route. In addition, the probe is able to selectively detect sulfide anion in totally aqueous media (100% water); and it is also capable of permeating the cell membrane and tracing sulfide anion in live cells. Moreover, with a relatively short response time, this probe is also very sensitive with a low detection limit of 150 nM.
Fig. 1. Schematic illustration for the structure of the probe and its selective detection of sulfide anion. (The probe: 102 mg/mL in pH 7.4 HEPES buffered water, sulfide anion: 8 105 M).
2.2. Synthesis of the probe The probe was synthesized through a two-step reaction. First, methoxypolyethylene glycol amine (average molecular weight 2000) (0.4 g, 0.2 mmol) and fluorescein isothiocyanate (79 mg, 0.2 mmol) were dissolved in 5 mL DMF in a flask. The reaction mixture was stirred under N2 at room temperature overnight, afterwards the solvent was removed under reduced pressure. The residue was dissolved in 3 mL CH2Cl2, and then 30 mL diethyl ether was added to precipitate the product. The mixture solution was centrifuged (10,000 r/s, 8 min). And the intermediate product 1 was obtained as a solid (442 mg, 92%). 1H NMR (CDCl3, 400 MHz) d ppm: 3.37 (methoxy protons of PEG), 3.63 (methylene protons of PEG backbone), 6.5e8.2 (fluorescein aromatic protons). Second, 1 (430 mg, 0.18 mmol) and N,N-diisopropylethylamine (65 mg, 0.5 mmol) were dissolved in 5 mL CH2Cl2 in a flask, the mixture was stirred under N2. Then 2,4-dinitrobenzenesulfonyl chloride (134 mg, 0.5 mmol) was added into the above solution dropwise. The solution was stirred at room temperature overnight. After that, the solution was filtered, 30 mL of CH2Cl2 was added into the filtrate, and the filtrate was then washed with deionized water. The combined organic phase was dried over anhydrous MgSO4. Then the solvent was evaporated under vacuum and the residue was dissolved in 3 mL CH2Cl2, and then 30 mL diethyl ether was added to precipitate the product. The mixture solution was centrifuged (10,000 r/s, 8 min). And the product (the probe) was obtained as a yellow solid (452 mg, 96%). 1H NMR (CDCl3, 400 MHz) d ppm: 3.37 (methoxy protons of PEG), 3.63 (methylene protons of PEG backbone), 6.5e8.2 (fluorescein aromatic protons), 8.3e8.7 (dinitrobenzene protons). 2.3. Cytotoxicity
2. Experimental
The cell line, L929 (murine aneuploid fibro-sarcoma cell) was incubated in RPMI1640 medium supplemented with 10% fetal bovine serum (FBS) at 37 C with 5% CO2. The cytotoxicity of the probe against L929 cells was assessed by MTT assay according to ISO 10993-5.
2.1. Materials and reagents
2.4. Cell incubation and imaging
Methoxypolyethylene glycol amine (average molecular weight around 2000, PEG2000), fluorescein isothiocyanate, N,N-diisopropylethylamine, 2,4-dinitrobenzenesulfonyl chloride, sodium salts of anion (S2, F, Cl, Br, I, CO2 3 ; 2 2 2 NO and 4(2-hydroxyethyl)-13 ; S2 O3 ; SO3 ; HSO3 ; SO4 ), piperazineethanesulfonic acid (HEPES) were purchased from Aldrich. The purified water used in this study was the tripledistilled water which was further treated by ion exchange columns and then by a Milli-Q water purification system. N, NDimethyl-formamide (DMF) was dried with CaH2 and vacuum distilled. Methanol and dichloromethane were analytically pure solvents and distilled before use.
Two cell lines, Hela (human cervical cancer cell) and L929 (murine aneuploid fibrosarcoma cell), were incubated in RPMI1640 medium supplemented with 10% Fetal Bovine Serum (FBS, Invitrogen). One day before imaging, cells were passed and plated on 30-mm glass culture dishes. For the experiments, cells were washed with RPMI1640, incubated in RPMI1640 medium containing N-methylmaleimide (which is a trapping reagent of thiol species) for 20 min and then the probe at 37 C under 5% CO2 for 2 h, and then treated with Na2S (2 mM and 8 mM respectively) for 30 min. After that, the cells were washed with PBS for three times and then imaged on an Olympus IX71 inverted fluorescence microscope equipped with a DP72 color CCD (blue light excitation).
Please cite this article in press as: Zheng F, et al., A water-soluble, low-cytotoxic and sensitive fluorescent probe based on poly(ethylene glycol) for detecting sulfide anion in aqueous media and imaging inside live cells, Polymer (2013), http://dx.doi.org/10.1016/j.polymer.2013.08.021
F. Zheng et al. / Polymer xxx (2013) 1e7
For the cells being treated with the probe only (without Na2S), one day before imaging, cells were passed and plated on 30-mm glass culture dishes and allowed to grow to 50e70% confluence. Afterwards, cells were washed with RPMI1640, incubated in RPMI1640 medium containing the probe at 37 C under 5% CO2 for 2 h, the cells were washed with PBS for three times and then imaged on an Olympus IX71 inverted fluorescence microscope. 2.5. Measurements 1
H NMR spectra were recorded on a Bruker Avance 400 MHz NMR spectrometer. Fluorescence spectra were recorded on a Hitachi F-4600 fluorescence spectrophotometer. Absorbance measurements were carried out using a Hitachi U-3010 UVevis spectrophotometer. MALDI-TOF mass spectra were measured by using UltrafleXtreme MALDI-TOF system. 3. Results and discussion 3.1. Synthesis of the probe The probe was readily synthesized through a two-step reaction. First, an amine-terminated PEG (methoxypolyethylene glycol amine, with the average molecular weight of 1780) was reacted with fluorescein isothiocyanate to produce an intermediate product, and the hydroxyl groups on fluorescein moiety were then reacted with 2,4-dinitrobenzenesulfonyl chloride to form the probe. The synthetic route is shown in Scheme 1. The intermediate product and the synthesized probe were characterized by 1H NMR and the matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (Figs. S1 and S2). Unlike a smallmolecular compound which has a definite molecular weight, a polymer sample is actually a mixture of polymer chains with different numbers of repeat units. The average molecular weight of the probe is determined to be 2430, and the polydispersity index is 1.1. In addition, due to the highly efficient reaction between isothiocyanate group and primary amine, as well as the highly efficient reaction between the hydroxyl groups on fluorescein moiety and 2,4-dinitrobenzenesulfonyl chloride, the synthetic reactions of the probe readily take place, and the probe can be easily reproducible. 3.2. Fluorescent turn-on sensing for sulfide anion in totally aqueous media (100% water) The fluorescence spectra of the probe in aqueous solution upon addition of varied amounts of sulfide anions are shown in Fig. 2A. A working curve is also established by plotting the increment of emission intensities at 518 nm vs. sulfide anion concentration, as
3
shown in Fig. 2B. It is clear that, the probe is essentially nonfluorescent in aqueous media (pH 7.4 HEPES buffered water), while the addition of increasing concentrations of sulfide anion elicits a dramatic change in the emission spectra, and an intense new peak around 518 nm can be observed; and at the sulfide anion concentration of 15 mM, the fluorescence enhancement at 518 nm is enhanced up to 340-fold, as shown in Fig. 2. When the sulfide amount is at low concentration range (<5 mM), the fluorescence intensity increases relatively slow. As the sulfide concentration is further increased, the fluorescence intensity increases steadily with the increasing concentration of the added sulfide anions; while when the sulfide concentration is higher than 35 mM, the fluorescence intensity increases slowly with the increasing concentration of sulfide, and finally the curve of fluorescence intensity vs. Csulfide levels off. Furthermore, the significant fluorescence amplification can also be observed visually by the emission of the probe solution from dark to bright green (in web version) upon introduction of sulfide anion as displayed in Fig. 1. Moreover, the detection limit toward sulfide anion is determined to be 150 nM (Fig. S3). To confirm that the fluorescence sensing response of the probe to sulfide anion is indeed due to the cleavage of the 2,4dinitrobenzenesulfonyl group, MALDI-TOF mass spectrometry was used to investigate the reaction of the probe with sulfide anion (since both the probe and the product resulted from the reaction of the probe and sulfide anion are polymer compounds, HPLC can’t be used to monitor the reaction). Into the probe (102 mg/mL) solution in pH 7.4 HEPES buffered water, Na2S (10 105 M) was added under stirring, 10 min later the water was evaporated under vacuum, and the solid residue (reaction product) was purified and then used for mass spectrometric measurement. The MALDI-TOF MS spectra for both the probe and the product are given in Fig. 3. It can be seen that, the molecular weight difference (w231) between the product and the corresponding probe equals the molecular weight of the 2,4dinitrobenzenesulfonyl group. This analysis strongly suggests that upon addition of Na2S, the 2,4-dinitrobenzenesulfonyl group is indeed cleaved from the probe and the fluorescein moiety is restored. For this sensing strategy, in the absence of the sulfide anion, the incorporation of 2,4-dinitrobenzenesulfonate ester group results in the quenching of fluorescence of the probe; while in the presence of sulfide anion, the cleavage of 2,4-dinitrobenzenesulfonyl will restore the fluorescein moiety, thereby leading to the fluorescence enhancement of the probe. 3.3. Selectivity and anti-interference performance of the sensing system To evaluate the selectivity of the probe towards sulfide anion, the fluorescence response of the probe in the presence of some
Scheme 1. Synthesis route for the probe.
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Fig. 2. (A) Fluorescence spectra of the probe (102 mg/mL) in the presence of different amounts of sulfide anion in pH 7.4 HEPES buffered water. (B) Fluorescence intensity of the probe (102 mg/mL) at 518 nm as a function of sulfide anion concentration in pH 7.4 HEPES buffered water. (lexc ¼ 490 nm) The spectra were recorded 10 min after addition of sulfide anion.
2 2 anions including F, Cl, Br, I, CO2 3 ; NO3 ; S2 O3 ; SO3 ; 2 HSO ; SO were recorded. And the comparison of the fluorescence 3 4 intensities at 518 nm is shown in Fig. 4A. It is obvious that, only sulfide anion elicits a significant fluorescence intensity enhancement, whereas the addition of other anions leads to almost no fluorescence change, even though the concentration of these anions is much higher than that of S2. These results confirm that the probe shows an excellent selectivity toward S2 over other competitive anion. This is probably because, sulfide anion is a highly effective nucleophile and reacts readily with sulfonate esters. On the other hand, the interference of the above species was also tested. Fig. 4B shows the fluorescence response of the probe towards S2 in the presence of other competitive anions. It is obvious that, generally the co-existence of these species does not interfere with the reaction of sulfide anion with the probe as well as the subsequent fluorescence enhancement. These results suggest that the probe can function as a highly selective probe for S2. Moreover, we also compared the fluorescence response of the probe over time in the presence of S2, three thiols (glutathione, cysteine or homocysteine), as shown in Fig. S4. It is clear that, in the presence of sulfide anion, the fluorescence intensity of the probe solution quickly reaches a plateau in about 10 min; while with the respective addition of the three thiols, the fluorescence intensity of the probe solution increases much slower, and will only reach the plateau after several hours. The results indicate that, the reaction between S2 and the probe is complete in about 10 min, which is much faster than that with the three thiols. This is why we choose 10 min as the reaction length for all fluorescence measurements. Compared to thiols, sulfide anion is a much more effective
nucleophile and reacts readily with sulfonate esters. Hence, the biologically-relevant thiols like glutathione (GSH), cysteine (Cys) and homocysteine (Hcy) react with sulfonate ester more slowly than sulfide. It can be seen from this figure that, as for GSH, even at the high concentration of 1 mM (ten-fold of that of sulfide anion), GSH still couldn’t lead to the fluorescence enhancement to the extent as sulfide does. Furthermore, we also investigated possible interference by other biologically relevant substances, including various amino acids and ascorbic acid, as shown in Fig. S5. It is clear that these biologically relevant compounds do not lead to any fluorescence change, even though the concentration of these anions is much higher than that of S2. This ensures that, the probe has quite good selectivity for sulfide anion over abundant biologically-relevant thiols and other compounds. 3.4. Sulfide anion sensing in running water To evaluate the efficacy of this probe’s application in real sample, the probe was applied to monitoring sulfide anion in running water. As shown in Table 1, this probe works quite well in running water with quite good recovery. The results given in Table 1 also show that the detected concentrations of the sulfide anion are in good agreement with those added in the samples. In addition, the composition of the running water does not significantly interfere with the detection of sulfide anion, indicating the capability of this probe for being used in water quality monitoring. According to World Health Organization, the maximum recommended sulfide concentration in drinking water is less than 500 mg/L (about
Fig. 3. Mass spectra of the as-prepared probe (A) and the purified reaction product upon addition of sulfide anion (B).
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Fig. 4. (A) Fluorescence intensities at 518 nm for the probe (102 mg/mL) in the presence of 15 mM S2 and 100 mM different anions respectively (lexc ¼ 490 nm). (B) Fluorescence intensities at 518 nm for the probe (102 mg/mL) in the presence of 15 mM of S2 and with the addition of 100 mM of different anions (lexc ¼ 490 nm). All fluorescence intensities were measured in pH 7.4 HEPES buffered water and 10 min after the addition of anions.
15 mM);7 hence with the detection limit of 0.15 mM, this probe can be used in the detection for sulfide anion in water quality monitoring. This probe is mainly intended to be used for detecting sulfide levels in running water, and the only possible interferent for the sensing e biothiols are rarely found in ground water; hence the sensor has the potential for being used in water quality monitoring. 3.5. Imaging of sulfide anion in live cells To investigate the biocompatibility of the probe, the cytotoxicity of the probe was evaluated using L929 cell line by MTT assay in compliance with ISO 10993-5. And the results are shown in Fig. 5. The probe shows a cell viability of 98% at the test concentration of 102 mg/mL. Even at the concentration of 5 102 mg/mL, the cell viability can still reach 94%. This indicates that the probe generally has no cytotoxicity, probably due to the incorporation of the biocompatible PEG. The feasibility of the probe for fluorescence imaging of sulfide anion in the living cells was also investigated. The favorable features of the probe include excitation and emission in the visible region, a significant fluorescence turn-on signal, high sensitivity, high selectivity, low cytotoxicity, and functioning well at physiological pH. These desirable characters are conducive to imaging S2 in living cells. Hence, we explored the capability of the probe to track sulfide level in two commonly-used cell lines e Hela (human cervical cancer cell) and L929 (murine aneuploid fibrosarcoma cell). To exclude the effects of any endogenous biothiols, N-methylmaleimide, a known trapping reagent of thiol species, was incubated with the cells before the cells were treated with the sensor. Fig. 6 and Fig. S6 display the fluorescent microscope images for the two kinds of cells incubated with the probe before and after being treated with different concentrations of S2. Fluorescence
microscope images of both Hela and L929 live cells loaded with the probe for 2 h at 37 C show no fluorescence intracellularly, as shown in Fig. 6A and D. The control experiment on cells without the probe gives no fluorescence either under the same exposure condition. By contrast, the intracellular fluorescence for the probestained cells exposed to 2 mM and 8 mM of S2 for 30 min at 37 C gives rise to bright green fluorescence, as shown in Fig. 6B, C, E and F (The concentrations (2 mM or 8 mM) were not the concentrations for the intracellular sulfide, but those for the sulfide donor in the culture media. Generally, only a small portion of sulfide donor can be internalized by the cells). These data establish that the probe is cell membrane permeable and is able to respond to sulfide anion inside living cells, as well as can be utilized to track sulfide level change inside live cells. 4. Conclusion In summary, we have successfully constructed a water-soluble and sensitive fluorescent turn-on sensing system for sulfide anion, which is usable in totally aqueous media. The sensing system features simplicity in synthesis, fast-responding, sensitive and selective detection of sulfide anion. Furthermore, the sensing system has good cell membrane permeability as well as very low cytotoxicity. The incorporation of 2,4-dinitrobenzenesulfonate ester group onto fluorescein can effectively quench the fluorescence of the fluorophore; while upon addition of sulfide anion, the sulfidemediated cleavage of 2,4-dinitrobenzenesulfonyl group occurs, resulting in the recovery of the strong fluorescence of the
Table 1 Determination of sulfide anion in running water. Samples
Amount of sulfide anion (mol L1)
Running water
0 1.00 1.50 2.00 2.50 3.00
Added
Recovery (%)
Found 105 106 105 105 105
e 1.02 1.55 2.03 2.57 3.11
105 105 105 105 105
e 102 103.3 101.5 102.8 103.7
In the test solutions, running water was 10-fold diluted. The measurements were conducted 10 min after the addition of sulfide.
Fig. 5. Cytotoxic effects against L929 cells upon 24 h of incubation. Control: L929 cells in the absence of the probe; others: L929 cells in the presence of the probe (102 mg/ mL, 2 102 mg/mL, 5 102 mg/mL respectively).
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Fig. 6. Fluorescence imaging of HeLa and L929 cells incubated with the probe before (A and D) and after (B and E: 2 mM S2; C and F: 8 mM S2) being treated with sulfide.
fluorescein moiety. Moreover, the sensing system can be used for imaging sulfide anion inside live cells. And this probe exhibits a low detection limit of 0.15 mM. This approach offers some useful insights for realizing technically-simple fluorescent turn-on sensing in the detection assays for other analytes. Acknowledgment This work was supported by the National Key Basic Research Program of China (Project No. 2013CB834702) and NSFC (Project No. 21025415, 21174040). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.polymer.2013.08.021. References [1] de Silva AP, Gunaratne HQN, Gunnlaugsson T, Huxley AJM, McCoy CP, Rademacher JT, et al. Chem Rev 1997;97:1515e66. [2] Esteban-Gomez D, Platas-Iglesias C, de Blas A, Fabbrizzi L, Rodriguez-Blas T. Chem Eur J 2008;14:5829e38. [3] Martínez-Manez R, Sancenon F. Chem Rev 2003;103:4419e76. [4] Hydrogen sulfide (environmental health criteria, no. 19). Geneva: World Health Organization; 1981. [5] Gosselin RE, Smith RP, Hodge HC, Braddock J. In: Clinical toxicology of commercial products. 5th ed. Baltimore, MD: Williams and Wilkins; 1984III-198e 202. [6] Patwardhan SA, Abhyankar SM. Colourage 1988;35:15e8. [7] WHO. Guidelines for drinking-water quality. 2nd ed. Geneva: World Health Organization; 1996. [8] Meyer B, Ward K, Koshlap K, Peter L. Inorg Chem 1983;22:2345e50. [9] Li L, Rose P, Moore PK. Annu Rev Pharmacol Toxicol 2011;51:169e87. [10] Boehning D, Snyder SH. Annu Rev Neurosci 2003;26:105e31. [11] Eto K, Asada T, Arima K, Makifuchi T, Kimura H. Biochem Biophys Res Commun 2002;293:1485e8. [12] Kamoun P, Belardinelli MC, Chabli A, Lallouchi K, Chadefaux-Vekemans B. Am J Med Genet 2003;116:310e1. [13] Balasubramanian S, Pugalenthi V. Wat Res 2000;34:4201e6. [14] Bings NH, Bogaerts A, Broekaert JAC. Anal Chem 2008;80:4317e47.
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Please cite this article in press as: Zheng F, et al., A water-soluble, low-cytotoxic and sensitive fluorescent probe based on poly(ethylene glycol) for detecting sulfide anion in aqueous media and imaging inside live cells, Polymer (2013), http://dx.doi.org/10.1016/j.polymer.2013.08.021