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A highly sensitive rapid-response fluorescent probe for specifically tracking endogenous labile Fe2+ in living cells and zebrafish
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A highly sensitive rapid-response fluorescent probe for specifically tracking endogenous labile Fe2þ in living cells and zebrafish Xue Zhang a, Yanan Chen a, Xinyu Cai a, Caiyun Liu a, *, Pan Jia a, Zilu Li a, Hanchuang Zhu a, Yamin Yu a, Kun Wang a, Xiwei Li a, Wenlong Sheng b, ***, Baocun Zhu a, ** a
School of Water Conservancy and Environment, University of Jinan, Shandong Provincial Engineering Technology Research Center for Ecological Carbon Sink and Capture Utilization, Jinan, 250022, China Biology Institute, Qilu University of Technology, Shandong Academy of Sciences, Jinan, 250103, China
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A R T I C L E I N F O
A B S T R A C T
Keywords: Fluorescent probe Nitroxide Hydroxylamine Ferrous ion (Fe2þ) Bioimaging
Iron plays an essential role in the chemical transformation of cells via the transition of multiple oxidation states of iron. Misregulation of iron may lead to the disorder of reactive oxygen species (ROS) catalyzed by iron, which is associated with diverse diseases. Therefore, the monitoring of labile iron in vivo is crucial for revealing its diverse functions in the biosystems. In this work, a novel “off-on” fluorescent probe NT-Fe was designed to detect ferrous ion (Fe2þ) in both aqueous solutions and biological systems, based on the reduction of nitroxides to hydroxyl amine. Probe NT-Fe manifested highly selective and sensitive detection (DL ¼ 89 nM) of Fe2þ. In addition, probe NT-Fe can detect Fe2þ within 50 s, which is conducive to real-time detection of labile iron in the biological system. Moreover, the intracellular Fe2þ can be tracked with probe NT-Fe in living cells effectively. Most of all, as far as we know, probe NT-Fe has been used for the first time to monitor Fe2þ in zebrafish.
1. Introduction Iron is an essential element in cellular events and the most abundant transition metal in the human body [1,2]. An adult contains 3–4 g of iron which is much higher than other transition metal, and iron mainly exists in the form of hemoglobin active center and ferritin. In practice, only approximately 1% of iron species are protein-free or weakly-protein-bound iron, and some iron is involved in the formation of reactive oxygen species (ROS) as a catalyst in Fenton reaction [3]. Therefore, iron homeostasis is beneficial for the normal operation of numerous biological processes. In addition, iron plays an important role in the biological process, such as oxygen transition, enzymatic reaction, and DNA synthesis [2,4]. The disorder of iron metabolism may affect the production of ROS accumulated in cells and destroy the normal ho meostasis of biological system. For instance, excessive production of hydroxyl radicals in Fenton reaction could seriously damage cells and further lead to organ dysfunction [5]. According to previous reports, excess iron may be associated with a variety of diseases, such as cancer, anemia, nephritis, and neurodegenerative diseases, which are ascribed
to the overproduction of ROS [6,7]. Consequently, intricate systems need to be created to maintain cellular iron homeostasis [8,9], and it is critical to explore the mechanism of iron in complex life system. In living organisms, the main existing forms of iron are ferrous ion (Fe2þ) and ferric ion (Fe3þ). What’s more, because of the reductive cellular environment, more and more evidences suggest that Fe2þ is higher than Fe3þ in cells. In recent years, fluorescent probes are ex pected to be a promising tool for further study of various species in living cells [10–21]. Substantial numbers of fluorescent probes have been re ported for the selective response to Fe3þ [22–29]. Nevertheless, fluo rescent probes for Fe2þ are still rare, let alone in vivo imaging applications. So far, several synthetic theories could be used to construct “turn-on” fluorescent probes for the detection of Fe2þ. Nagasawa and colleagues have synthetized a series of “turn-on” fluorescent probes for the selective detection of Fe2þ, and explored the physiological and pathological functions of Fe2þ, based on N-oxide chemistry [30–39]. Very recently, Ye et al. developed a novel N-oxide based fluorescent probe for selective detection of Fe2þ in living cells with two-photon excitation [40]. Chang and colleagues reported another novel
* Corresponding author. ** Corresponding author. *** Corresponding author. E-mail addresses: [email protected] (C. Liu), [email protected] (W. Sheng), [email protected] (B. Zhu). https://doi.org/10.1016/j.dyepig.2019.108065 Received 20 September 2019; Received in revised form 9 November 2019; Accepted 21 November 2019 Available online 22 November 2019 0143-7208/© 2019 Elsevier Ltd. All rights reserved.
Please cite this article as: Xue Zhang, Dyes and Pigments, https://doi.org/10.1016/j.dyepig.2019.108065
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fluorescent probe used to detect labile Fe2þ on the basis of cleavable oxidative C–O bond [41]. Subsequently, Chang et al. reported another fluorescent probe for Fe2þ via Fe2þ-triggered peroxide cleavage reaction [42,43]. Li et al. developed two fluorescent probes for detecting Fe2þ employing 4’-(aminomethylphenyl)-2,2’,6’,2’’-terpyridine (Tpy) as the responsive group [44,45]. Long et al. reported a “turn-on” probe for Fe2þ based on cyclization reaction [46]. There are also several probes that have been synthetized based on the different reaction mechanisms [47–50]. These imaging probes can be successfully applied to detect and image Fe2þ in living cells. However, there are still some challenges in the sensitive detection and real-time imaging of Fe2þ in living cells. What’s more, no fluorescent probe has been reported to detect Fe2þ in vivo (e.g. zebrafish). Therefore, reasonable design of novel fluorescent probes for sensitive and rapid detection of in vivo Fe2þ is urgent and necessary. Reported investigations have demonstrated that paramagnetic nitroxide is a highly efficient quencher owing to the electron exchange interaction between the ground state nitroxide and the excited state fluorescent dyes [51–53]. The reduction of the nitroxide to the diamagnetic hydroxylamine by Fe2þ would eliminate the above-mentioned quenching process, indicating the successful con struction of “turn-on” fluorescent probe for Fe2þ [54,55]. So, we chose 4-amino-2,2,6,6-tetramethylpiperidine-1-oxyl (4-amino-TEMPO) con taining nitroxide moiety as recognition receptor of Fe2þ. 4-Amino-1, 8-naphthalimide was extensively adopted as fluorophore to construct various fluorescent probes because of its excellent photophysical prop erties and good biocompatibility [56]. Therefore, a new fluorescent probe NT-Fe was reasonably constructed (Scheme 1). As expected, probe NT-Fe displayed ultrasensitivity and its detection limit is 89 nM. Besides, the selectivity of probe NT-Fe is remarkable, even for other relative species such as transition metals, ROS and so on. Importantly, ultra-fast response speed allows probe NT-Fe with real-time character istics to detect endogenous and exogenous Fe2þ in the biosystems.
preparation processes are displayed in the Supporting Information. 2.2. Synthesis of probe NT-Fe 4-Chloro-1,8-naphthalimide (546 mg, 2 mmol) was dissolved in methyl glycol (10 mL), then 4-amino-2,2,6,6-tetramethylpiperidine-1oxyl (1026 mg, 3 mmol) and N,N-diisopropylethylamine (DIPEA, 1.55 g, 6 mmol) were added. The mixture was stirred for 24 h at 140 � C. After the solution was cooled to room temperature, the mixture was dissolved in dichloromethane (DCM) and washed by water. Then, the dried DCM was removed by a rotary evaporate. The residue was purified by silica column to get the yellow solid. Because of the paramagnetism of probe NT-Fe, its HRMS date has been provided to replace NMR data. HRMS (ESI): Calcd for C25H33N3O3 [MþH]þ 423.2516; Found, 423.2513. IR (KBr) ν: 3370, 2960, 2925, 2849, 1684, 1639, 1576, 1545, 1385, 1349, 1310, 1240, 1177, 1104, 1077, 772, 758 cm-1. Elemental Analysis: Calcd C, 71.06%; H, 7.63%; N, 9.94%. Found C, 70.86%; H, 7.95%; N, 9.27%. 3. Results and discussion 3.1. Characteristic spectra of probe NT-Fe The spectroscopic performances of probe NT-Fe to Fe2þ were eval uated in 20 mM phosphate buffered solution (PBS)/ethanol (9:1 v/v, pH 7.4). As designed, probe NT-Fe containing nitroxide moiety displayed weak fluorescence, which is attributed to the highly effective intra molecular electron exchange between the ground state nitroxide and the excited state 1,8-naphthalimide. In the reaction of probe NT-Fe (5 μM) with 1 equiv Fe2þ, probe NT-Fe showed a fast response (<50 s) (Fig. 1). With the addition of Fe2þ, probe NT-Fe also showed a significant fluo rescence enhancement at 540 nm (Fig. S1). The absorption spectrum was also carried out in the presence of Fe2þ (Fig. 2). To the best of our knowledge, the response speed of probe NT-Fe for detecting Fe2þ is fastest among the reported Fe2þ-specific probes (Table S1), which is crucial for achieving the real-time determination of Fe2þ in the biolog ical systems [61]. In the presence of Fe2þ, nitroxide radical of probe NTFe was reduced to diamagnetic hydroxylamine, eliminating the quenching pathway, and restoring the strong fluorescence of 1,8-naph thalimide (Scheme 2). This response mechanism has been further confirmed by HRMS (see the Supporting Information).
2. Experimental section 2.1. General information for spectroscopic studies and bioimaging studies Unless otherwise stated, the spectra were carried out in aqueous solution (PBS, 20 mM, pH 7.4, 10% ethanol), and the slit widths were 2 nm for both excitation and emission. The spectrum was recorded at 25 � C after the analyte was added to the solution for 1 min. In the bio imaging of Fe2þ, HeLa cells and 5-day-old zebrafish were selected as biological samples. The detailed bioimaging chemicals, instruments and
3.2. Sensitivity and quantification of Fe2þ Upon the increasing amount of Fe2þ (0–5 μM) in probe NT-Fe solu tion, the fluorescence peak increased gradually at 540 nm in 20 mM PBS
Fig. 1. Time-course of probe NT-Fe (5 μM) for tracking Fe2þ (5 μM).
Scheme 1. Synthesis of fluorescent probe NT-Fe. 2
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Fig. 2. Absorption responses of probe NT-Fe (5 μM) in the presence of Fe2þ (10 μM).
Fig. 3. (a) The fluorescence spectra of probe NT-Fe (5 μM) with the continuous addition of Fe2þ (0–5 μM). (b) The linear relationship of fluorescence intensities at 540 nm with Fe2þ (0–5 μM).
2þ
Scheme 2. Mechanism of fluorescent probe NT-Fe for the detection of Fe
enhancement, while no significant changes were observed in the pres ence of other species. Therefore, probe NT-Fe showed the highly se lective property in detecting Fe2þ, suggesting that probe NT-Fe can be accurately applied to Fe2þ detection without interference in the bio logical systems.
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buffer of pH 7.4 (Fig. 3). The fluorescence intensity shows an excellent linear relationship in the presence of (0–5 μM) Fe2þ (linear equation: y ¼ 100182 � [Fe2þ] (μM) þ 172083, correlation coefficient: R2 ¼ 0.9914), and the detection limit of probe NT-Fe for Fe2þ was detected to be 89 nM based on the equation DL ¼ 3σ/k. Therefore, probe NT-Fe can be applied to detect Fe2þ quantitatively and sensitively, which is also central for monitoring the changes of Fe2þ levels in biosystems (Table S1). 3.3. Selectivity to Fe2þ We detected the selectivity of probe NT-Fe towards Fe2þ over other relevant metals. Given that there are negligible fluorescence changes, probe NT-Fe is selective response to Fe2þ over transition, alkali and alkaline earth metal ions, such as Naþ, Kþ, Ca2þ, Mg2þ, Cd2þ, Co2þ, Ni2þ, Zn2þ, Fe3þ, Cu2þ, Cr3þ, Hg2þ, and Pb2þ. Then, we evaluate the effect of reductive species, such as Na2S, Na2SO3, glutathione (GSH), cysteine (Cys). Besides, reactive oxygen species, as highly reactive spe cies, are also used to test its effects on the fluorescence intensity of the probe. As is depicted in Fig. 4, only Fe2þ caused obvious fluorescence
Fig. 4. The fluorescence responses of probe NT-Fe (5 μM) to various analytes (50 μM except for otherwise stated). 3
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3.4. Imaging of Fe2þ in living cells We then evaluated the bioimaging applications of probe NT-Fe for tracking Fe2þ in living cells by using confocal fluorescence microscopy. The cytotoxicity of probe NT-Fe was firstly tested before bioimaging, and its low-toxicity indicated its good biocompatibility (Fig. 5). HeLa cells were incubated with probe NT-Fe, showing moderate fluorescence enhancement compared with the control cells (Fig. 6a1-a3 and b1-b3). Then, HeLa cells were pretreated with ferrous ammonium sulfate and further incubated with probe NT-Fe. Because of the obvious fluores cence enhancement, probe NT-Fe can be used to detect exogenous Fe2þ (Fig. 6c1-c3). To confirm the high selectivity of probe NT-Fe toward Fe2þ in living cells, the cells were pretreated with 2,20 -bipyridine (Bpy) and Fe2þ, and then incubated with the probe. The decrease in fluores cence intensity signal demonstrated that probe NT-Fe can be applied to detect labile Fe2þ in living cells (Fig. 6d1-d3). Therefore, it is endoge nous Fe2þ leads to the obvious fluorescence enhancement. Finally, the effect of superoxide anion (O2 ) on the fluorescence intensity of the probe was also tested in cells (Fig. S2). The fluorescence intensity of HeLa cells pretreated with O2 did not change significantly. The other group cells were stimulated by phorbol 12-myristate 13-acetate (PMA) to induce endogenous O2 , but the fluorescence did not change. As a result, the difference between the above cells fully demonstrated probe NT-Fe can be used to detect Fe2þ effectively. 3.5. Imaging of Fe2þ in zebrafish According to the satisfactory applications of probe NT-Fe in the detection of Fe2þ in live cells, we chose zebrafish to study its usability in vivo. As shown in Fig. 7b1-b3 and 7c1-c3, Fe2þ-pretreated zebrafish exhibited stronger green fluorescence than zebrafish only treated with probe NT-Fe, for probe NT-Fe could react with exogenous Fe2þ to form hydroxylamine-derived compound. Bpy has been demonstrated to serve as a tissue-permeable Fe2þ-selective chelating agent, and it was also adopted in bioimaging applications in vivo. The weak fluorescence signal of zebrafish incubated with Bpy and Fe2þ confirmed the successful determination of in vivo Fe2þ (Fig. 7d1-d3). Based on the above imaging experiments of zebrafish, probe NT-Fe could be used as a powerful tool for tracking in vivo Fe2þ. 4. Conclusions In conclusion, we have developed a novel fluorescent probe to selectively detect Fe2þ in the biosystems, based on the reduction of
Fig. 6. Fluorescence imaging of Fe2þ in live HeLa cells: (a2) control cells; (b2) the cells incubated with probe NT-Fe (10 μM); (c2) the cells pretreated with Fe2þ (100 μM) and then treated with probe NT-Fe (10 μM); (d2) the cells cotreated with Bpy (1 mM) and Fe2þ (100 μM) and then treated with probe NTFe (10 μM); (a1-d1) bright-field images of (a2-d2); (a3-d3) merged images of (a2-d2); (e) Relative fluorescence intensities for images (a2-d2).
nitroxide to hydroxylamine. Probe NT-Fe can be used to detect Fe2þ within 50 s, and its response speed to Fe2þ is fastest among the reported probes. Additionally, probe NT-Fe can sensitively determinate Fe2þ in the range of 0–5 μM with the detection limit of 89 nM. More impor tantly, the imaging studies of probe NT-Fe have proven that its ability for monitoring labile Fe2þ in living cells and zebrafish. Thus, we
Fig. 5. HeLa viability with different concentration NT-Fe. 4
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expected that probe NT-Fe described in this work can be extended to explore the physiological and pathological roles of Fe2þ in biological systems. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements We gratefully acknowledge financial support from the National Natural Science Foundation of China (21607053 and 21777053), Shandong Provincial Natural Science Foundation (ZR2017MB014 and ZR2018PC013), and Youth Fund of Shandong Academy of Science (2019QN001). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.dyepig.2019.108065. References [1] Hentze MW, Muckenthaler MU, Galy B, Camaschella C. Two to tango: regulation of mammalian iron metabolism. Cell 2010;142:24–38. [2] Theil EC, Goss DJ. Living with iron (and oxygen): questions and answers about iron homeostasis. Chem Rev 2009;109:4568–79. [3] Biaglow JE, Kachur AV. The generation of hydroxyl radicals in the reaction of molecular oxygen with polyphosphate complexes of ferrous ion. Radiat Res 1997; 148:181–7. [4] Ganz T. Systemic iron homeostasis. Physiol Rev 2013;93:1721–41. [5] Dixon SJ, Stockwell BR. The role of iron and reactive oxygen species in cell death. Nat Chem Biol 2014;10:9–17. [6] Imlay JA, Chin SM, Linn S. Toxic DNA damage by hydrogen peroxide through the Fenton reaction in vivo and in vitro. Science 1988;240:640–2. [7] Toyokuni S. Role of iron in carcinogenesis: cancer as a ferrotoxic disease. Cancer Sci 2009;100:9–16. [8] Salahudeen AA, Bruick RK. Maintaining mammalian iron and oxygen homeostasis: sensors, regulation, and cross-talk. Ann N Y Acad Sci 2009;1177:30–8. [9] Crichton RR, Wilmet S, Legssyer R, Ward RJ. Molecular and cellular mechanisms of iron homeostasis and toxicity in mammalian cells. J Inorg Biochem 2002;91:9–18. [10] Domaille DW, Que EL, Chang CJ. Corrigendum: synthetic fluorescent sensors for studying the cell biology of metals. Nat Chem Biol 2008;4:168–75. [11] Niu H, Chen K, Xu J, Zhu X, Cao W, Wang Z, Ye Y, Zhao Y. Mitochondria-targeted fluorescent probes for oxidative stress imaging. Sens Actuators B Chem 2019;299: 126938. [12] Duan Q, Jia P, Zhuang Z, Liu C, Zhang X, Wang Z, Sheng W, Li Z, Zhu H, Zhu B, Zhang X. Rational design of a hepatoma-specific fluorescent probe for HOCl and its bioimaging applications in living HepG2 cells. Anal Chem 2019;91:2163–8. [13] Qin F, Zhang Y, Zhu J, Li Y, Cao W, Ye Y. A mitochondrial-targeted fluorescent probe to sense pH and HOCl in living cells. Sens Actuators B Chem 2019;291: 207–15. [14] Liu C, Li Z, Yu C, Chen Y, Liu D, Zhuang Z, Jia P, Zhu H, Zhang X, Yu Y, Zhu B, Sheng W. Development of a concise rhodamine-formylhydrazine type fluorescent probe for highly specific and ultrasensitive tracing of basal HOCl in live cells and zebrafish. ACS Sens 2019;48:2156–63. [15] Yang X, Liu W, Tang J, Li P, Weng H, Ye Y, Xian M, Tang B, Zhao Y. A multi-signal mitochondria-targeted fluorescent probe for real-time visualization of cysteine metabolism in living cells and animals. Chem Commun 2018;54:11387–90. [16] Que EL, Domaille DW, Chang CJ. Metals in neurobiology: probing their chemistry and biology with molecular imaging. Chem Rev 2008;108:1517–49. [17] Niu H, Zhang Y, Zhao F, Mo S, Cao W, Ye Y, Zhao Y. Reductive stress imaging in the endoplasmic reticulum by using living cells and zebrafish. Chem Commun 2019; 55:9629–32. [18] Liu C, Zhang X, Li Z, Chen Y, Zhuang Z, Jia P, Zhu H, Yu Y, Zhu B, Sheng W. Novel dimethylhydrazine-derived spirolactam fluorescent chemodosimeter for tracing basal peroxynitrite in live cells and zebrafish. J Agric Food Chem 2019;67: 6407–13. [19] McRae R, Bagchi P, Sumalekshmy S, Fahrni CJ. In situ imaging of metals in cells and tissues. Chem Rev 2009;109:4780–827. [20] Haas KL, Franz KJ. Application of metal coordination chemistry to explore and manipulate cell biology. Chem Rev 2009;109:4921–60. [21] Li G, Ma S, Tang J, Ye Y. Lysosome-targeted two-photon fluorescent probes for rapid detection of H2S in live cells. New J Chem 2019;43:1267–74. [22] Sahoo SK, Sharma D, Bera RK, Crisponi G, Callan JF. Iron(III) selective molecular and supramolecular fluorescent probes. Chem Soc Rev 2012;41:7195–227.
Fig. 7. Fluorescence imaging of Fe2þ in zebrafish: (a2) control zebrafish; (b2) the zebrafish incubated with probe NT-Fe (10 μM); (c2) the zebrafish pretreated with Fe2þ (100 μM) and then treated with probe NT-Fe (10 μM); (d2) the zebrafish cotreated with Bpy (1 mM) and Fe2þ (100 μM), and then treated with probe NT-Fe (10 μM); (a1-d1) bright-field images of (a2-d2); (a3-d3) merged images of (a2-d2). (e) Relative fluorescence intensities for images (a2-d2).
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Dyes and Pigments journal homepage: http://www.elsevier.com/locate/dyepig
A highly sensitive rapid-response fluorescent probe for specifically tracking endogenous labile Fe2þ in living cells and zebrafish Xue Zhang a, Yanan Chen a, Xinyu Cai a, Caiyun Liu a, *, Pan Jia a, Zilu Li a, Hanchuang Zhu a, Yamin Yu a, Kun Wang a, Xiwei Li a, Wenlong Sheng b, ***, Baocun Zhu a, ** a
School of Water Conservancy and Environment, University of Jinan, Shandong Provincial Engineering Technology Research Center for Ecological Carbon Sink and Capture Utilization, Jinan, 250022, China Biology Institute, Qilu University of Technology, Shandong Academy of Sciences, Jinan, 250103, China
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A R T I C L E I N F O
A B S T R A C T
Keywords: Fluorescent probe Nitroxide Hydroxylamine Ferrous ion (Fe2þ) Bioimaging
Iron plays an essential role in the chemical transformation of cells via the transition of multiple oxidation states of iron. Misregulation of iron may lead to the disorder of reactive oxygen species (ROS) catalyzed by iron, which is associated with diverse diseases. Therefore, the monitoring of labile iron in vivo is crucial for revealing its diverse functions in the biosystems. In this work, a novel “off-on” fluorescent probe NT-Fe was designed to detect ferrous ion (Fe2þ) in both aqueous solutions and biological systems, based on the reduction of nitroxides to hydroxyl amine. Probe NT-Fe manifested highly selective and sensitive detection (DL ¼ 89 nM) of Fe2þ. In addition, probe NT-Fe can detect Fe2þ within 50 s, which is conducive to real-time detection of labile iron in the biological system. Moreover, the intracellular Fe2þ can be tracked with probe NT-Fe in living cells effectively. Most of all, as far as we know, probe NT-Fe has been used for the first time to monitor Fe2þ in zebrafish.
1. Introduction Iron is an essential element in cellular events and the most abundant transition metal in the human body [1,2]. An adult contains 3–4 g of iron which is much higher than other transition metal, and iron mainly exists in the form of hemoglobin active center and ferritin. In practice, only approximately 1% of iron species are protein-free or weakly-protein-bound iron, and some iron is involved in the formation of reactive oxygen species (ROS) as a catalyst in Fenton reaction [3]. Therefore, iron homeostasis is beneficial for the normal operation of numerous biological processes. In addition, iron plays an important role in the biological process, such as oxygen transition, enzymatic reaction, and DNA synthesis [2,4]. The disorder of iron metabolism may affect the production of ROS accumulated in cells and destroy the normal ho meostasis of biological system. For instance, excessive production of hydroxyl radicals in Fenton reaction could seriously damage cells and further lead to organ dysfunction [5]. According to previous reports, excess iron may be associated with a variety of diseases, such as cancer, anemia, nephritis, and neurodegenerative diseases, which are ascribed
to the overproduction of ROS [6,7]. Consequently, intricate systems need to be created to maintain cellular iron homeostasis [8,9], and it is critical to explore the mechanism of iron in complex life system. In living organisms, the main existing forms of iron are ferrous ion (Fe2þ) and ferric ion (Fe3þ). What’s more, because of the reductive cellular environment, more and more evidences suggest that Fe2þ is higher than Fe3þ in cells. In recent years, fluorescent probes are ex pected to be a promising tool for further study of various species in living cells [10–21]. Substantial numbers of fluorescent probes have been re ported for the selective response to Fe3þ [22–29]. Nevertheless, fluo rescent probes for Fe2þ are still rare, let alone in vivo imaging applications. So far, several synthetic theories could be used to construct “turn-on” fluorescent probes for the detection of Fe2þ. Nagasawa and colleagues have synthetized a series of “turn-on” fluorescent probes for the selective detection of Fe2þ, and explored the physiological and pathological functions of Fe2þ, based on N-oxide chemistry [30–39]. Very recently, Ye et al. developed a novel N-oxide based fluorescent probe for selective detection of Fe2þ in living cells with two-photon excitation [40]. Chang and colleagues reported another novel
* Corresponding author. ** Corresponding author. *** Corresponding author. E-mail addresses: [email protected] (C. Liu), [email protected] (W. Sheng), [email protected] (B. Zhu). https://doi.org/10.1016/j.dyepig.2019.108065 Received 20 September 2019; Received in revised form 9 November 2019; Accepted 21 November 2019 Available online 22 November 2019 0143-7208/© 2019 Elsevier Ltd. All rights reserved.
Please cite this article as: Xue Zhang, Dyes and Pigments, https://doi.org/10.1016/j.dyepig.2019.108065
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fluorescent probe used to detect labile Fe2þ on the basis of cleavable oxidative C–O bond [41]. Subsequently, Chang et al. reported another fluorescent probe for Fe2þ via Fe2þ-triggered peroxide cleavage reaction [42,43]. Li et al. developed two fluorescent probes for detecting Fe2þ employing 4’-(aminomethylphenyl)-2,2’,6’,2’’-terpyridine (Tpy) as the responsive group [44,45]. Long et al. reported a “turn-on” probe for Fe2þ based on cyclization reaction [46]. There are also several probes that have been synthetized based on the different reaction mechanisms [47–50]. These imaging probes can be successfully applied to detect and image Fe2þ in living cells. However, there are still some challenges in the sensitive detection and real-time imaging of Fe2þ in living cells. What’s more, no fluorescent probe has been reported to detect Fe2þ in vivo (e.g. zebrafish). Therefore, reasonable design of novel fluorescent probes for sensitive and rapid detection of in vivo Fe2þ is urgent and necessary. Reported investigations have demonstrated that paramagnetic nitroxide is a highly efficient quencher owing to the electron exchange interaction between the ground state nitroxide and the excited state fluorescent dyes [51–53]. The reduction of the nitroxide to the diamagnetic hydroxylamine by Fe2þ would eliminate the above-mentioned quenching process, indicating the successful con struction of “turn-on” fluorescent probe for Fe2þ [54,55]. So, we chose 4-amino-2,2,6,6-tetramethylpiperidine-1-oxyl (4-amino-TEMPO) con taining nitroxide moiety as recognition receptor of Fe2þ. 4-Amino-1, 8-naphthalimide was extensively adopted as fluorophore to construct various fluorescent probes because of its excellent photophysical prop erties and good biocompatibility [56]. Therefore, a new fluorescent probe NT-Fe was reasonably constructed (Scheme 1). As expected, probe NT-Fe displayed ultrasensitivity and its detection limit is 89 nM. Besides, the selectivity of probe NT-Fe is remarkable, even for other relative species such as transition metals, ROS and so on. Importantly, ultra-fast response speed allows probe NT-Fe with real-time character istics to detect endogenous and exogenous Fe2þ in the biosystems.
preparation processes are displayed in the Supporting Information. 2.2. Synthesis of probe NT-Fe 4-Chloro-1,8-naphthalimide (546 mg, 2 mmol) was dissolved in methyl glycol (10 mL), then 4-amino-2,2,6,6-tetramethylpiperidine-1oxyl (1026 mg, 3 mmol) and N,N-diisopropylethylamine (DIPEA, 1.55 g, 6 mmol) were added. The mixture was stirred for 24 h at 140 � C. After the solution was cooled to room temperature, the mixture was dissolved in dichloromethane (DCM) and washed by water. Then, the dried DCM was removed by a rotary evaporate. The residue was purified by silica column to get the yellow solid. Because of the paramagnetism of probe NT-Fe, its HRMS date has been provided to replace NMR data. HRMS (ESI): Calcd for C25H33N3O3 [MþH]þ 423.2516; Found, 423.2513. IR (KBr) ν: 3370, 2960, 2925, 2849, 1684, 1639, 1576, 1545, 1385, 1349, 1310, 1240, 1177, 1104, 1077, 772, 758 cm-1. Elemental Analysis: Calcd C, 71.06%; H, 7.63%; N, 9.94%. Found C, 70.86%; H, 7.95%; N, 9.27%. 3. Results and discussion 3.1. Characteristic spectra of probe NT-Fe The spectroscopic performances of probe NT-Fe to Fe2þ were eval uated in 20 mM phosphate buffered solution (PBS)/ethanol (9:1 v/v, pH 7.4). As designed, probe NT-Fe containing nitroxide moiety displayed weak fluorescence, which is attributed to the highly effective intra molecular electron exchange between the ground state nitroxide and the excited state 1,8-naphthalimide. In the reaction of probe NT-Fe (5 μM) with 1 equiv Fe2þ, probe NT-Fe showed a fast response (<50 s) (Fig. 1). With the addition of Fe2þ, probe NT-Fe also showed a significant fluo rescence enhancement at 540 nm (Fig. S1). The absorption spectrum was also carried out in the presence of Fe2þ (Fig. 2). To the best of our knowledge, the response speed of probe NT-Fe for detecting Fe2þ is fastest among the reported Fe2þ-specific probes (Table S1), which is crucial for achieving the real-time determination of Fe2þ in the biolog ical systems [61]. In the presence of Fe2þ, nitroxide radical of probe NTFe was reduced to diamagnetic hydroxylamine, eliminating the quenching pathway, and restoring the strong fluorescence of 1,8-naph thalimide (Scheme 2). This response mechanism has been further confirmed by HRMS (see the Supporting Information).
2. Experimental section 2.1. General information for spectroscopic studies and bioimaging studies Unless otherwise stated, the spectra were carried out in aqueous solution (PBS, 20 mM, pH 7.4, 10% ethanol), and the slit widths were 2 nm for both excitation and emission. The spectrum was recorded at 25 � C after the analyte was added to the solution for 1 min. In the bio imaging of Fe2þ, HeLa cells and 5-day-old zebrafish were selected as biological samples. The detailed bioimaging chemicals, instruments and
3.2. Sensitivity and quantification of Fe2þ Upon the increasing amount of Fe2þ (0–5 μM) in probe NT-Fe solu tion, the fluorescence peak increased gradually at 540 nm in 20 mM PBS
Fig. 1. Time-course of probe NT-Fe (5 μM) for tracking Fe2þ (5 μM).
Scheme 1. Synthesis of fluorescent probe NT-Fe. 2
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Fig. 2. Absorption responses of probe NT-Fe (5 μM) in the presence of Fe2þ (10 μM).
Fig. 3. (a) The fluorescence spectra of probe NT-Fe (5 μM) with the continuous addition of Fe2þ (0–5 μM). (b) The linear relationship of fluorescence intensities at 540 nm with Fe2þ (0–5 μM).
2þ
Scheme 2. Mechanism of fluorescent probe NT-Fe for the detection of Fe
enhancement, while no significant changes were observed in the pres ence of other species. Therefore, probe NT-Fe showed the highly se lective property in detecting Fe2þ, suggesting that probe NT-Fe can be accurately applied to Fe2þ detection without interference in the bio logical systems.
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buffer of pH 7.4 (Fig. 3). The fluorescence intensity shows an excellent linear relationship in the presence of (0–5 μM) Fe2þ (linear equation: y ¼ 100182 � [Fe2þ] (μM) þ 172083, correlation coefficient: R2 ¼ 0.9914), and the detection limit of probe NT-Fe for Fe2þ was detected to be 89 nM based on the equation DL ¼ 3σ/k. Therefore, probe NT-Fe can be applied to detect Fe2þ quantitatively and sensitively, which is also central for monitoring the changes of Fe2þ levels in biosystems (Table S1). 3.3. Selectivity to Fe2þ We detected the selectivity of probe NT-Fe towards Fe2þ over other relevant metals. Given that there are negligible fluorescence changes, probe NT-Fe is selective response to Fe2þ over transition, alkali and alkaline earth metal ions, such as Naþ, Kþ, Ca2þ, Mg2þ, Cd2þ, Co2þ, Ni2þ, Zn2þ, Fe3þ, Cu2þ, Cr3þ, Hg2þ, and Pb2þ. Then, we evaluate the effect of reductive species, such as Na2S, Na2SO3, glutathione (GSH), cysteine (Cys). Besides, reactive oxygen species, as highly reactive spe cies, are also used to test its effects on the fluorescence intensity of the probe. As is depicted in Fig. 4, only Fe2þ caused obvious fluorescence
Fig. 4. The fluorescence responses of probe NT-Fe (5 μM) to various analytes (50 μM except for otherwise stated). 3
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3.4. Imaging of Fe2þ in living cells We then evaluated the bioimaging applications of probe NT-Fe for tracking Fe2þ in living cells by using confocal fluorescence microscopy. The cytotoxicity of probe NT-Fe was firstly tested before bioimaging, and its low-toxicity indicated its good biocompatibility (Fig. 5). HeLa cells were incubated with probe NT-Fe, showing moderate fluorescence enhancement compared with the control cells (Fig. 6a1-a3 and b1-b3). Then, HeLa cells were pretreated with ferrous ammonium sulfate and further incubated with probe NT-Fe. Because of the obvious fluores cence enhancement, probe NT-Fe can be used to detect exogenous Fe2þ (Fig. 6c1-c3). To confirm the high selectivity of probe NT-Fe toward Fe2þ in living cells, the cells were pretreated with 2,20 -bipyridine (Bpy) and Fe2þ, and then incubated with the probe. The decrease in fluores cence intensity signal demonstrated that probe NT-Fe can be applied to detect labile Fe2þ in living cells (Fig. 6d1-d3). Therefore, it is endoge nous Fe2þ leads to the obvious fluorescence enhancement. Finally, the effect of superoxide anion (O2 ) on the fluorescence intensity of the probe was also tested in cells (Fig. S2). The fluorescence intensity of HeLa cells pretreated with O2 did not change significantly. The other group cells were stimulated by phorbol 12-myristate 13-acetate (PMA) to induce endogenous O2 , but the fluorescence did not change. As a result, the difference between the above cells fully demonstrated probe NT-Fe can be used to detect Fe2þ effectively. 3.5. Imaging of Fe2þ in zebrafish According to the satisfactory applications of probe NT-Fe in the detection of Fe2þ in live cells, we chose zebrafish to study its usability in vivo. As shown in Fig. 7b1-b3 and 7c1-c3, Fe2þ-pretreated zebrafish exhibited stronger green fluorescence than zebrafish only treated with probe NT-Fe, for probe NT-Fe could react with exogenous Fe2þ to form hydroxylamine-derived compound. Bpy has been demonstrated to serve as a tissue-permeable Fe2þ-selective chelating agent, and it was also adopted in bioimaging applications in vivo. The weak fluorescence signal of zebrafish incubated with Bpy and Fe2þ confirmed the successful determination of in vivo Fe2þ (Fig. 7d1-d3). Based on the above imaging experiments of zebrafish, probe NT-Fe could be used as a powerful tool for tracking in vivo Fe2þ. 4. Conclusions In conclusion, we have developed a novel fluorescent probe to selectively detect Fe2þ in the biosystems, based on the reduction of
Fig. 6. Fluorescence imaging of Fe2þ in live HeLa cells: (a2) control cells; (b2) the cells incubated with probe NT-Fe (10 μM); (c2) the cells pretreated with Fe2þ (100 μM) and then treated with probe NT-Fe (10 μM); (d2) the cells cotreated with Bpy (1 mM) and Fe2þ (100 μM) and then treated with probe NTFe (10 μM); (a1-d1) bright-field images of (a2-d2); (a3-d3) merged images of (a2-d2); (e) Relative fluorescence intensities for images (a2-d2).
nitroxide to hydroxylamine. Probe NT-Fe can be used to detect Fe2þ within 50 s, and its response speed to Fe2þ is fastest among the reported probes. Additionally, probe NT-Fe can sensitively determinate Fe2þ in the range of 0–5 μM with the detection limit of 89 nM. More impor tantly, the imaging studies of probe NT-Fe have proven that its ability for monitoring labile Fe2þ in living cells and zebrafish. Thus, we
Fig. 5. HeLa viability with different concentration NT-Fe. 4
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expected that probe NT-Fe described in this work can be extended to explore the physiological and pathological roles of Fe2þ in biological systems. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements We gratefully acknowledge financial support from the National Natural Science Foundation of China (21607053 and 21777053), Shandong Provincial Natural Science Foundation (ZR2017MB014 and ZR2018PC013), and Youth Fund of Shandong Academy of Science (2019QN001). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.dyepig.2019.108065. References [1] Hentze MW, Muckenthaler MU, Galy B, Camaschella C. Two to tango: regulation of mammalian iron metabolism. Cell 2010;142:24–38. [2] Theil EC, Goss DJ. Living with iron (and oxygen): questions and answers about iron homeostasis. Chem Rev 2009;109:4568–79. [3] Biaglow JE, Kachur AV. The generation of hydroxyl radicals in the reaction of molecular oxygen with polyphosphate complexes of ferrous ion. Radiat Res 1997; 148:181–7. [4] Ganz T. Systemic iron homeostasis. Physiol Rev 2013;93:1721–41. [5] Dixon SJ, Stockwell BR. The role of iron and reactive oxygen species in cell death. Nat Chem Biol 2014;10:9–17. [6] Imlay JA, Chin SM, Linn S. Toxic DNA damage by hydrogen peroxide through the Fenton reaction in vivo and in vitro. Science 1988;240:640–2. [7] Toyokuni S. Role of iron in carcinogenesis: cancer as a ferrotoxic disease. Cancer Sci 2009;100:9–16. [8] Salahudeen AA, Bruick RK. Maintaining mammalian iron and oxygen homeostasis: sensors, regulation, and cross-talk. Ann N Y Acad Sci 2009;1177:30–8. [9] Crichton RR, Wilmet S, Legssyer R, Ward RJ. Molecular and cellular mechanisms of iron homeostasis and toxicity in mammalian cells. J Inorg Biochem 2002;91:9–18. [10] Domaille DW, Que EL, Chang CJ. Corrigendum: synthetic fluorescent sensors for studying the cell biology of metals. Nat Chem Biol 2008;4:168–75. [11] Niu H, Chen K, Xu J, Zhu X, Cao W, Wang Z, Ye Y, Zhao Y. Mitochondria-targeted fluorescent probes for oxidative stress imaging. Sens Actuators B Chem 2019;299: 126938. [12] Duan Q, Jia P, Zhuang Z, Liu C, Zhang X, Wang Z, Sheng W, Li Z, Zhu H, Zhu B, Zhang X. Rational design of a hepatoma-specific fluorescent probe for HOCl and its bioimaging applications in living HepG2 cells. Anal Chem 2019;91:2163–8. [13] Qin F, Zhang Y, Zhu J, Li Y, Cao W, Ye Y. A mitochondrial-targeted fluorescent probe to sense pH and HOCl in living cells. Sens Actuators B Chem 2019;291: 207–15. [14] Liu C, Li Z, Yu C, Chen Y, Liu D, Zhuang Z, Jia P, Zhu H, Zhang X, Yu Y, Zhu B, Sheng W. Development of a concise rhodamine-formylhydrazine type fluorescent probe for highly specific and ultrasensitive tracing of basal HOCl in live cells and zebrafish. ACS Sens 2019;48:2156–63. [15] Yang X, Liu W, Tang J, Li P, Weng H, Ye Y, Xian M, Tang B, Zhao Y. A multi-signal mitochondria-targeted fluorescent probe for real-time visualization of cysteine metabolism in living cells and animals. Chem Commun 2018;54:11387–90. [16] Que EL, Domaille DW, Chang CJ. Metals in neurobiology: probing their chemistry and biology with molecular imaging. Chem Rev 2008;108:1517–49. [17] Niu H, Zhang Y, Zhao F, Mo S, Cao W, Ye Y, Zhao Y. Reductive stress imaging in the endoplasmic reticulum by using living cells and zebrafish. Chem Commun 2019; 55:9629–32. [18] Liu C, Zhang X, Li Z, Chen Y, Zhuang Z, Jia P, Zhu H, Yu Y, Zhu B, Sheng W. Novel dimethylhydrazine-derived spirolactam fluorescent chemodosimeter for tracing basal peroxynitrite in live cells and zebrafish. J Agric Food Chem 2019;67: 6407–13. [19] McRae R, Bagchi P, Sumalekshmy S, Fahrni CJ. In situ imaging of metals in cells and tissues. Chem Rev 2009;109:4780–827. [20] Haas KL, Franz KJ. Application of metal coordination chemistry to explore and manipulate cell biology. Chem Rev 2009;109:4921–60. [21] Li G, Ma S, Tang J, Ye Y. Lysosome-targeted two-photon fluorescent probes for rapid detection of H2S in live cells. New J Chem 2019;43:1267–74. [22] Sahoo SK, Sharma D, Bera RK, Crisponi G, Callan JF. Iron(III) selective molecular and supramolecular fluorescent probes. Chem Soc Rev 2012;41:7195–227.
Fig. 7. Fluorescence imaging of Fe2þ in zebrafish: (a2) control zebrafish; (b2) the zebrafish incubated with probe NT-Fe (10 μM); (c2) the zebrafish pretreated with Fe2þ (100 μM) and then treated with probe NT-Fe (10 μM); (d2) the zebrafish cotreated with Bpy (1 mM) and Fe2þ (100 μM), and then treated with probe NT-Fe (10 μM); (a1-d1) bright-field images of (a2-d2); (a3-d3) merged images of (a2-d2). (e) Relative fluorescence intensities for images (a2-d2).
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