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A near-infrared ratiometric fluorescent probe with large stokes based on isophorone for rapid detection of ClO− and its bioimaging in cell and mice
Sensors & Actuators: B. Chemical 287 (2019) 453–458
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A near-infrared ratiometric fluorescent probe with large stokes based on isophorone for rapid detection of ClO− and its bioimaging in cell and mice Yongfei Huanga, Yongbin Zhangb, Fangjun Huob, Jianbin Chaob, Caixia Yina,
T
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a
Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education, Key Laboratory of Materials for Energy Conversion and Storage of Shanxi Province, Institute of Molecular Science, Shanxi University, Taiyuan 030006, China b Key Laboratory of Functional Molecules of Shanxi Province, Research Institute of Applied Chemistry, Shanxi University, Taiyuan 030006, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Near infrared Ratiometric probe Rapid HOCl/ClOIn vivo imaging
The reactive oxygen species (ROS) including hypochlorous acid (HOCl), on the one hand they can regulate physiological function in our living organisms, on the other hand abnormal concentration of ROS would induce a series of biomacromolecules damage and further endanger virtually all aspects of human health. Especially it can be directly related to cancer. Thus to elucidate and visualize their concentration is very imminent. Before many fluorescent probes were used to detect HOCl. In recent years, near-infrared or ratiometric fluorescent developed prosperously. The strategy of combining near-infrared and ratiometric fluorescent probe is overwhelming in the design of fluorescent probes. In this work, we successfully designed and obtained a near-infrared and ratiometric fluorescent probe with large stokes by reacting isophorone with coumarin, which can monitor HOCl high efficiently with quick response and low detection limit. In addition, cell imaging experiments show that the probe can identify endogenous and exogenous ClO− successfully, and nude mouse imaging experiments show that the probe can detect exogenous ClO−. It is possible that the probe can be applied in early clinical diagnosis.
1. Introduction In our daily life, hypochlorous acid (HOCl) and hypochlorite (ClO−) are widely used due to their strong oxidizing properties. The treatment of bacteria in drinking water, the bleaching of paper and industrial textiles are inseparable from the role of this strong oxidant [1,2]. As a reactive oxygen species (ROS), hypochlorous acid (HOCl) also plays an important role in physiological and pathological processes [3–6]. Abnormal concentration of HOCl will cause many diseases, such as cardiovascular disease [7], kidney disease [8], inflammatory disease [9] and some cancers [10]. Therefore, rapid detection of hypochlorous acid (HOCl) in vivo is very helpful in treating diseases. In recent years, fluorescent probes have attracted the attention of researchers because of their fast response, high selectivity and high sensitivity [11–22]. So far, the molecular structure of fluorescent probe for detecting hypochlorous acid (HOCl) changes because of hypochlorous acid’s strong oxidizing property, which causes the change of fluorescence spectroscopy. Common fluorophores mainly include the following categories: rhodamine, it is widely used in the construction of probes due to its excellent photophysical properties and high quantum yields [23–26], fluorescein, as a dye, which provides a good way for fluorescent probe design due to its good water solubility and excellent
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fluorescence emission [27–29], BODIPY derivative, which is used in probe synthesis due to high symmetry and stable structure and exhibiting a strong absorption in the UV–vis spectrum [30–33], cyanine, has a complex conjugated system and a high molar absorption coefficient to enable detection in the near-infrared region [34,35], naphthalimide, is widely used in the synthesis of probes because of its high fluorescence quantum yield [36–38]. Unfortunately, most probes have poor solubility, poor water solubility, and low fluorescence quantum yield. Therefore, it has attracted attention that designing a NIR ratiometric fluorescent probe. As is known to all, coumarin derivatives, as a class of fluorophones, have excellent optical properties such as light stability and high fluorescence quantum yield, and have been widely studied as fluorescence reagents and chemical sensors [39–41]. It is worth mentioning that although the isophorone replaced by malononitrile does not have fluorescence, it will have red shift emission and a large stokes shift when it connected with coumarin derivatives. The work was first synthetized and reported by Jiuyan Li et al in 2003 [42], but unfortunately it has not been used in molecular recognition and bioimaging applications. A few literatures have reported that C]C double bond can be broken by ClO− oxidation to achieve specific recognition of ClO− [43–46]. Based on this mechanism, a NIR ratiometric fluorescent probe
Corresponding author. E-mail address: [email protected] (C. Yin).
https://doi.org/10.1016/j.snb.2019.02.075 Received 17 January 2019; Received in revised form 17 February 2019; Accepted 18 February 2019 Available online 18 February 2019 0925-4005/ © 2019 Elsevier B.V. All rights reserved.
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(named ICC) is synthesized. Compared with reported ClO- probe, in this work we successfully designed and easily obtained a near-infrared and ratiometric fluorescent probe with large stokes by direct condensation reaction between isophorone and coumarin [47–49]. ICC has a good light stability and has a red emission peak at 685 nm. It produces a blue emission peak at 486 nm with a large stokes shift when ClO- is added (Table S1). In addition, the low detection limit (0.17 μM) and short response time (less than 100 s) all indicate that ICC can detect both endogenous and exogenous ClO−. 2. Experimental 2.1. Materials and instruments The chemicals and reagents used in the experiment were purchased commercially and could be used in the experiment without purification. All solutions were prepared using deionized water prepared by the school. UV–vis spectroscopy was performed using HITACHI U-3900 spectrophotometer, and fluorescence spectroscopy was performed using HITACHI F-7000 spectrophotometer. Synthetic intermediates and probes were characterized by 1H NMR and 13C NMR using a Bruker AVANCE-600 MHz spectrometer, followed by mass spectrometry using an AB Triple TOF 5600plus System (AB SCIEX, Framingham, USA). The final bioimaging application were measured the Zeiss LSM880 Airyscan confocal laser scanning microscope.
Fig. 1. UV–vis absorption spectroscopy of ICC (10μM) in the presence of different amounts of ClO− in DMSO/PBS (1/1, v/v, pH = 7.4) system.
prepared from KH2PO4 and Na2HPO4. 3. Results and discussion 3.1. UV–vis spectroscopy To investigate the optical response of ICC to ClO−, we first analyzed the UV–vis absorption spectra in DMSO/PBS (1/1, v/v, pH = 7.4). As shown in Fig. 1, ICC exhibited strong absorption at 370 nm and 530 nm. Upon addition of ClO−, the absorption peaks gradually decreased at 370 nm and 530 nm, while the absorption peak gradually at 420 nm increased and an equal absorption point appeared at 450 nm. We concluded that ICC reacted with ClO- to form a new substance. The color of the solution went from pink to colorless when ClO− was added. This phenomenon could indicate that ICC can be used as a "naked eye" to identify the colorimetric probe of ClO−.
2.2. Synthesis The method of the reference was used to synthesize compound 1 and compound 2 [50,51]. Synthesis of ICC (Scheme 1). In a 25.0 ml round bottom flask, compound 1 (0.245 g, 1.00 mmol) and compound 2 (0.186 g, 1.00 mmol) were dissolved in ethanol (10.0 ml). Piperidine (0.150 ml) was added and the mixture was refluxed for 8 h. The reaction was cooled to room temperature, the mixture was filtrated, washed with anhydrous ethanol, and dried to obtain dark green solid (0.240 g, yield 58.5%).1H NMR (600 MHz, DMSO-d6) δ 8.30 (s, 1 H), 7.49-7.41 (m, 2 H), 7.19 (d, J = 16.1 Hz, 1 H), 6.78 (d, J = 9.0 Hz, 1 H), 6.73 (s, 1 H), 6.58 (s, 1 H), 3.48 (q, J = 6.8 Hz, 4 H), 3.33 (s, 2 H), 2.60 (s, 2 H), 1.15 (t, J = 6.9 Hz, 6 H), 1.02 (s, 6 H).13C NMR (150 MHz, DMSO-d6) δ 170.43, 160.57, 156.51, 151.96, 142.70, 133.09, 130.83, 129.40, 122.60, 115.04, 114.47, 113.67, 110.38, 109.03, 96.80, 75.93, 44.79, 42.73, 40.52, 38.44, 32.15, 27.87, 12.86. ESI-MS m/z:[M−H]− calcd for 412.2031; Found 412.2029 (Fig. S1).
3.2. Fluorescence spectroscopy Next, we performed a fluorescence spectroscopy study on ICC and ClO− in DMSO/PBS (1/1, v/v, pH = 7.4). As shown in Fig. 2, ICC showed a strong red emission at 685 nm when the excitation wavelength was 530 nm (Φ = 0.22). At the same time, ICC showed weak emission at 486 nm with 420 nm excitation. With the addition of ClO−, the peak began to decrease at 685 nm and increase at 486 nm. When ClO− was added to 469.0 μM, the reaction basically completed (Φ = 0.17). From this, it could be confirmed that ICC can be used as a ratiometric fluorescent probe for recognizing ClO−. Moreover, we studied the sensitivity of ICC. A good linear relationship between F486 nm/F685 nm and [ClO−] was observed in the 0–14 μM range (R2 = 0.9966) (Fig. 3). The linear equation was: F486 − nm/F685 nm = 0.024 + 0.019 × [ClO ]. Subsequently, the detection limit (3σ/slope) was calculated to be 0.17 μM. The above studies showed that ICC can accurately and sensitively detect trace concentrations of ClO−.
2.3. Spectral test preparation The ICC was dissolved in DMSO to make a 2.0 mM stock solution. After screening, we selected DMSO/PBS (1/1, v/v, pH = 7.4) as the test system, the amount of the probe was 10.0 μM. Stock solutions of 0.10 M 16 substances including 9 cations (Na+, K+, Mg2+, Cu2+, Zn2+, Fe2+, Fe3+, Al3+, Ca2+) existing widely in serum and oxygenated substance (NO2−, NO3−, ClO2−, ClO3−, ClO4−, MnO4−, H2O2) were prepared by direct dissolution in deionized water. Different PBS buffers were
Scheme 1. The synthesis of the ICC. 454
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Fig. 2. Fluorescent spectral changes of ICC (10.0 μM) upon addition of ClO− in DMSO/PBS (1/1, v/v, pH = 7.4) system, channel 1: λex = 420 nm, Slit: 2.5 nm/5 nm; channel 2: λex = 530 nm, Slit: 5 nm/10 nm. Insert: visual solution color change under illumination with a 365 nm UV lamp.
(Na+, K+, Mg2+, Cu2+, Zn2+, Fe2+, Fe3+, Al3+, Ca2+, NO2-, NO3-, ClO2-, ClO3-, ClO4-, MnO4-, H2O2). It could be found that every substance did not disturb the detection of ClO- (Fig. S3).
3.4. Response time The speed of the reaction determined whether probe can be better applied to the detection of actual samples. In this regard, we explored the kinetic studies of ICC in the presence of ClO−. As shown in Fig. S4, the reaction was finished less than 100 s after adding 469 μM ClO−. We concluded that ICC can be used as a ratiometric fluorescent probe for rapid recognition of ClO−.
3.5. Reaction mechanism Fig. 3. The linear relationship between F486 nm/F685 nm and the concentration of ClO− (0–14.0 μM) in DMSO/PBS (1/1, v/v, pH = 7.4) system.
The response mechanism of the reaction between ICC and ClO− was studied by ESI mass spectrum (Scheme 2). After ICC was treated with ClO−, we found the corresponding mass spectra peak of compound 1 (calcd for 268.0950; found 268.0941, Fig. S5a), which relied on the oxidation cleavage of the C = C caused by ClO−, so as to achieve specific fluorescence changes (Table S2). Due to the strong oxidizing property of hypochlorous acid, the unsaturated C = C is broken, and the conjugated system is broken, and the fluorescent emission took place hypochromatic shift with respect to compound 1.We tested the absorption and fluorescence spectra of compound 1 and the results were consistent with our hypothetical mechanism (Fig. S5b/c).
3.3. pH and selectivity study For the study of the practical applicability of ICC, we tested the fluorescence intensity ratio (F486 nm/F685 nm) at different pH (2–12), which showed no change for the ICC. On the other side, fluorescence intensity ratio (F486 nm/F685 nm) started to increase at pH 5 and reached a maximum at pH 7 when ICC was reacted with ClO−. Therefore, we could know that ICC can be used in physiological conditions. We studied the fluorescence spectra of the reaction of ICC with other analytes and the reaction of ICC with ClO− in the presence of other analyte each
Scheme 2. Proposed detection mechanism of ICC toward ClO−.
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Fig. 4. Theoretical calculation of HOMO / LUMO energy gaps of ICC and compound 1. Fig. 5. Confocal images of ICC responds to ClO− in HpG-2 cells. Bright field imaging (a1-c1): a1: HpG-2 cells were incubated with ICC (10.0 μM) for 15 min. b1: HpG-2 cells were incubated with ICC (10.0 μM) for 15 min prior to incubated with ClO- (5.0 μM) for 15 min. c1: HpG-2 cells were incubated with LPS (1.0 μg/mL) for 12 h prior to incubated with ICC (10.0 μM) for 15 min; (a2-c2) shows the fluorescence imaging from the blue channel; (a3-c3) shows the fluorescence imaging from the red channel; (a4-c4) overlap of (a2c2) and (a3-c3). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
3.6. Theoretical calculation
the theoretical basis by density functional theory (DFT) and B3LYP / 6–31 + G (d, p) method. The highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) of the two substances and the energy gap between them are obtained respectively. From Fig. 4, it could be analyzed that the energy gap of
To understand the optical properties of ICC, we calculated energy gap by using the Gaussian 09 program. Firstly, we optimized the structure of ICC and the structure of compound 1, and then calculated 456
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Fig. 6. In vivo images of nude mice (a) injected with ICC (20.0 μM) (b) injected with ICC (20.0 μM) after injected with ClO− (50.0 μM) for 5–30 min; λex = 530 nm, λem = 600–700 nm.
4. Conclusions
compound 1 is higher than the energy gap of ICC. This phenomenon was consistent with the blue shift of the UV–vis spectrum. Through theoretical calculations, the relationship between the change of electronic structure and the change of optical properties was effectively explained.
In summary, a near-infrared ratiometric fluorescent probe ICC was successfully synthesized for detection of ClO−. ICC can rapidly and specifically recognize ClO− with a response detection limit of 0.17 μM. Also it can be widely used as a "naked eye" probe for detecting ClO− due to color changes. Cell imaging experiments show that ICC can identify endogenous and exogenous ClO− in HepG-2 cells. More importantly, ICC has the potential to detect exogenous ClO− in nude mice. We hope that ICC can be used in early diagnosis and pathological studies of clinical diseases.
3.7. Cytotoxicity analysis We conducted cytotoxicity studies for subsequent cellular imaging applications. ICC at different concentrations (0, 2.5, 5.0, 10.0, 30.0, and 50.0 μM) and HpG-2 cells were incubated for 5/10 h in the medium, and CCK-8 was added and incubated for 1 h. As shown in Fig. S6, the survival rate of cells was above 0.8, which indicated that ICC is lowtoxic to cells and can be used for biological imaging.
Acknowledgments We thank the National Natural Science Foundation of China (No. 21672131, 21775096, 21705102), Shanxi Province Foundation for Returness (No. 2017-026), the Shanxi Province Science Foundation for Youths (No. 201701D221061), the Natural Science Foundation of Shanxi Province (No. 201701D121018), Shanxi Province "1331 project" key innovation team construction plan cultivation team (2018-CT-1), and Scientific Instrument Center of Shanxi University (201512).
3.8. Cell imaging The above series of tests confirmed that ICC has a good ratio response to the detection of ClO−. Cell experiments of the NIR fluorescent probe ICC were carried out in HepG-2. Since the fluorescence spectra showed two emission peaks at 486 nm and 685 nm, we set green channel 1 and red channel 2 for observation. First, we incubated HepG2 cells added in ICC for 15 min (as shown in Fig. 5a1–a4). It could be seen that the red channel emits intense red light. Subsequently, we added ClO- to incubate HepG-2 cells for 15 min. From Fig. 5b2, it could be seen that the cells emit significant fluorescence in channel 1. Hereby ICC can detect the ratio of exogenous ClO− at the cellular level. To illustrate ICC response to endogenous ClO−, we first incubated HepG-2 cells with LPS (a lipopolysaccharide that stimulates cells to produce hypochlorous acid) for 12 h and then continued to incubate with ICC for 15 min. As can be seen from Fig. 5c2, there were obvious fluorescent signals collected in channel 1. The above cell imaging experiments showed that as a ratiometric probe, both endogenous and exogenous ClO− could be detected simultaneously.
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3.9. Nude mice imaging For further application of ICC in bioimaging, ICC (20.0 μM) was injected intraperitoneally of mice and signal acquisition was performed by using vivo imaging system (Fig. 6). It could be observed that the injected area has a strong fluorescent signal. Subsequently, ClO− (50.0 μM) was injected into the abdominal cavity of the mice, and the fluorescent signal gradually weakened with the passage of time (0–30 min). Due to background interference of mice, we only collected fluorescence signals in the 600–700 nm regions. Therefore, the probe was able to detect exogenous ClO− in mice. 457
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Soc. 136 (2014) 12820–12823. [32] S.R. Liu, S.P. Wu, Hypochlorous acid turn-on fluorescent probe based on oxidation of diphenyl selenide, Org. Lett. 15 (2013) 878–881. [33] E. Wang, H. Qiao, Y.M. Zhou, L.F. Pang, F. Yu, J.L. Zhang, T.S. Ma, A novel “turnon” fluorogenic probe for sensing hypochlorous acid based on BODIPY, RSC Adv. 5 (2015) 73040–73045. [34] M.T. Sun, H. Yu, H.J. Zhu, F. Ma, S. Zhang, D.J. Huang, S.H. Wang, Oxidative cleavage-based near-infrared fluorescent probe for hypochlorous acid detection and myeloperoxidase activity evaluation, Anal. Chem. 86 (2014) 671–677. [35] G.H. Cheng, J.L. Fan, W. Sun, J.F. Cao, C. Hu, X.J. Peng, A near-infrared fluorescent probe for selective detection of HClO based on Se-sensitized aggregation of
Yongfei Huang obtained his BSc in chemistry from Lvliang University in 2016. Now he is studying his doctor degree in Institute of Molecular Science at Shanxi University. His current research interest is material chemistry. Yongbin Zhang obtained his Doctor Degree in chemistry from Shanxi University in 2015. Now he is an Associate Professor in Research Institute of Applied Chemistry at Shanxi University major in organic chemistry. His current research interests are organic synthesis, molecular recognition. Fangjun Huo obtained his Doctor Degree in chemistry from Shanxi University in 2007. Now he is a Professor in Research Institute of Applied Chemistry at Shanxi University major in organic chemistry. His current research interests are sensors, supramolecular chemistry. Jianbin Chao is a Professor in Scientific Instrument Center at Shanxi University major in analytical chemistry. His current research interests are molecular recognition, supramolecular chemistry. Caixia Yin obtained her Doctor Degree in chemistry from Shanxi University in 2005. Now she is a Professor in Institute of Molecular Science at Shanxi University major in inorganic chemistry. Her current research interests are molecular recognition, sensors chemistry.
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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
A near-infrared ratiometric fluorescent probe with large stokes based on isophorone for rapid detection of ClO− and its bioimaging in cell and mice Yongfei Huanga, Yongbin Zhangb, Fangjun Huob, Jianbin Chaob, Caixia Yina,
T
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a
Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education, Key Laboratory of Materials for Energy Conversion and Storage of Shanxi Province, Institute of Molecular Science, Shanxi University, Taiyuan 030006, China b Key Laboratory of Functional Molecules of Shanxi Province, Research Institute of Applied Chemistry, Shanxi University, Taiyuan 030006, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Near infrared Ratiometric probe Rapid HOCl/ClOIn vivo imaging
The reactive oxygen species (ROS) including hypochlorous acid (HOCl), on the one hand they can regulate physiological function in our living organisms, on the other hand abnormal concentration of ROS would induce a series of biomacromolecules damage and further endanger virtually all aspects of human health. Especially it can be directly related to cancer. Thus to elucidate and visualize their concentration is very imminent. Before many fluorescent probes were used to detect HOCl. In recent years, near-infrared or ratiometric fluorescent developed prosperously. The strategy of combining near-infrared and ratiometric fluorescent probe is overwhelming in the design of fluorescent probes. In this work, we successfully designed and obtained a near-infrared and ratiometric fluorescent probe with large stokes by reacting isophorone with coumarin, which can monitor HOCl high efficiently with quick response and low detection limit. In addition, cell imaging experiments show that the probe can identify endogenous and exogenous ClO− successfully, and nude mouse imaging experiments show that the probe can detect exogenous ClO−. It is possible that the probe can be applied in early clinical diagnosis.
1. Introduction In our daily life, hypochlorous acid (HOCl) and hypochlorite (ClO−) are widely used due to their strong oxidizing properties. The treatment of bacteria in drinking water, the bleaching of paper and industrial textiles are inseparable from the role of this strong oxidant [1,2]. As a reactive oxygen species (ROS), hypochlorous acid (HOCl) also plays an important role in physiological and pathological processes [3–6]. Abnormal concentration of HOCl will cause many diseases, such as cardiovascular disease [7], kidney disease [8], inflammatory disease [9] and some cancers [10]. Therefore, rapid detection of hypochlorous acid (HOCl) in vivo is very helpful in treating diseases. In recent years, fluorescent probes have attracted the attention of researchers because of their fast response, high selectivity and high sensitivity [11–22]. So far, the molecular structure of fluorescent probe for detecting hypochlorous acid (HOCl) changes because of hypochlorous acid’s strong oxidizing property, which causes the change of fluorescence spectroscopy. Common fluorophores mainly include the following categories: rhodamine, it is widely used in the construction of probes due to its excellent photophysical properties and high quantum yields [23–26], fluorescein, as a dye, which provides a good way for fluorescent probe design due to its good water solubility and excellent
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fluorescence emission [27–29], BODIPY derivative, which is used in probe synthesis due to high symmetry and stable structure and exhibiting a strong absorption in the UV–vis spectrum [30–33], cyanine, has a complex conjugated system and a high molar absorption coefficient to enable detection in the near-infrared region [34,35], naphthalimide, is widely used in the synthesis of probes because of its high fluorescence quantum yield [36–38]. Unfortunately, most probes have poor solubility, poor water solubility, and low fluorescence quantum yield. Therefore, it has attracted attention that designing a NIR ratiometric fluorescent probe. As is known to all, coumarin derivatives, as a class of fluorophones, have excellent optical properties such as light stability and high fluorescence quantum yield, and have been widely studied as fluorescence reagents and chemical sensors [39–41]. It is worth mentioning that although the isophorone replaced by malononitrile does not have fluorescence, it will have red shift emission and a large stokes shift when it connected with coumarin derivatives. The work was first synthetized and reported by Jiuyan Li et al in 2003 [42], but unfortunately it has not been used in molecular recognition and bioimaging applications. A few literatures have reported that C]C double bond can be broken by ClO− oxidation to achieve specific recognition of ClO− [43–46]. Based on this mechanism, a NIR ratiometric fluorescent probe
Corresponding author. E-mail address: [email protected] (C. Yin).
https://doi.org/10.1016/j.snb.2019.02.075 Received 17 January 2019; Received in revised form 17 February 2019; Accepted 18 February 2019 Available online 18 February 2019 0925-4005/ © 2019 Elsevier B.V. All rights reserved.
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(named ICC) is synthesized. Compared with reported ClO- probe, in this work we successfully designed and easily obtained a near-infrared and ratiometric fluorescent probe with large stokes by direct condensation reaction between isophorone and coumarin [47–49]. ICC has a good light stability and has a red emission peak at 685 nm. It produces a blue emission peak at 486 nm with a large stokes shift when ClO- is added (Table S1). In addition, the low detection limit (0.17 μM) and short response time (less than 100 s) all indicate that ICC can detect both endogenous and exogenous ClO−. 2. Experimental 2.1. Materials and instruments The chemicals and reagents used in the experiment were purchased commercially and could be used in the experiment without purification. All solutions were prepared using deionized water prepared by the school. UV–vis spectroscopy was performed using HITACHI U-3900 spectrophotometer, and fluorescence spectroscopy was performed using HITACHI F-7000 spectrophotometer. Synthetic intermediates and probes were characterized by 1H NMR and 13C NMR using a Bruker AVANCE-600 MHz spectrometer, followed by mass spectrometry using an AB Triple TOF 5600plus System (AB SCIEX, Framingham, USA). The final bioimaging application were measured the Zeiss LSM880 Airyscan confocal laser scanning microscope.
Fig. 1. UV–vis absorption spectroscopy of ICC (10μM) in the presence of different amounts of ClO− in DMSO/PBS (1/1, v/v, pH = 7.4) system.
prepared from KH2PO4 and Na2HPO4. 3. Results and discussion 3.1. UV–vis spectroscopy To investigate the optical response of ICC to ClO−, we first analyzed the UV–vis absorption spectra in DMSO/PBS (1/1, v/v, pH = 7.4). As shown in Fig. 1, ICC exhibited strong absorption at 370 nm and 530 nm. Upon addition of ClO−, the absorption peaks gradually decreased at 370 nm and 530 nm, while the absorption peak gradually at 420 nm increased and an equal absorption point appeared at 450 nm. We concluded that ICC reacted with ClO- to form a new substance. The color of the solution went from pink to colorless when ClO− was added. This phenomenon could indicate that ICC can be used as a "naked eye" to identify the colorimetric probe of ClO−.
2.2. Synthesis The method of the reference was used to synthesize compound 1 and compound 2 [50,51]. Synthesis of ICC (Scheme 1). In a 25.0 ml round bottom flask, compound 1 (0.245 g, 1.00 mmol) and compound 2 (0.186 g, 1.00 mmol) were dissolved in ethanol (10.0 ml). Piperidine (0.150 ml) was added and the mixture was refluxed for 8 h. The reaction was cooled to room temperature, the mixture was filtrated, washed with anhydrous ethanol, and dried to obtain dark green solid (0.240 g, yield 58.5%).1H NMR (600 MHz, DMSO-d6) δ 8.30 (s, 1 H), 7.49-7.41 (m, 2 H), 7.19 (d, J = 16.1 Hz, 1 H), 6.78 (d, J = 9.0 Hz, 1 H), 6.73 (s, 1 H), 6.58 (s, 1 H), 3.48 (q, J = 6.8 Hz, 4 H), 3.33 (s, 2 H), 2.60 (s, 2 H), 1.15 (t, J = 6.9 Hz, 6 H), 1.02 (s, 6 H).13C NMR (150 MHz, DMSO-d6) δ 170.43, 160.57, 156.51, 151.96, 142.70, 133.09, 130.83, 129.40, 122.60, 115.04, 114.47, 113.67, 110.38, 109.03, 96.80, 75.93, 44.79, 42.73, 40.52, 38.44, 32.15, 27.87, 12.86. ESI-MS m/z:[M−H]− calcd for 412.2031; Found 412.2029 (Fig. S1).
3.2. Fluorescence spectroscopy Next, we performed a fluorescence spectroscopy study on ICC and ClO− in DMSO/PBS (1/1, v/v, pH = 7.4). As shown in Fig. 2, ICC showed a strong red emission at 685 nm when the excitation wavelength was 530 nm (Φ = 0.22). At the same time, ICC showed weak emission at 486 nm with 420 nm excitation. With the addition of ClO−, the peak began to decrease at 685 nm and increase at 486 nm. When ClO− was added to 469.0 μM, the reaction basically completed (Φ = 0.17). From this, it could be confirmed that ICC can be used as a ratiometric fluorescent probe for recognizing ClO−. Moreover, we studied the sensitivity of ICC. A good linear relationship between F486 nm/F685 nm and [ClO−] was observed in the 0–14 μM range (R2 = 0.9966) (Fig. 3). The linear equation was: F486 − nm/F685 nm = 0.024 + 0.019 × [ClO ]. Subsequently, the detection limit (3σ/slope) was calculated to be 0.17 μM. The above studies showed that ICC can accurately and sensitively detect trace concentrations of ClO−.
2.3. Spectral test preparation The ICC was dissolved in DMSO to make a 2.0 mM stock solution. After screening, we selected DMSO/PBS (1/1, v/v, pH = 7.4) as the test system, the amount of the probe was 10.0 μM. Stock solutions of 0.10 M 16 substances including 9 cations (Na+, K+, Mg2+, Cu2+, Zn2+, Fe2+, Fe3+, Al3+, Ca2+) existing widely in serum and oxygenated substance (NO2−, NO3−, ClO2−, ClO3−, ClO4−, MnO4−, H2O2) were prepared by direct dissolution in deionized water. Different PBS buffers were
Scheme 1. The synthesis of the ICC. 454
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Fig. 2. Fluorescent spectral changes of ICC (10.0 μM) upon addition of ClO− in DMSO/PBS (1/1, v/v, pH = 7.4) system, channel 1: λex = 420 nm, Slit: 2.5 nm/5 nm; channel 2: λex = 530 nm, Slit: 5 nm/10 nm. Insert: visual solution color change under illumination with a 365 nm UV lamp.
(Na+, K+, Mg2+, Cu2+, Zn2+, Fe2+, Fe3+, Al3+, Ca2+, NO2-, NO3-, ClO2-, ClO3-, ClO4-, MnO4-, H2O2). It could be found that every substance did not disturb the detection of ClO- (Fig. S3).
3.4. Response time The speed of the reaction determined whether probe can be better applied to the detection of actual samples. In this regard, we explored the kinetic studies of ICC in the presence of ClO−. As shown in Fig. S4, the reaction was finished less than 100 s after adding 469 μM ClO−. We concluded that ICC can be used as a ratiometric fluorescent probe for rapid recognition of ClO−.
3.5. Reaction mechanism Fig. 3. The linear relationship between F486 nm/F685 nm and the concentration of ClO− (0–14.0 μM) in DMSO/PBS (1/1, v/v, pH = 7.4) system.
The response mechanism of the reaction between ICC and ClO− was studied by ESI mass spectrum (Scheme 2). After ICC was treated with ClO−, we found the corresponding mass spectra peak of compound 1 (calcd for 268.0950; found 268.0941, Fig. S5a), which relied on the oxidation cleavage of the C = C caused by ClO−, so as to achieve specific fluorescence changes (Table S2). Due to the strong oxidizing property of hypochlorous acid, the unsaturated C = C is broken, and the conjugated system is broken, and the fluorescent emission took place hypochromatic shift with respect to compound 1.We tested the absorption and fluorescence spectra of compound 1 and the results were consistent with our hypothetical mechanism (Fig. S5b/c).
3.3. pH and selectivity study For the study of the practical applicability of ICC, we tested the fluorescence intensity ratio (F486 nm/F685 nm) at different pH (2–12), which showed no change for the ICC. On the other side, fluorescence intensity ratio (F486 nm/F685 nm) started to increase at pH 5 and reached a maximum at pH 7 when ICC was reacted with ClO−. Therefore, we could know that ICC can be used in physiological conditions. We studied the fluorescence spectra of the reaction of ICC with other analytes and the reaction of ICC with ClO− in the presence of other analyte each
Scheme 2. Proposed detection mechanism of ICC toward ClO−.
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Fig. 4. Theoretical calculation of HOMO / LUMO energy gaps of ICC and compound 1. Fig. 5. Confocal images of ICC responds to ClO− in HpG-2 cells. Bright field imaging (a1-c1): a1: HpG-2 cells were incubated with ICC (10.0 μM) for 15 min. b1: HpG-2 cells were incubated with ICC (10.0 μM) for 15 min prior to incubated with ClO- (5.0 μM) for 15 min. c1: HpG-2 cells were incubated with LPS (1.0 μg/mL) for 12 h prior to incubated with ICC (10.0 μM) for 15 min; (a2-c2) shows the fluorescence imaging from the blue channel; (a3-c3) shows the fluorescence imaging from the red channel; (a4-c4) overlap of (a2c2) and (a3-c3). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
3.6. Theoretical calculation
the theoretical basis by density functional theory (DFT) and B3LYP / 6–31 + G (d, p) method. The highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) of the two substances and the energy gap between them are obtained respectively. From Fig. 4, it could be analyzed that the energy gap of
To understand the optical properties of ICC, we calculated energy gap by using the Gaussian 09 program. Firstly, we optimized the structure of ICC and the structure of compound 1, and then calculated 456
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Fig. 6. In vivo images of nude mice (a) injected with ICC (20.0 μM) (b) injected with ICC (20.0 μM) after injected with ClO− (50.0 μM) for 5–30 min; λex = 530 nm, λem = 600–700 nm.
4. Conclusions
compound 1 is higher than the energy gap of ICC. This phenomenon was consistent with the blue shift of the UV–vis spectrum. Through theoretical calculations, the relationship between the change of electronic structure and the change of optical properties was effectively explained.
In summary, a near-infrared ratiometric fluorescent probe ICC was successfully synthesized for detection of ClO−. ICC can rapidly and specifically recognize ClO− with a response detection limit of 0.17 μM. Also it can be widely used as a "naked eye" probe for detecting ClO− due to color changes. Cell imaging experiments show that ICC can identify endogenous and exogenous ClO− in HepG-2 cells. More importantly, ICC has the potential to detect exogenous ClO− in nude mice. We hope that ICC can be used in early diagnosis and pathological studies of clinical diseases.
3.7. Cytotoxicity analysis We conducted cytotoxicity studies for subsequent cellular imaging applications. ICC at different concentrations (0, 2.5, 5.0, 10.0, 30.0, and 50.0 μM) and HpG-2 cells were incubated for 5/10 h in the medium, and CCK-8 was added and incubated for 1 h. As shown in Fig. S6, the survival rate of cells was above 0.8, which indicated that ICC is lowtoxic to cells and can be used for biological imaging.
Acknowledgments We thank the National Natural Science Foundation of China (No. 21672131, 21775096, 21705102), Shanxi Province Foundation for Returness (No. 2017-026), the Shanxi Province Science Foundation for Youths (No. 201701D221061), the Natural Science Foundation of Shanxi Province (No. 201701D121018), Shanxi Province "1331 project" key innovation team construction plan cultivation team (2018-CT-1), and Scientific Instrument Center of Shanxi University (201512).
3.8. Cell imaging The above series of tests confirmed that ICC has a good ratio response to the detection of ClO−. Cell experiments of the NIR fluorescent probe ICC were carried out in HepG-2. Since the fluorescence spectra showed two emission peaks at 486 nm and 685 nm, we set green channel 1 and red channel 2 for observation. First, we incubated HepG2 cells added in ICC for 15 min (as shown in Fig. 5a1–a4). It could be seen that the red channel emits intense red light. Subsequently, we added ClO- to incubate HepG-2 cells for 15 min. From Fig. 5b2, it could be seen that the cells emit significant fluorescence in channel 1. Hereby ICC can detect the ratio of exogenous ClO− at the cellular level. To illustrate ICC response to endogenous ClO−, we first incubated HepG-2 cells with LPS (a lipopolysaccharide that stimulates cells to produce hypochlorous acid) for 12 h and then continued to incubate with ICC for 15 min. As can be seen from Fig. 5c2, there were obvious fluorescent signals collected in channel 1. The above cell imaging experiments showed that as a ratiometric probe, both endogenous and exogenous ClO− could be detected simultaneously.
Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.snb.2019.02.075. References [1] M.A. Shannon, P.W. Bohn, M. Elimelech, J.G. Georgiadis, B.J. Marinas, A.M. Mayes, Science and technology for water purification in the coming decades, Nature 452 (2008) 301–310. [2] J. Zhang, X.R. Yang, A simple yet effective chromogenic reagent for the rapid estimation of bromate and hypochlorite in drinking water, Analyst 138 (2013) 434–437. [3] J.R. Stone, S. Yang, Hydrogen peroxide: a signaling messenger, Antioxid. Redox Signal. 8 (2006) 243–270. [4] C.C. Winterbourn, Reconciling the chemistry and biology of reactive oxygen species, Nat. Chem. Biol. 4 (2008) 278–286. [5] G.C. Bittner, C.R. Bertozzi, C.J. Chang, Strategy for dual-analyte luciferin imaging: in vivo bioluminescence detection of hydrogen peroxide and caspase activity in a murine model of acute inflammation, J. Am. Chem. Soc. 135 (2013) 1783–1795. [6] K.B. Li, L. Dong, S. Zhang, W. Shi, W.P. Jia, D.M. Han, Fluorogenic boronate-based probe-lactulose complex for full-aqueous analysis of peroxynitrite, Talanta 165 (2017) 593–597. [7] L.J. Hazell, L. Arnold, D. Flowers, G. Waeg, E. Malle, R. Stocker, Presence of hypochlorite-modified proteins in human atherosclerotic lesions, J. Clin. Investig. 97 (1996) 1535–1544. [8] E. Malle, T. Buch, H.J. Grone, Myeloperoxidase in kidney disease, Kidney Int. 64 (2003) 1956–1967. [9] Y. Maruyama, B. Lindholm, P. Stenvinkel, Inflammation and oxidative stress in
3.9. Nude mice imaging For further application of ICC in bioimaging, ICC (20.0 μM) was injected intraperitoneally of mice and signal acquisition was performed by using vivo imaging system (Fig. 6). It could be observed that the injected area has a strong fluorescent signal. Subsequently, ClO− (50.0 μM) was injected into the abdominal cavity of the mice, and the fluorescent signal gradually weakened with the passage of time (0–30 min). Due to background interference of mice, we only collected fluorescence signals in the 600–700 nm regions. Therefore, the probe was able to detect exogenous ClO− in mice. 457
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Yongfei Huang obtained his BSc in chemistry from Lvliang University in 2016. Now he is studying his doctor degree in Institute of Molecular Science at Shanxi University. His current research interest is material chemistry. Yongbin Zhang obtained his Doctor Degree in chemistry from Shanxi University in 2015. Now he is an Associate Professor in Research Institute of Applied Chemistry at Shanxi University major in organic chemistry. His current research interests are organic synthesis, molecular recognition. Fangjun Huo obtained his Doctor Degree in chemistry from Shanxi University in 2007. Now he is a Professor in Research Institute of Applied Chemistry at Shanxi University major in organic chemistry. His current research interests are sensors, supramolecular chemistry. Jianbin Chao is a Professor in Scientific Instrument Center at Shanxi University major in analytical chemistry. His current research interests are molecular recognition, supramolecular chemistry. Caixia Yin obtained her Doctor Degree in chemistry from Shanxi University in 2005. Now she is a Professor in Institute of Molecular Science at Shanxi University major in inorganic chemistry. Her current research interests are molecular recognition, sensors chemistry.
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