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Reversible “turn-off-on” fluorescence response of Fe(III) towards Rhodamine B based probe in vivo and plant tissues
Accepted Manuscript Reversible “turn-off-on” fluorescence response of Fe(III) towards Rhodamine B based probe in vivo and plant tissues Fan Song, Xiaotao Shao, Jing Zhu, Xiaofeng Bao, Lei Du, Chun Kan PII: DOI: Reference:
S0040-4039(19)30362-4 https://doi.org/10.1016/j.tetlet.2019.04.025 TETL 50740
To appear in:
Tetrahedron Letters
Received Date: Revised Date: Accepted Date:
12 January 2019 9 April 2019 13 April 2019
Please cite this article as: Song, F., Shao, X., Zhu, J., Bao, X., Du, L., Kan, C., Reversible “turn-off-on” fluorescence response of Fe(III) towards Rhodamine B based probe in vivo and plant tissues, Tetrahedron Letters (2019), doi: https://doi.org/10.1016/j.tetlet.2019.04.025
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Reversible “turn-off-on” fluorescence response of Fe(III) towards Rhodamine B based probe in vivo and plant tissues Fan Song a, Xiaotao Shao a, Jing Zhu b, Xiaofeng Bao c, Lei Du a, Chun Kan a,* a
College of Science, Nanjing Forestry University, 159 LongpanRoad, Nanjing 210037, China Department of Pharmacy, Jiangsu Key Laboratory for Pharmacology and Safety Evaluation of Chinese Materia Medica, Nanjing University of Chinese Medicine, 138 XianlinDadao, Nanjing 210023, China c School of Environmental and Biological Engineering, Jiangsu Key Laboratory of Chemical Pollution Control and Resources Reuse, Nanjing University of Science and Technology, 200 Xiaolinwei, Nanjing 210094, China b
ABSTRACT A Fe3+-specific probe (N-TC) based on Rhodamine B was designed and synthesized. N-TC has a good spectral response to Fe3+ in the EtOH/H2O solution (1:1, v/v, HEPES, 0.5mM, pH=7.38) with low detection limits and high binding constants. N-TC displays the reversible “turn-off-on” fluorescence response with 1:1 binding stoichiometry. It is further proven to be practical in sensitively monitoring trace Fe3+ in environmental water specimens. Biological experiments demonstrated that N-TC can be respectively used as a probe for detection of Fe3+ in living cells，animals and plant tissues.
Keywords: Fluorescent probe; Water sample; Imaging; In vivo
Introduction Metal ions are found everywhere in nature and play a key role in maintaining the physiological balance of living organisms [1-3]. As a result of human production and activities, a large amount of metal ions is generated. Thus, the atmosphere, water and soil in the environment begin to be seriously polluted, which in turn poses a serious hazard to the health of the organism [4-11]. Therefore, designing a chemical sensor that can detect metal ions is imminent. Because of its excellent light stability and relatively large molar absorption coefficient, Rhodamine B has become an important material for designing fluorescent probes and plays an extremely important role in the detection of metal ions in the environment. In addition, Rhodamine B also possesses excellent biocompatibility and permeability, as well as low cytotoxicity, allowing its detection range to extend from the living organism to the organism [12-16]. Now Rhodamine B-type probes have been widely used in medical diagnostics [17,18], environmental chemistry [19], materials science and other fields [20-26]. Iron is abundant metal elements in the earth and plays an important role in the development of 1
modern society [27-30]. More importantly, iron is the most important more than 10 kinds of trace elements necessary for the human body in. Iron is widely distributed in the human body and is found in almost all organs, including blood, muscle, spleen, liver and bone marrow [31-37]. Iron is an important component of hemoglobin, myoglobin and various enzymes. It is also an essential trace element in human physiological processes, such as cell metabolism, oxygen metabolism, electron transfer and DNA and RNA synthesis, proton transfer, maintenance of cell osmotic pressure and acid-base balance in the regulation of physiological activity [38-44]. When the body iron content deviates from normal or abnormal distribution, human health will be endangered. Iron deficiency or excessive iron content in the human body can impair normal physiological function [45-48]. Therefore, real-time detection of iron ions plays an important role in biological and human health. Herein, we designed a reversible “turn-off-on” fluorescent probes based on Rhodamine B for 3+ Fe , which was named N-TC. N-TC was successfully used as a selective and sensitive fluorescent and colorimetric probe for Fe3+. N-TC is further proven to be practical in sensitively monitoring trace Fe3+ in environmental water specimens. Most importantly, biological experiments showed that N-TC can be used as a fluorescent probe for the detection of Fe3+ in living cells, zebrafish and plant tissues.
Results and discussion Synthesis and characterization The N-TC synthesis procedure is illustrated in Scheme 1. The intermediates 1 was synthesized by using rhodamine B and ethylenediamine as reactants. Reaction of 1 with pyridine-3-sulfonyl chloride afforded the probe N-TC. The structures of compound 1 and N-TC were confirmed by 1H NMR, 13C NMR and ESI-MS (Supplementary Material). Taking into account the solubility of probe in component solvent system, EtOH/H2O (1:1, v/v, HEPES, 0.5mM, pH=7.38) for N-TC in sensing Fe3+ was chosen as a test solution. N-TC was compared with Fe3+ by UV-vis absorption and fluorescence emission to get the detailed concentration of probes used in sensing Fe3+ was 20μM. Due to induction of probe by Fe3+, the structure of
spironolactam is constantly destroyed, the five-membered ring opens and produces strong fluorescence. Evaluation of pH response of N-TC by acid-base titration (Fig. 1A). The results show that N-TC was insensitive to pH from 6.0 to 12.0 in the EtOH/H2O (1:1, v/v, HEPES, 0.5mM, pH=7.38) solution and may have been able to sense Fe3+ under approximately physiological conditions with very low background fluorescence. Moreover, the response time of N-TC in sensing Fe3+ was investigated (Fig. 1B). In fluorescence emission spectra, with the increase of time, the fluorescence intensity of N-TC at 582 nm gradually increased and until maintained stable, which indicates that N-TC are sensitive probes for Fe3+. In order to further understand the interaction between N-TC and Fe3+, the Fe3+ titration against N-TC was also monitored by the fluorescence spectra (Fig. S5). In absence of Fe3+, N-TC had no clear 2
fluorescent intensity at 582 nm (λex = 560nm). With the increasing concentration of Fe3+, the fluorescence is obviously enhanced. The measurement solution underwent color change from colorlessness to the light pink and reach saturation thereafter. The change of N-TC towards Fe3+ can be ascribed to the formation of the ring-opened amide form of N-TC upon Fe3+ binding. Thus, N-TC can act as quantitative determination methods of measuring Fe3+ concentrations. The association constant Ka of N-TC/Fe3+ complex could be calculated based on the revised Benesi-Hildebrand equation as 1.15×104 M-1 (Fig. S6A). On basis of fluorescence titration, the detection limit of N-TC for Fe3+ also could be determined to be 0.201μM (Fig. S6B). Thus, N-TC has higher association constant and lower detection limit.
Scheme 1. Synthesis of N-TC.
Fig. 1. (A) Effect of pH on the fluorescence of N-TC (20μM) in EtOH/H2O (1:1, v/v, HEPES, 0.5mM, pH=7.38) in the absence and presence of Fe3+ (100μM). (B) Fluorescence spectra of N-TC (20μM) with Fe3+ (100μM) in EtOH/H2O (1:1, v/v, HEPES, 0.5mM, pH=7.38) solution. The excitation and emission wavelengths were 560 nm and 582 nm, respectively. Inset: (B) Plot of the fluorescence intensities at 582 nm over a period of 100 min. Ions selectivity To evaluate the selectivity of N-TC, common metal cations (Fe3+, Fe2+, Al3+, Ba2+, Ca2+, Co2+, Cd2+, Cr3+, Cu2+, Hg2+, Mg2+, Mn2+, Na+, Ni2+, Pb2+ and Zn2+) was tested in aqueous solutions by UV–vis absorption spectroscopy and fluorescence spectrometry. The UV–vis absorption spectroscopy of N-TC showed best strongest absorption behavior to Fe3+ along with significant color changes of test solution (Fig. 2A). However, under the same condition, no significant response was observed after other metal ions were implanted. As far as N-TC is concerned, only 3
absorption behavior to Al3+ of other cations induced a much weaker change than Fe3+. Without the addition of metal ions in the fluorescence spectrum, N-TC displayed no obvious fluorescence and showed the form of spirolactam in the experimental system. When Fe3+ was added, the fluorescence of N-TC was significantly enhanced. When other metal ions were added respectively, no obvious fluorescence enhancement was revealed. Only other few cations (Al3+) induced a much weaker fluorescent intensity change than Fe3+ (Fig. 2B). The results indicated that the interaction between N-TC and Fe3+ resulted from the formation of the strongly fluorescent ring-opened N-TC- Fe3+ complex. Thus, N-TC had distinctive selectivity to Fe3+ and N-TC could be served as highly specific-Fe3+ sensing probe. To further confirm the selectivity of N-TC to Fe3+, the fluorescence competition experiments were also conducted by additionally mixing Fe3+ to N-TC solution in presence of other metal ions (Fig. S7). No significant variation in the emission of the N-TC-Fe3+ complex was observed when compared with the results obtained with or without other metal ions. The detection of Fe 3+ is not interfered with other metal ions. The above results show that N-TC could be used as selective Fe3+ fluorescent probe. We further studied the effects of various anions on the rupture of the N-TC-Fe3+ complex to regenerate N-TC. Fluorescence spectroscopic experiments of the N-TC-Fe3+ complex was performed in the presence of different anions such as SO42-, Br-, BrO3-, B4O72-, F-, H2PO4-, NO3-, S2O82- and IO3- (Fig. S8). The results showed that the fluorescence intensity of the N-TC-Fe3+ complex didn’t change significantly after adding various anions to the solution containing N-TC-Fe3+ complex, thus these anions failed to produce any discernible spectral change under the identical conditions. The above conclusion indicates that the N-TC-Fe3+ complex can still exist stably when anions are present.
Fig. 2. (A) UV–vis absorbance spectra of N-TC (20μM) in EtOH/H2O (1:1, v/v, HEPES, 0.5mM, pH=7.38) in the absence and presence of 5 equiv. of various metal ions: Fe3+, Fe2+, Al3+, Ba2+, Ca2+, Co2+, Cd2+, Cr3+, Cu2+, Hg2+, Mg2+, Mn2+, Na+, Ni2+, Pb2+ and Zn2+. The absorption wavelength of N-TC was 558 nm. (B) Fluorescence spectra of N-TC (20μM) in EtOH/H2O (1:1, v/v, HEPES, 0.5mM, pH=7.38) in the absence and presence of 5 equiv. various metal ions. The excitation and emission wavelengths were 560 nm and 582 nm, respectively. 4
Proposed sensing mechanism The reversibility between N-TC and Fe3+ was determined with the Na4P2O7-adding experiment by UV-vis (Fig. 3A) and fluorescence method (Fig. 3B). The addition of Na4P2O7 can restore the color and fluorescence to the initial state of N-TC. Then, an excess of Fe3+ is respectively added to the above solution again, and N-TC would return to the “On-state” in both absorption and fluorescence signals. Re-titrating both N-TC with Fe3+ and Na4P2O7 for four cycles could still obtained good “Off-On” fluorescence signal response, which indicated that the sensing process between N-TC and Fe3+ was reversible. The experiment of the relationship between reversibility and pH was also tested. It can be seen that pH is less than 9 all the time after N-TC with Fe3+ and Na4P2O7 was retitrated for four cycles. According to Fig. 1A, the fluorescence intensity of its complex is strong and can remain stable at pH<9 after the addition of Fe3+. Thus, the measured experimental results are effective. Job’s plot experiments were conducted that the maximum value of absorption of N-TC/Fe3+ appeared at 0.5 (Fig. S9). Thus, the binding stoichiometry between N-TC and Fe3+ was 1:1 in EtOH/H2O (1:1, v/v, HEPES, 0.5mM, pH=7.38). 1 H NMR titration experiments were carried out in CD3OD:D2O (1:1, v/v), Fe3+ and their complex with N-TC can affect the proton signals that are close to the Fe3+ binding site. As shown in Fig. S10, the proton signals of He, Hf, Hg displayed apparent downfield shifts due to the Fe3+ ion induced ring-opening process of the rhodamine B spirocycle. The peaks of Hh and Hi protons also show significant shifts. This change is due to the coordination of N-TC with Fe3+. The hydrogen on pyridine ring also shifts to a certain extent, and the peak pattern changes from multiple peaks to a wide single peak. Thus, we coordinated the N atom on the pyridine ring with Fe3+. In this way, a stable six-membered ring and a seven-membered ring can be formed. From above conclusions, a stable sensing mechanism of N-TC-Fe3+ could be summarized as Scheme 2. To further confirm the mechanism, the ESI–MS result of the N-TC/Fe3+ species was also provided (Fig. S11).
Fig. 3. Reversibility of Fe3+ to N-TC by Na4P2O7. Examined (A) N-TC by UV-vis absorption titration. Pink line: free probe (20μM), purple line: probe + Fe3+ + Na4P2O7 (20μM), black line: probe + Fe3+ + Na4P2O7 + Fe3+. (B) Retitrated N-TC with Fe3+ and Na4P2O7 for four cycles by 5
fluorescence method and the effect of pH on retitrating N-TC with Fe3+ and Na4P2O7 for four cycles.
Scheme 2. Proposed sensing mechanism of N-TC with Fe3+. Performance in environmental water samples Samples of pure water and lake water were both collected to study the performance of N-TC in the detection of Fe3+ in environmental water samples. To determine the recovery of Fe3+, the linear plotting of fluorescence intensity of N-TC as a function of Fe3+ concentration (0~200μΜ) in pure water was firstly carried out. (Fig. 4A). The lake specimens were spiked with Fe3+ of different concentration, and the recovered Fe3+ concentrations were measured by fluorescent method (red for Zixi Lake or green for Zixia Lake) (Fig. 4B). Considering the complexity of the environmental water sample, the recovery of Fe3+ obtained by N-TC was little bit lower than that in pure water, but both still keep high recovery, which indicated that N-TC can be used for monitoring trace Fe3+ in environmental water.
Fig. 4. (A) Plotting the fluorescence spectra of N-TC (20μM) as a function of Fe3+ concentration (0~200μM) in pure water. (B) Recovered Fe3+ concentration in Zixi Lake and Zixia Lake determined by the low-concentration linear plotting of N-TC. Cell imaging N-TC and its complex with Fe3+ remain stable in the range of 6~12 pH wide, which implied that N-TC would have the potential to visualize Fe3+ in living cells. MTT assay was used to 6
detect the cytotoxicity of N-TC in MCF-7 cells (Fig. S12). The result of MTT test showed that N-TC had no cytotoxicity at low concentrations, but had a relatively high cytotoxicity when the concentration of N-TC reached 10μM and still could have more than 80% cell viability. Owing to their favorable molecular properties, N-TC should be suited for fluorescence imaging in living cells. According to MTT test, 10μM was then chosen as the suitable concentration for N-TC in the further confocal fluorescence imaging experiment. After culturing with N-TC, no fluorescence was observed by confocal fluorescence microscopy from the red channel (Fig. 5C1). Remarkable red fluorescence was observed in N-TC medium, when 20μM Fe3+ were extra added (Fig. 5C2). Fluorescence captured from the blue channel implied that MCF-7 cells still stayed in good cell states after staining with N-TC (Fig. 5B). The merged images indicated that the areas of red fluorescence and blue fluorescence could complement with each other, and also revealed that Fe3+ mainly existed in the cytoplasm of the cells sensing by N-TC (Fig. 5D). The bright-field images confirmed that the cells were viable throughout the imaging experiments (Fig. 5A). Thus, N-TC had good cell-membrane permeability and could both be applied in fluorescence imaging Fe3+ in living cells.
Fig. 5. Confocal fluorescence images of N-TC in MCF-7 cells. Cells pretreated with N-TC (10μM) incubated with Fe3+ (20μM) for 2h. (A) bright field, (B) blue channel, (C) red channel, (D) the overlay images. Scale bar: 50μM. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) Animal and plant tissue imaging Zebrafish is a highly valuable model for in vivo imaging. We then examined the feasibility of detecting Fe3+ by N-TC in zebrafish. As shown in Fig. 6, the zebrafish itself did not show fluorescence, and there is almost no fluorescence after incubation for 30 min in an aqueous solution containing N-TC. Then, significant fluorescence was observed after incubation of zebrafish for 30 min with Fe3+. These results indicate that N-TC can detect Fe3+ in living zebrafish. In plant tissue imaging, soybean root samples showed low fluorescence before 7
immersion in N-TC. After soaking with N-TC for 30 min, the samples of soybean root showed obvious red fluorescence (Fig. 7). All the results proved histocompatibility of N-TC which could be applied to detect Fe3+ in plant tissues.
Fig. 6. Fluorescence images of Fe3+ in zebrafish using N-TC (λex=560 nm). (1) Merged image of zebrafish incubated with probe (20μM) for 30 min, (2) merged image of probe-loaded zebrafish incubated with Fe3+ (20μM) for 30 min, (A) bright field, (B) fluorescent images, (C) the overlay images.
Fig. 7. Fluorescence images of soybean root tissues treated with N-TC (λex=560 nm). (1) Merged image of soybean incubated with Fe3+ (20μM) for 30 min, (2) merged image of soybean roots incubated with probe (10μM) for 30 min, (A) bright field, (B) fluorescent images, (C) the overlay images.
Conclusion 8
In summary, we developed a new reversible “turn-off-on” fluorescence probes (N-TC) for sensing Fe3+ based on rhodamine B derivative. N-TC exhibited the excellent selectivity and sensitivity for Fe3+ in aqueous media. N-TC exhibited sensing mechanism towards Fe3+ with 1:1 binding stoichiometry. We found N-TC has higher association constant and lower detection limit. Moreover, N-TC was successfully applied in detecting trace Fe3+ in lake water with good recovery efficiency. Besides, Biological experiments demonstrated their potential value of applications in living cells, animals and plant tissues.
Experimental Materials and instruments For details, see the Supplementary Material. Compound 1 was synthesized in Supplementary Material. Synthesis of N-TC To a solution of compound 1 (160mg, 0.33mmol) in 30mL of dry CH2Cl2 was added pyridine-3-sulfonyl chloride (56mg, 0.32mmol) and triethylamine (40μL, 0.3mmol). The mixture was stirred at room temperature for 3h, and the solvent was removed in a vacuum. The residue was dissolved in CH2Cl2 and then washed with H2O. The organic phase was dried with Magnesium sulfate and then concentrated. The residue was purified by column chromatography using CH2Cl2/ethyl acetate (97:3, v/v) as an eluent to afford pink solid (104mg, 48%). 1H NMR (CDCl3), δ8.95 (d, J=2.1Hz, 1H), 8.72 (dd, J=5.0, 4.7Hz, 1H), 8.09 (m, 1H), 7.88 (dd, J=6.6, 5.3Hz, 1H), 7.46 (t, J1=4.7Hz, J2=3.9Hz, 2H), 7.36 (dd, J=8.0, 7.9Hz, 1H), 7.04 (dd, J=6.6, 5.1Hz, 1H), 6.46 (s, 1H), 6.35 (s, 2H), 6.19 (s, 4H), 3.33 (dd, J=7.0, 6.9Hz, 8H), 3.20 (t, J1=5.2Hz, J2=5.1Hz, 2H), 2.80 (t, J1=5.2Hz, J2=4.6Hz, 2H), 1.19 (m, 12H) ppm. 13C NMR (CDCl3), δ169.83, 153.43, 153.28, 152.83, 149.01, 148.06,136.94, 134.80, 133.02, 130.30, 128.37, 128.29, 123.93, 123.59, 122.93, 108.32, 104.28, 97.76, 65.72, 44.41, 43.93, 40.08, 29.73, 29.35, 12.59 ppm. ESI-MS (M+H)+ found, 626.27; calculated for C35H39N5O3S2, 625.79. Spectral properties test The stock solution of N-TC (1mM) was prepared in EtOH/H2O (1:1, v/v, HEPES, 0.5mM, pH=7.38) solution. The stock solutions (1mM) of the chloride or nitrate salts of Fe3+, Fe2+, Al3+, Ba2+, Ca2+, Co2+, Cd2+, Cr3+, Cu2+, Hg2+, Mg2+, Mn2+, Na+, Ni2+, Pb2+ and Zn2+ in water were prepared as well. Working solutions of N-TC was freshly prepared by diluting the highly concentrated stock solution to the desired concentration prior to the spectroscopic measurements. In all of the spectroscopy experiments, the spectral data of N-TC were recorded 60 min after the addition of the ions. To investigate the metal ion selectivity, the test samples were prepared by placing 10 equiv. of the cation stock solution in 3 mL of the N-TC solution (20μM). For the 9
fluorescence measurements, excitation was provided at 560nm, and the emission was collected from 564 to 700nm. Na4P2O7 was respectively chosen as the chelating agent with Fe3+ to confirm the reversibility of N-TC in both UV–vis absorption and fluorescence methods. Pure water samples were used directly, and lake water samples were collected from Zixi Lake of Nanjing Forestry University and Zixia Lake of Nanjing University of Science and Technology which were passed through the microfiltration membrane before use. Cell culture and fluorescence imaging MCF-7 cells were purchased from the American type culture collection (ATCC) and cultured in Dulbecco’s modified Eagle’s medium (high glucose) supplemented with 10% FBS (fetal bovine serum), 100 units/mL penicillin, and 100 units/mL streptomycin in a 5% CO2/95% air incubator at 37℃. MCF-7 cells were seeded at 1.5×105 cells per well in 24-well flat-bottomed plates in an atmosphere of 5% CO2/95% air at 37℃ and incubated for 24h before treatment. For living cell imaging, cells were incubated with 10μM N-TC in culture medium for 5h in an air incubator at 37℃. In the control experiment, cells were pretreated with 100μM Fe3+ for 1h and then incubated with 10μM N-TC in culture medium for an additional 2h. Fluorescence imaging was performed after washing the cells with PBS 3 times and was recorded using the red channel, blue channel and merged channel under an inverted fluorescence microscope. Animal and plant tissue imaging Zebrafish was purchased from Kaiji Biotechnology Co., Ltd. (Nanjing, China) and respectively incubated in aqueous solution containing N-TC for 30 min and were imaged by confocal microscopy. After incubation with Fe3+ for 30 min, imaging was performed. Fluorescence imaging was recorded using the red channel and merged channel under a confocal microscopy. The seeds of soybean disinfected by 70% ethanol and 3% sodium hypochlorite solution were cultured on Petri dishes at room temperature and 70% relative humidity. The control group was supplied with distilled water; the experimental groups were treated with Fe3+ (20μM). After a week, the roots were collected and immersed in solution containing N-TC (10μM) for 30 min. After three times washing with PBS, the samples were imaged by confocal microscopy. The control group was incubated with 10μM probe N-TC for 30 min. Cytotoxicity assay The cytotoxicity of N-TC was evaluated by an MTT (3-(4,5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide) assay using MCF-7 cells. Cytotoxicity was determined by an MTT colorimetric method using four parallel wells at each time point. MCF-7 cells were cultured in 96-well plates at a density of 5×103 cells per well and were incubated at 37℃ in 5% CO2/95% air for 24h.Then, cells were incubated with different concentrations of N-TC (0-90μM) for 24h, followed by further incubation with 5 mg/mL of MTT for 4h at 37℃. The supernatant 10
was then discarded, and DMSO (150μL/well) was added to dissolve the formazan. Finally, the absorbance was measured at 550 nm with a microplate reader (Infinite M200 Pro). Acknowledgement This work was supported by the National Natural Science Foundation of China (50772048, 81371616), Jiangsu Six Talent Peak Award (2015SWYY013), Natural Science Fund for Colleges and Universities in Jiangsu Province (09KJD430007). This research was also supported by Key Project of Jiangsu Province for Fundamental Research and Development (BE2018717), Scientific Research Foundation of Nanjing Forestry University, Undergraduate Scientific and Technological Innovation Project of Nanjing Forestry University, Practical Innovation Training Program for College Students of Nanjing Forestry University (2018NFUSPITP582). We thank the testing instruments and technical support provided by Advanced analysis and testing center of Nanjing Forestry University.
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Highlights
A Fe3+-specific probe based on Rhodamine B was designed and synthesized.
The probe displays the reversible “turn-off-on” fluorescence change.
The probe is feasible for monitoring trace Fe3+ in environmental water samples.
The probe can be used for detection of Fe3+ in cells, animals and plant tissues.
14
Graphical Abstract
15
S0040-4039(19)30362-4 https://doi.org/10.1016/j.tetlet.2019.04.025 TETL 50740
To appear in:
Tetrahedron Letters
Received Date: Revised Date: Accepted Date:
12 January 2019 9 April 2019 13 April 2019
Please cite this article as: Song, F., Shao, X., Zhu, J., Bao, X., Du, L., Kan, C., Reversible “turn-off-on” fluorescence response of Fe(III) towards Rhodamine B based probe in vivo and plant tissues, Tetrahedron Letters (2019), doi: https://doi.org/10.1016/j.tetlet.2019.04.025
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Reversible “turn-off-on” fluorescence response of Fe(III) towards Rhodamine B based probe in vivo and plant tissues Fan Song a, Xiaotao Shao a, Jing Zhu b, Xiaofeng Bao c, Lei Du a, Chun Kan a,* a
College of Science, Nanjing Forestry University, 159 LongpanRoad, Nanjing 210037, China Department of Pharmacy, Jiangsu Key Laboratory for Pharmacology and Safety Evaluation of Chinese Materia Medica, Nanjing University of Chinese Medicine, 138 XianlinDadao, Nanjing 210023, China c School of Environmental and Biological Engineering, Jiangsu Key Laboratory of Chemical Pollution Control and Resources Reuse, Nanjing University of Science and Technology, 200 Xiaolinwei, Nanjing 210094, China b
ABSTRACT A Fe3+-specific probe (N-TC) based on Rhodamine B was designed and synthesized. N-TC has a good spectral response to Fe3+ in the EtOH/H2O solution (1:1, v/v, HEPES, 0.5mM, pH=7.38) with low detection limits and high binding constants. N-TC displays the reversible “turn-off-on” fluorescence response with 1:1 binding stoichiometry. It is further proven to be practical in sensitively monitoring trace Fe3+ in environmental water specimens. Biological experiments demonstrated that N-TC can be respectively used as a probe for detection of Fe3+ in living cells，animals and plant tissues.
Keywords: Fluorescent probe; Water sample; Imaging; In vivo
Introduction Metal ions are found everywhere in nature and play a key role in maintaining the physiological balance of living organisms [1-3]. As a result of human production and activities, a large amount of metal ions is generated. Thus, the atmosphere, water and soil in the environment begin to be seriously polluted, which in turn poses a serious hazard to the health of the organism [4-11]. Therefore, designing a chemical sensor that can detect metal ions is imminent. Because of its excellent light stability and relatively large molar absorption coefficient, Rhodamine B has become an important material for designing fluorescent probes and plays an extremely important role in the detection of metal ions in the environment. In addition, Rhodamine B also possesses excellent biocompatibility and permeability, as well as low cytotoxicity, allowing its detection range to extend from the living organism to the organism [12-16]. Now Rhodamine B-type probes have been widely used in medical diagnostics [17,18], environmental chemistry [19], materials science and other fields [20-26]. Iron is abundant metal elements in the earth and plays an important role in the development of 1
modern society [27-30]. More importantly, iron is the most important more than 10 kinds of trace elements necessary for the human body in. Iron is widely distributed in the human body and is found in almost all organs, including blood, muscle, spleen, liver and bone marrow [31-37]. Iron is an important component of hemoglobin, myoglobin and various enzymes. It is also an essential trace element in human physiological processes, such as cell metabolism, oxygen metabolism, electron transfer and DNA and RNA synthesis, proton transfer, maintenance of cell osmotic pressure and acid-base balance in the regulation of physiological activity [38-44]. When the body iron content deviates from normal or abnormal distribution, human health will be endangered. Iron deficiency or excessive iron content in the human body can impair normal physiological function [45-48]. Therefore, real-time detection of iron ions plays an important role in biological and human health. Herein, we designed a reversible “turn-off-on” fluorescent probes based on Rhodamine B for 3+ Fe , which was named N-TC. N-TC was successfully used as a selective and sensitive fluorescent and colorimetric probe for Fe3+. N-TC is further proven to be practical in sensitively monitoring trace Fe3+ in environmental water specimens. Most importantly, biological experiments showed that N-TC can be used as a fluorescent probe for the detection of Fe3+ in living cells, zebrafish and plant tissues.
Results and discussion Synthesis and characterization The N-TC synthesis procedure is illustrated in Scheme 1. The intermediates 1 was synthesized by using rhodamine B and ethylenediamine as reactants. Reaction of 1 with pyridine-3-sulfonyl chloride afforded the probe N-TC. The structures of compound 1 and N-TC were confirmed by 1H NMR, 13C NMR and ESI-MS (Supplementary Material). Taking into account the solubility of probe in component solvent system, EtOH/H2O (1:1, v/v, HEPES, 0.5mM, pH=7.38) for N-TC in sensing Fe3+ was chosen as a test solution. N-TC was compared with Fe3+ by UV-vis absorption and fluorescence emission to get the detailed concentration of probes used in sensing Fe3+ was 20μM. Due to induction of probe by Fe3+, the structure of
spironolactam is constantly destroyed, the five-membered ring opens and produces strong fluorescence. Evaluation of pH response of N-TC by acid-base titration (Fig. 1A). The results show that N-TC was insensitive to pH from 6.0 to 12.0 in the EtOH/H2O (1:1, v/v, HEPES, 0.5mM, pH=7.38) solution and may have been able to sense Fe3+ under approximately physiological conditions with very low background fluorescence. Moreover, the response time of N-TC in sensing Fe3+ was investigated (Fig. 1B). In fluorescence emission spectra, with the increase of time, the fluorescence intensity of N-TC at 582 nm gradually increased and until maintained stable, which indicates that N-TC are sensitive probes for Fe3+. In order to further understand the interaction between N-TC and Fe3+, the Fe3+ titration against N-TC was also monitored by the fluorescence spectra (Fig. S5). In absence of Fe3+, N-TC had no clear 2
fluorescent intensity at 582 nm (λex = 560nm). With the increasing concentration of Fe3+, the fluorescence is obviously enhanced. The measurement solution underwent color change from colorlessness to the light pink and reach saturation thereafter. The change of N-TC towards Fe3+ can be ascribed to the formation of the ring-opened amide form of N-TC upon Fe3+ binding. Thus, N-TC can act as quantitative determination methods of measuring Fe3+ concentrations. The association constant Ka of N-TC/Fe3+ complex could be calculated based on the revised Benesi-Hildebrand equation as 1.15×104 M-1 (Fig. S6A). On basis of fluorescence titration, the detection limit of N-TC for Fe3+ also could be determined to be 0.201μM (Fig. S6B). Thus, N-TC has higher association constant and lower detection limit.
Scheme 1. Synthesis of N-TC.
Fig. 1. (A) Effect of pH on the fluorescence of N-TC (20μM) in EtOH/H2O (1:1, v/v, HEPES, 0.5mM, pH=7.38) in the absence and presence of Fe3+ (100μM). (B) Fluorescence spectra of N-TC (20μM) with Fe3+ (100μM) in EtOH/H2O (1:1, v/v, HEPES, 0.5mM, pH=7.38) solution. The excitation and emission wavelengths were 560 nm and 582 nm, respectively. Inset: (B) Plot of the fluorescence intensities at 582 nm over a period of 100 min. Ions selectivity To evaluate the selectivity of N-TC, common metal cations (Fe3+, Fe2+, Al3+, Ba2+, Ca2+, Co2+, Cd2+, Cr3+, Cu2+, Hg2+, Mg2+, Mn2+, Na+, Ni2+, Pb2+ and Zn2+) was tested in aqueous solutions by UV–vis absorption spectroscopy and fluorescence spectrometry. The UV–vis absorption spectroscopy of N-TC showed best strongest absorption behavior to Fe3+ along with significant color changes of test solution (Fig. 2A). However, under the same condition, no significant response was observed after other metal ions were implanted. As far as N-TC is concerned, only 3
absorption behavior to Al3+ of other cations induced a much weaker change than Fe3+. Without the addition of metal ions in the fluorescence spectrum, N-TC displayed no obvious fluorescence and showed the form of spirolactam in the experimental system. When Fe3+ was added, the fluorescence of N-TC was significantly enhanced. When other metal ions were added respectively, no obvious fluorescence enhancement was revealed. Only other few cations (Al3+) induced a much weaker fluorescent intensity change than Fe3+ (Fig. 2B). The results indicated that the interaction between N-TC and Fe3+ resulted from the formation of the strongly fluorescent ring-opened N-TC- Fe3+ complex. Thus, N-TC had distinctive selectivity to Fe3+ and N-TC could be served as highly specific-Fe3+ sensing probe. To further confirm the selectivity of N-TC to Fe3+, the fluorescence competition experiments were also conducted by additionally mixing Fe3+ to N-TC solution in presence of other metal ions (Fig. S7). No significant variation in the emission of the N-TC-Fe3+ complex was observed when compared with the results obtained with or without other metal ions. The detection of Fe 3+ is not interfered with other metal ions. The above results show that N-TC could be used as selective Fe3+ fluorescent probe. We further studied the effects of various anions on the rupture of the N-TC-Fe3+ complex to regenerate N-TC. Fluorescence spectroscopic experiments of the N-TC-Fe3+ complex was performed in the presence of different anions such as SO42-, Br-, BrO3-, B4O72-, F-, H2PO4-, NO3-, S2O82- and IO3- (Fig. S8). The results showed that the fluorescence intensity of the N-TC-Fe3+ complex didn’t change significantly after adding various anions to the solution containing N-TC-Fe3+ complex, thus these anions failed to produce any discernible spectral change under the identical conditions. The above conclusion indicates that the N-TC-Fe3+ complex can still exist stably when anions are present.
Fig. 2. (A) UV–vis absorbance spectra of N-TC (20μM) in EtOH/H2O (1:1, v/v, HEPES, 0.5mM, pH=7.38) in the absence and presence of 5 equiv. of various metal ions: Fe3+, Fe2+, Al3+, Ba2+, Ca2+, Co2+, Cd2+, Cr3+, Cu2+, Hg2+, Mg2+, Mn2+, Na+, Ni2+, Pb2+ and Zn2+. The absorption wavelength of N-TC was 558 nm. (B) Fluorescence spectra of N-TC (20μM) in EtOH/H2O (1:1, v/v, HEPES, 0.5mM, pH=7.38) in the absence and presence of 5 equiv. various metal ions. The excitation and emission wavelengths were 560 nm and 582 nm, respectively. 4
Proposed sensing mechanism The reversibility between N-TC and Fe3+ was determined with the Na4P2O7-adding experiment by UV-vis (Fig. 3A) and fluorescence method (Fig. 3B). The addition of Na4P2O7 can restore the color and fluorescence to the initial state of N-TC. Then, an excess of Fe3+ is respectively added to the above solution again, and N-TC would return to the “On-state” in both absorption and fluorescence signals. Re-titrating both N-TC with Fe3+ and Na4P2O7 for four cycles could still obtained good “Off-On” fluorescence signal response, which indicated that the sensing process between N-TC and Fe3+ was reversible. The experiment of the relationship between reversibility and pH was also tested. It can be seen that pH is less than 9 all the time after N-TC with Fe3+ and Na4P2O7 was retitrated for four cycles. According to Fig. 1A, the fluorescence intensity of its complex is strong and can remain stable at pH<9 after the addition of Fe3+. Thus, the measured experimental results are effective. Job’s plot experiments were conducted that the maximum value of absorption of N-TC/Fe3+ appeared at 0.5 (Fig. S9). Thus, the binding stoichiometry between N-TC and Fe3+ was 1:1 in EtOH/H2O (1:1, v/v, HEPES, 0.5mM, pH=7.38). 1 H NMR titration experiments were carried out in CD3OD:D2O (1:1, v/v), Fe3+ and their complex with N-TC can affect the proton signals that are close to the Fe3+ binding site. As shown in Fig. S10, the proton signals of He, Hf, Hg displayed apparent downfield shifts due to the Fe3+ ion induced ring-opening process of the rhodamine B spirocycle. The peaks of Hh and Hi protons also show significant shifts. This change is due to the coordination of N-TC with Fe3+. The hydrogen on pyridine ring also shifts to a certain extent, and the peak pattern changes from multiple peaks to a wide single peak. Thus, we coordinated the N atom on the pyridine ring with Fe3+. In this way, a stable six-membered ring and a seven-membered ring can be formed. From above conclusions, a stable sensing mechanism of N-TC-Fe3+ could be summarized as Scheme 2. To further confirm the mechanism, the ESI–MS result of the N-TC/Fe3+ species was also provided (Fig. S11).
Fig. 3. Reversibility of Fe3+ to N-TC by Na4P2O7. Examined (A) N-TC by UV-vis absorption titration. Pink line: free probe (20μM), purple line: probe + Fe3+ + Na4P2O7 (20μM), black line: probe + Fe3+ + Na4P2O7 + Fe3+. (B) Retitrated N-TC with Fe3+ and Na4P2O7 for four cycles by 5
fluorescence method and the effect of pH on retitrating N-TC with Fe3+ and Na4P2O7 for four cycles.
Scheme 2. Proposed sensing mechanism of N-TC with Fe3+. Performance in environmental water samples Samples of pure water and lake water were both collected to study the performance of N-TC in the detection of Fe3+ in environmental water samples. To determine the recovery of Fe3+, the linear plotting of fluorescence intensity of N-TC as a function of Fe3+ concentration (0~200μΜ) in pure water was firstly carried out. (Fig. 4A). The lake specimens were spiked with Fe3+ of different concentration, and the recovered Fe3+ concentrations were measured by fluorescent method (red for Zixi Lake or green for Zixia Lake) (Fig. 4B). Considering the complexity of the environmental water sample, the recovery of Fe3+ obtained by N-TC was little bit lower than that in pure water, but both still keep high recovery, which indicated that N-TC can be used for monitoring trace Fe3+ in environmental water.
Fig. 4. (A) Plotting the fluorescence spectra of N-TC (20μM) as a function of Fe3+ concentration (0~200μM) in pure water. (B) Recovered Fe3+ concentration in Zixi Lake and Zixia Lake determined by the low-concentration linear plotting of N-TC. Cell imaging N-TC and its complex with Fe3+ remain stable in the range of 6~12 pH wide, which implied that N-TC would have the potential to visualize Fe3+ in living cells. MTT assay was used to 6
detect the cytotoxicity of N-TC in MCF-7 cells (Fig. S12). The result of MTT test showed that N-TC had no cytotoxicity at low concentrations, but had a relatively high cytotoxicity when the concentration of N-TC reached 10μM and still could have more than 80% cell viability. Owing to their favorable molecular properties, N-TC should be suited for fluorescence imaging in living cells. According to MTT test, 10μM was then chosen as the suitable concentration for N-TC in the further confocal fluorescence imaging experiment. After culturing with N-TC, no fluorescence was observed by confocal fluorescence microscopy from the red channel (Fig. 5C1). Remarkable red fluorescence was observed in N-TC medium, when 20μM Fe3+ were extra added (Fig. 5C2). Fluorescence captured from the blue channel implied that MCF-7 cells still stayed in good cell states after staining with N-TC (Fig. 5B). The merged images indicated that the areas of red fluorescence and blue fluorescence could complement with each other, and also revealed that Fe3+ mainly existed in the cytoplasm of the cells sensing by N-TC (Fig. 5D). The bright-field images confirmed that the cells were viable throughout the imaging experiments (Fig. 5A). Thus, N-TC had good cell-membrane permeability and could both be applied in fluorescence imaging Fe3+ in living cells.
Fig. 5. Confocal fluorescence images of N-TC in MCF-7 cells. Cells pretreated with N-TC (10μM) incubated with Fe3+ (20μM) for 2h. (A) bright field, (B) blue channel, (C) red channel, (D) the overlay images. Scale bar: 50μM. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) Animal and plant tissue imaging Zebrafish is a highly valuable model for in vivo imaging. We then examined the feasibility of detecting Fe3+ by N-TC in zebrafish. As shown in Fig. 6, the zebrafish itself did not show fluorescence, and there is almost no fluorescence after incubation for 30 min in an aqueous solution containing N-TC. Then, significant fluorescence was observed after incubation of zebrafish for 30 min with Fe3+. These results indicate that N-TC can detect Fe3+ in living zebrafish. In plant tissue imaging, soybean root samples showed low fluorescence before 7
immersion in N-TC. After soaking with N-TC for 30 min, the samples of soybean root showed obvious red fluorescence (Fig. 7). All the results proved histocompatibility of N-TC which could be applied to detect Fe3+ in plant tissues.
Fig. 6. Fluorescence images of Fe3+ in zebrafish using N-TC (λex=560 nm). (1) Merged image of zebrafish incubated with probe (20μM) for 30 min, (2) merged image of probe-loaded zebrafish incubated with Fe3+ (20μM) for 30 min, (A) bright field, (B) fluorescent images, (C) the overlay images.
Fig. 7. Fluorescence images of soybean root tissues treated with N-TC (λex=560 nm). (1) Merged image of soybean incubated with Fe3+ (20μM) for 30 min, (2) merged image of soybean roots incubated with probe (10μM) for 30 min, (A) bright field, (B) fluorescent images, (C) the overlay images.
Conclusion 8
In summary, we developed a new reversible “turn-off-on” fluorescence probes (N-TC) for sensing Fe3+ based on rhodamine B derivative. N-TC exhibited the excellent selectivity and sensitivity for Fe3+ in aqueous media. N-TC exhibited sensing mechanism towards Fe3+ with 1:1 binding stoichiometry. We found N-TC has higher association constant and lower detection limit. Moreover, N-TC was successfully applied in detecting trace Fe3+ in lake water with good recovery efficiency. Besides, Biological experiments demonstrated their potential value of applications in living cells, animals and plant tissues.
Experimental Materials and instruments For details, see the Supplementary Material. Compound 1 was synthesized in Supplementary Material. Synthesis of N-TC To a solution of compound 1 (160mg, 0.33mmol) in 30mL of dry CH2Cl2 was added pyridine-3-sulfonyl chloride (56mg, 0.32mmol) and triethylamine (40μL, 0.3mmol). The mixture was stirred at room temperature for 3h, and the solvent was removed in a vacuum. The residue was dissolved in CH2Cl2 and then washed with H2O. The organic phase was dried with Magnesium sulfate and then concentrated. The residue was purified by column chromatography using CH2Cl2/ethyl acetate (97:3, v/v) as an eluent to afford pink solid (104mg, 48%). 1H NMR (CDCl3), δ8.95 (d, J=2.1Hz, 1H), 8.72 (dd, J=5.0, 4.7Hz, 1H), 8.09 (m, 1H), 7.88 (dd, J=6.6, 5.3Hz, 1H), 7.46 (t, J1=4.7Hz, J2=3.9Hz, 2H), 7.36 (dd, J=8.0, 7.9Hz, 1H), 7.04 (dd, J=6.6, 5.1Hz, 1H), 6.46 (s, 1H), 6.35 (s, 2H), 6.19 (s, 4H), 3.33 (dd, J=7.0, 6.9Hz, 8H), 3.20 (t, J1=5.2Hz, J2=5.1Hz, 2H), 2.80 (t, J1=5.2Hz, J2=4.6Hz, 2H), 1.19 (m, 12H) ppm. 13C NMR (CDCl3), δ169.83, 153.43, 153.28, 152.83, 149.01, 148.06,136.94, 134.80, 133.02, 130.30, 128.37, 128.29, 123.93, 123.59, 122.93, 108.32, 104.28, 97.76, 65.72, 44.41, 43.93, 40.08, 29.73, 29.35, 12.59 ppm. ESI-MS (M+H)+ found, 626.27; calculated for C35H39N5O3S2, 625.79. Spectral properties test The stock solution of N-TC (1mM) was prepared in EtOH/H2O (1:1, v/v, HEPES, 0.5mM, pH=7.38) solution. The stock solutions (1mM) of the chloride or nitrate salts of Fe3+, Fe2+, Al3+, Ba2+, Ca2+, Co2+, Cd2+, Cr3+, Cu2+, Hg2+, Mg2+, Mn2+, Na+, Ni2+, Pb2+ and Zn2+ in water were prepared as well. Working solutions of N-TC was freshly prepared by diluting the highly concentrated stock solution to the desired concentration prior to the spectroscopic measurements. In all of the spectroscopy experiments, the spectral data of N-TC were recorded 60 min after the addition of the ions. To investigate the metal ion selectivity, the test samples were prepared by placing 10 equiv. of the cation stock solution in 3 mL of the N-TC solution (20μM). For the 9
fluorescence measurements, excitation was provided at 560nm, and the emission was collected from 564 to 700nm. Na4P2O7 was respectively chosen as the chelating agent with Fe3+ to confirm the reversibility of N-TC in both UV–vis absorption and fluorescence methods. Pure water samples were used directly, and lake water samples were collected from Zixi Lake of Nanjing Forestry University and Zixia Lake of Nanjing University of Science and Technology which were passed through the microfiltration membrane before use. Cell culture and fluorescence imaging MCF-7 cells were purchased from the American type culture collection (ATCC) and cultured in Dulbecco’s modified Eagle’s medium (high glucose) supplemented with 10% FBS (fetal bovine serum), 100 units/mL penicillin, and 100 units/mL streptomycin in a 5% CO2/95% air incubator at 37℃. MCF-7 cells were seeded at 1.5×105 cells per well in 24-well flat-bottomed plates in an atmosphere of 5% CO2/95% air at 37℃ and incubated for 24h before treatment. For living cell imaging, cells were incubated with 10μM N-TC in culture medium for 5h in an air incubator at 37℃. In the control experiment, cells were pretreated with 100μM Fe3+ for 1h and then incubated with 10μM N-TC in culture medium for an additional 2h. Fluorescence imaging was performed after washing the cells with PBS 3 times and was recorded using the red channel, blue channel and merged channel under an inverted fluorescence microscope. Animal and plant tissue imaging Zebrafish was purchased from Kaiji Biotechnology Co., Ltd. (Nanjing, China) and respectively incubated in aqueous solution containing N-TC for 30 min and were imaged by confocal microscopy. After incubation with Fe3+ for 30 min, imaging was performed. Fluorescence imaging was recorded using the red channel and merged channel under a confocal microscopy. The seeds of soybean disinfected by 70% ethanol and 3% sodium hypochlorite solution were cultured on Petri dishes at room temperature and 70% relative humidity. The control group was supplied with distilled water; the experimental groups were treated with Fe3+ (20μM). After a week, the roots were collected and immersed in solution containing N-TC (10μM) for 30 min. After three times washing with PBS, the samples were imaged by confocal microscopy. The control group was incubated with 10μM probe N-TC for 30 min. Cytotoxicity assay The cytotoxicity of N-TC was evaluated by an MTT (3-(4,5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide) assay using MCF-7 cells. Cytotoxicity was determined by an MTT colorimetric method using four parallel wells at each time point. MCF-7 cells were cultured in 96-well plates at a density of 5×103 cells per well and were incubated at 37℃ in 5% CO2/95% air for 24h.Then, cells were incubated with different concentrations of N-TC (0-90μM) for 24h, followed by further incubation with 5 mg/mL of MTT for 4h at 37℃. The supernatant 10
was then discarded, and DMSO (150μL/well) was added to dissolve the formazan. Finally, the absorbance was measured at 550 nm with a microplate reader (Infinite M200 Pro). Acknowledgement This work was supported by the National Natural Science Foundation of China (50772048, 81371616), Jiangsu Six Talent Peak Award (2015SWYY013), Natural Science Fund for Colleges and Universities in Jiangsu Province (09KJD430007). This research was also supported by Key Project of Jiangsu Province for Fundamental Research and Development (BE2018717), Scientific Research Foundation of Nanjing Forestry University, Undergraduate Scientific and Technological Innovation Project of Nanjing Forestry University, Practical Innovation Training Program for College Students of Nanjing Forestry University (2018NFUSPITP582). We thank the testing instruments and technical support provided by Advanced analysis and testing center of Nanjing Forestry University.
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Highlights
A Fe3+-specific probe based on Rhodamine B was designed and synthesized.
The probe displays the reversible “turn-off-on” fluorescence change.
The probe is feasible for monitoring trace Fe3+ in environmental water samples.
The probe can be used for detection of Fe3+ in cells, animals and plant tissues.
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Graphical Abstract
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