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Dioxetane-based chemiluminescent probe for fluoride ion-sensing in aqueous solution and living imaging
Sensors & Actuators: B. Chemical 301 (2019) 127111
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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Dioxetane-based chemiluminescent probe for fluoride ion-sensing in aqueous solution and living imaging
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Bowen Gu, Chao Dong, Ruwei Shen, Jian Qiang, Tingwen Wei, Fang Wang, Sheng Lu , ⁎ Xiaoqiang Chen State Key Laboratory of Materials-Oriented Chemical Engineering, College of Chemical Engineering, Nanjing Tech University, Nanjing 211816, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Chemiluminescent probe Fluoride ion 1,2-Dioxetane Toothpaste Living mice In vivo imaging
Fluoride plays an important role in our daily life as it associates with a variety of biological and pathological processes. Hence, an analytic method that can detect fluoride ions under physiological conditions with high sensitivity and specificity, and fast response, is needed. Herein, we introduced a dioxetane-based chemiluminescent probe, CL-F, specifically designed for the detection of fluoride ions in physiological environment. The probe CL-F emitted strong green chemiluminescent light within 1 min when incubating with fluoride ions specifically. The linear range for sensing fluoride ions and limit of detection were determined to be 0–30 μM and 0.91 μM, respectively. The probe CL-F was then successfully applied in quantifying fluoride ions in toothpaste and in vivo imaging in living mice, demonstrating CL-F as a promising tool for sensing fluoride ion in vitro and in vivo.
1. Introduction Owing to the advances in molecular sensing approaches, the development of sensors with high sensitivity and selectivity for anions has attracted much attention due to their vital roles in chemical and biological processes [1–6]. Fluoride is one of the anions that are most relevant to our daily life. It is added as supplements in our daily goods, such as drinking water, milk, toothpaste and mouth rinses, due to its function in preventing tooth decay and osteoporosis [7,8]. However, excessive fluoride intake also leads to fluorosis, urolithiasis, or even cancer [9,10]. According to the U.S. Public Health Service, the optimal level of fluoride consumption is 1 mg per day, while the World Health Organization (WHO) recommends fluoride levels below 1.5 ppm in drinking water [11]. Since artificial sources of fluoride are also used in large-scale industrial applications, the produced industrial wastes would result in fluoride contamination in water sources, affecting our fluoride intake. Therefore, efficient and reliable sensing methods for fluoride levels in aqueous environment are highly demanded. The analytic approaches used for fluoride ion detection involve standard willard and winter methods, ion-selective electrode, and ion chromatography [12,13]. These conventional analytic methods have intrinsic drawbacks, such as complicated procedures, high cost and low mobility, which hinder the rapidness and convenience of fluoride detection. Recently, fluorescent chemosensors have received considerable
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attention due to their high sensitivity and specificity, and ease of operating, providing an alternative tool for the detection of fluoride ions [14–17]. But the inherent drawbacks of fluorescence assays, including photobleaching, phototoxicity and interference of autofluorescence from other species in the samples, restrict their practical applications. Comparing to fluorescence-based mechanisms, bio- and chemiluminescent assays can generate optical signals without excitation from external light sources, rendering them higher reliability, sensitivity and signal-to-noise ratios [18]. Therefore, bio- and chemiluminescent probes have been widely used for in vitro and in vivo imaging of various enzymes and analytes [19–22]. To date, extensive examples of bio- and chemiluminescent probes are designed based on two major mechanisms, firefly luciferase-luciferin system and adamantylidene − dioxetane model [23–28]. Despite holding advantages of sensitivity and high signal-to-noise ratio, firefly luciferase-luciferin systems require exogenous gene expression of luciferase to trigger the luminescence, elevating the complexity in practical applications [29]. The adamantylidene − dioxetane developed by Schaap is a classic molecular model to construct chemiluminenscent probes, which do not require the activation by oxidation due to the relative stable dioxetane moiety [30–32]. This system exhibits a good capability of emitting light in organic solvents with medium polarity, but performs badly under aqueous conditions [33]. The addition of a surfactant, enhancer Emerald-II, was found to amplify the luminescence in aqueous solution by providing a
Corresponding authors. E-mail addresses: [email protected] (S. Lu), [email protected], [email protected] (X. Chen).
https://doi.org/10.1016/j.snb.2019.127111 Received 23 July 2019; Received in revised form 5 September 2019; Accepted 6 September 2019 Available online 09 September 2019 0925-4005/ © 2019 Elsevier B.V. All rights reserved.
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imaging was obtained using an Automatic Chemiluminescence Image Analysis System in Tris buffer solution, after distinct densities of F− (0, 5, 15, 45, 60, 80 μM) were mixed into a 96-well plate. 2.4. Chemiluminescence test to toothpaste Four grams of toothpaste dissolved in 40 ml water, then aqueous solution was filtered with a filter to get 38 mL clear solution. 30 μL filtered aqueous solution of toothpaste was added into 2970 μL buffer solution containing 2 μM CLF. The control group was carried out in the same manner except that the clear solution was replaced with Tris buffer. Chemiluminescent intensity was acquired 30 s latter after incubation of CLF (2 μM) with clear solution in buffer. All tests were performed in Tris buffer solution.
Scheme 1. The preparation path of CL-F.
hydrophobic environment that reduces the water-induced quenching [34]. However, the toxicity of surfactants impedes the applications in biological fields. More recently, Shabat’s group improved the luminescence quantum yield of the adamantylidene − dioxetane probe in aqueous conditions by introducing an electron withdrawing group (EWG) [35]. Inspired by this, our group designed and synthesized a novel fluoride ion specific dioxetane probe CLF (Scheme 1), for fluoride sensing and quantification under aqueous conditions. This probe was shown to emit strong green light rapidly after incubating with fluoride ions in aqueous solutions, and possessed an outstanding limit of detection (LOD) of 0.91 μM. The feasibility of quantifying fluoride ions in toothpaste and live imaging in mice was proven in subsequent studies. This work provides a promising tool and strategy for fluoride sensing in aqueous environment.
2.5. MTT assay The cytotoxicity of CL-F toward living cells was checked via MTT assay. HeLa cells were added to 96-well culture plate incubated for 12 h. Next, CL-F (2, 5 and 10 μM) was mixed in the wells and then 20 μL of MTT (5.0 mg/mL) were poured into each well after incubation of 12 h. Four hours later, the supernatant was thrown away and 150 μL DMSO was added to each well. Finally, the absorbance of formazan solution was collected at 490 nm. 2.6. Chemiluminescence assays in living mice CL-F (0.2 mM) and F− (6 mM) were readied in advance. The images of living mice were gained by using an Automatic Chemiluminescence Image Analysis System. Firstly, a mixture solution containing 20 μM F−, 2 μM CL-F, and Tris−HCl buffer solution at the total volume of 50 μL was injected into the back of mice, the chemiluminescence was collected immediately. As control, a 50 μL solution was carried out in the same manner except that the fluoride ion was replaced with Tris buffer solution. All imaging investigations used anesthetized BALB-C mice.
2. Experimental section 2.1. Materials and instruments Unless otherwise stated, the experimental drug was purchased directly from the supplier and was not repurified. Chemical products were purified and separated by column chromatography. Bruker 400 Nuclear Magnetic Resonance Spectrometer was used to gain 1H NMR and 13C NMR spectra. Electrospray ionization mass spectra (ESI–MS) were performed by Agilent 1260 HPLC-6500 Q-TOF Mass Spectrometer. Chemiluminescent spectra were recorded by RF-5301PC fluorescence spectrophotometer. MTT assay were performed by Multiskan Go (51119200-VAN). The chemiluminescent images were obtained by using an Automatic Chemiluminescence Image Analysis System.
3. Results and discussions 3.1. Fluoride ion triggered chemiluminescence of CL-F The general mechanism of fluoride ion sensing of this probe is presented in Scheme 2. Removal of the triggering substrate by fluoride ion releases the phenolate-dioxetane intermediate (product 1), product 1 decomposes through a chemically initiated electron exchange luminescence (CIEEL) process to produce the electronically excited benzoate ester (product 2) [36,37]. This excited benzoate ester relaxes to its
2.2. Synthesis The synthetic route of probe CLF is illustrated in Scheme 1. Starting material compound a was obtained in the light of previous literatures [35,36]. Then the phenol group of compound a was protected with tertbutyldimethylsilyl (TBS) choride to gain compound b. Finally, our desired product CLF was obtained by the oxidation of b. The identity of compound b and product CLF was proved via 1H NMR, 13C NMR, and ESI–MS (Figure S9–Figure S14). 2.3. Chemiluminescence study in vitro A stock DMSO solution of 0.2 mM CL-F was prepared. 2960 μL of Tris buffer (50 mM, pH = 7.4), 10 μL of different concentrations of F−, 30 μL DMSO solution of 0.2 mM CL-F were added to test glass tube. A chemiluminescent kinetic curve was acquired by using time scanning module. With the response of probe CL-F to F−, diverse concentrations of F− (0, 5, 10, 15, 20, 30, 45, 70, 100 μM) was treated with 2 μM CL-F in Tris buffer solution, then tested the chemiluminescent intensity 30 s latter. The selectivity of CL-F towards various anions was illustrated by testing the chemiluminescent light emission. The chemiluminescent
Scheme 2. Activation pathway of probe CL-F for fluoride ion. 2
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Fig. 1. (A) Time scans of the chemiluminescent intensity of CL-F (2 μM) reacted with fluoride ion (20 μM) in Tris buffer (50 mM, pH = 7.4). The inset shows emission spectra of CL-F for various concentrations of fluoride. (B) Chemiluminescent intensity acquired 30 s latter after incubation of CL-F (2 μM) with various concentrations of fluoride ion in Tris buffer (50 mM, pH = 7.4). (C) Time-lapse images of probe within 50 min.
shown in Fig. 2B, an evident enhancement in chemiluminescent signal appeals in the presence of fluoride ion, while the signals are barely seen after incubating with other anions. The pH effect and selectivity data support that probe CL-F is suitable for fluoride ion detection at physiologically relevant conditions with a good specificity.
ground state and yields a green chemiluminescent signal. The identity of final product 3 was proved by ESI-MS (Figure S15). With the probe CL-F in hand, we first studied the kinetics of the fluoride-dependent chemiluminescent emission. Upon the addition of 20 μM of fluoride ion, the intensity of chemiluminescent light emitted from CL-F buffered solution (50 mM Tris, pH 7.4) as a function of time was measured. As shown in Fig. 1A, the chemiluminescent light intensity increases sharply when CL-F reacted with fluoride ions, and maximizes within 1 min, following by a slow decay over a time course of 50 min. This profile of CL-F represents a typical chemiluminescence kinetic behavior. The chemiluminescent kinetic process was also visualized in Fig. 1C. Chemiluminescent light was barely observed without the presence of fluoride ions (Figure S2), validating CL-F as a sensor for fluoride ions. Then we investigated the responsibility of probe CL-F to fluoride ions at a series of concentrations ranging from 0 to 100 μM. As shown in Fig. 1B, the chemiluminescent intensity increases with elevated fluoride ion concentrations. The enhancement of the chemiluminescence is linearly proportional to the increment of fluoride ion concentration in the range of 0–30 μM, indicating the dynamic range of probe CL-F for fluoride quantification. The LOD of fluoride (3σ/k) was determined to be 0.91 μM (∼17.3 ppb), where σ represents the standard deviation of blank measurement, while k represents the slope of the plot for the chemiluminescent intensity versus fluoride concentrations. The LOD is advantageous to those of previously reported methods, which are mostly beyond 25 ppb [38–42]. The outstanding performance could be attributed to the high chemiluminescent efficiency of probe CL-F.
3.3. Comparison to a conventional chemiluminescent probe To demonstrate the superior activity of probe CL-F in fluoride ion sensing, XCL-F, a fluoride ion specific chemiluminescent probe based on the mechanism of Schapp’s adamantylidene-dioxetane [30], was synthesized to make a comparison with CL-F. The synthetic route of XCL-F is shown in Scheme S1. As expected, after incubating with fluoride ions, XCL-F only emitted a moderate blue light in DMSO (Fig. 3A and Figure S3), but not in aqueous solution (Fig. 3B and C). After the addition of Emerald-II enhancer (10%, v/v), XCL-F was able to sense fluoride ions by emitting a green light (Figure S4). The chemiluminescent intensity was enhanced by 106-fold when compared to the case without the enhancer (Fig. 3B and C). However, the intensity of chemiluminescent signal generated from probe CL-F without the enhancer was approximate 6-fold higher than that of probe XCL-F with the presence of enhancer (Fig. 3D and E). The comparison suggests that our probe CL-F holds superior efficiency for sensing fluoride ions in aqueous environment. 3.4. Quantification of fluoride ions in toothpaste Since toothpaste is one of the most common fluoride sources in our daily lives, we then continued to quantify the concentration of fluoride ions in Crest toothpaste using probe CLF as an example of practical application. To prepare the test samples, four grams of toothpaste were dissolved in water, following by filtration to obtain a clear solution. 30 μL of the filtered aqueous solution was added into 2970 μL buffer solution containing 2 μM of CLF. The control group was conducted by substituting the toothpaste sample with a blank buffer solution. The toothpaste sample induced a chemiluminescent intensity of 34.5 (Fig. 4). Given the calibration curve obtained in Fig. 1B, the concentration of NaF in toothpaste was calculated to be 1.02 mg/g, with a difference less than 8% when compared to the content provided by the manufacturer, which is 1.1 mg/g. These results clearly show that CLF
3.2. pH influence and selectivity investigation Next, the pH effect on the response of probe CL-F toward fluoride ions was tested. As shown in Fig. 2A, the chemiluminescent intensity increases gradually from pH 4.0 to 8.0, reaching a maximum in the pH range from 7.5 to 8.0, whereas decreases along with further basified environment. These data indicate that CL-F can detect fluoride ion at physiological condition at its highest sensitivity. To further evaluate the specificity of CL-F probe for fluoride sensing, twelve common anions (Br−, C2O42-, I−, SO32-, N3−, NO2−, NO3−, SCN−, OH−, SO42-, H2PO4−,Cl−) were added into CL-F buffer solutions individually and the chemiluminesent responses were monitored. As 3
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Fig. 2. (A) Chemiluminescent intensity acquired 30 s latter after incubating CL-F (2 μM) with fluoride ions (20 μM) in buffer solutions at different pH values. (B) Chemiluminescent intensity acquired 30 s latter after incubating CL-F (2 μM) with fluoride ions (20 μM) and other anions (100 μM) in Tris buffer (50 mM, pH = 7.4). The inset shows chemiluminescent imaging of CL-F (5 μM) with fluoride (20 μM) and other anions (100 μM) in buffer solution containing 5% DMSO. All the anions mentioned above are derived from the sodium salt: NaBr, Na2C2O4, NaI, Na2SO3, NaN3, NaNO2, NaNO3, NaSCN, NaOH, Na2SO4, NaH2PO4, NaCl, NaF.
Fig. 3. (A) Chemiluminescent imaging after incubation of CL-F (5 μM) and XCL-F (5 μM) with fluoride ion (20 μM) in Tris buffer (50 mM, pH 7.4), 5% DMSO, and DMSO respectively. (B) Chemiluminescent intensity of probe XCL-F (5 μM) with or without Emerald-II enhancer (10%, v/v) in Tris buffer (50 mM, pH 7.4) containing 5% DMSO, in the presence of fluoride ion (20 μM), and (C) Chemiluminescent emission spectrum. (D) Chemiluminescent intensity of probe XCL-F (5 μM) enhanced by Emerald-II enhancer (10%, v/v) and probe CL-F (5 μM) in Tris buffer (50 mM, pH7.4) containing 5% DMSO, in the presence of fluoride ion (20 μM), and (E) Chemiluminescent emission spectrum.
Fig. 4. (A) Chemiluminescent intensity acquired 30 s latter after incubation of CL-F (2 μM) with 30 μL clarified aqueous of toothpaste in buffer solution, and (B) Chemiluminescent emission spectrum.
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Fig. 5. (A) In vitro images of CLF chemiluminescence after adding CLF (2 μM) to 0, 5, 15, 45, 60, and 80 μM of fluoride ion in Tris buffer solution (n = 3). (B) Chemiluminescent imaging of fluoride ion in vivo. (right) The mouse was injected with 50 μL of buffer solution containing CLF (2 μM) and fluoride ion (20 μM). (left) The control group was injected with 50 μL of Tris buffer (50 mM, pH = 7.4) solution solely containing CLF (2 μM).
probe is capable of quantitatively sensing fluoride in a practical scenario with much complexity.
online version, at doi:https://doi.org/10.1016/j.snb.2019.127111. References
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Encouraged by the strong signal response of probe CLF toward fluoride ions in vitro, we continued on evaluating its potential for detecting fluoride ions in vivo. Beforehand, we firstly studied the chemiluminescent imaging of CLF triggered by fluoride ions with an Automatic Chemiluminescence Image Analysis System under physiological conditions in vitro. Fluoride ions were added into the wells of a 96-well black plate containing CLF (2 μM) buffer solution with final concentrations of 0, 5, 15, 45, 60, and 80 μM, respectively. As shown in Fig. 5A, the light output exhibits an observable intensity when the concentration of fluoride ions is above 5 μM. Moreover, the experimental data shown that probe CLF has little toxicity to living cells when its concentration is lower than 10 μM (Figure S5). Subsequently, the live imaging of fluoride ions was conducted on anesthetized BALB-C mice model. A mixture buffer solution containing CLF (2 μM) and fluoride ion (20 μM) was injected to the back of the mice at the volume of 50 μL, and the chemiluminescent signal was monitored under the Automatic Chemiluminescence Image Analysis System. As shown in Fig. 5B, a strong light signal is observed at the injection site while the signal from the control group is negligible. These results demonstrate that probe CLF is capable of monitoring the presence of fluoride ions in vivo. 4. Conclusion In summary, we designed and prepared a fluoride ion specific chemiluminescent probe (CL-F) by adopting 1,2-dioxetane as the chemiluminescent platform and Si-O moiety as the recognition unit [43]. The high performance and specificity towards fluoride ions of this probe have been demonstrated in aqueous solution with a dynamic range from 0 to 30 μM and a LOD of 0.91 μM. We further demonstrated the potentials of CL-F in practical applications, including quantification of fluoride ions in toothpaste and imaging fluoride ions in living mice. The features reported here strongly suggest that CL-F is a promising tool for the detection of fluoride ion in vitro and in vivo. Acknowledgements This work was supported by the National Key Research and Development Program of China (2018YFA0902200), the National Natural Science Foundation of China (Nos. 21978131,21722605 and21878156), the Six Talent Peaks Project in Jiangsu Province (XCL034) and the Project of Priority Academic Program Development of Jiangsu Higher Education Institutions(PAPD). Appendix A. Supplementary data Supplementary material related to this article can be found, in the 5
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Bowen Gu obtained his B.S. Degree in Chemistry from Yancheng Normal University (China) in 2017. Subsequently, he joined Prof. Xiaoqiang Chen’s group at Nanjing Tech University (China) as a master student. His research interests focus on developing fluorescent chemosensors. Chao Dong obtained his B.S. Degree in Chemistry from Anhui Polytechnic University. Subsequently, he joined Prof. Ruwei Shen’s group at Nanjing Tech University (China) as a master student. His research interests focus on synthetic organic chemistry and catalysis. Ruwei Shen obtained his Ph.D. in chemistry from Zhejiang University in 2009, and then moved to National Institute of Advanced Industrial Science and Technology (AIST, Japan), where he worked as a postdoctoral fellow with Prof. Li-Biao Han. After a short stay in Prof. Nobuaki Kambe’ group as a visiting scholar at Osaka University (Japan), he joined the faculty of the college of chemical engineering at Nanjing Tech University (China) in 2012. He is currently an associate professor working in the fields of synthetic organic chemistry and catalysis. Jian Qiang obtained his B.S. Degree in Chemistry from Huainan Normal University. Subsequently, he joined Prof. Xiaoqiang Chen’s group at Nanjing Tech University (China) as a master student. His research interests focus on developing fluorescent chemosensors. Tingwen Wei obtained his B.S. degree from Nantong University (China) in 2013. Subsequently, he joined Prof. Xiaoqiang Chen’s group at Nanjing Tech University (China) as a Ph.D. student. His research interests focus on developing fluorescent chemosensors. Fang Wang obtained her B.S. degree from Yan Bian University (China) in 2005 and her master degree from the College of Pharmacy, Kyung Hee University (Korea)in 2008. Subsequently, she joined Prof. Juyoung Yoon’s group at Ewha Womans University (Korea) as a Ph.D. student and received her Ph.D. in 2012. Currently, she is working at the College of Chemical Engineering, Nanjing Tech University (China). Her research interests mainly focus on developing fluorescent chemosensors. Sheng Lu received his Ph.D. in the department of Chemical Engineering from University of Waterloo (Canada) in 2015. In 2017, he started his postdoctoral fellowship in Waterloo Institute of Nanotechnology (WIN) at University of Waterloo, and continued his academic career as an assistant professor at College of Chemical Engineering, Nanjing Tech University (China) from 2019. His current research interests mainly focus on self-assembly and chemosensors. Xiaoqiang Chen received his Ph.D. in Applied Chemistry from the Dalian University of Technology (China) in 2007. In 2008, he worked as a postdoctoral fellow at Ewha Womans University (Korea). In March 2010, he moved to the Nanjing Tech University (China), where he is currently a professor at the College of Chemical Engineering. His current research interests mainly focus on fluorescent chemosensors.
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Dioxetane-based chemiluminescent probe for fluoride ion-sensing in aqueous solution and living imaging
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Bowen Gu, Chao Dong, Ruwei Shen, Jian Qiang, Tingwen Wei, Fang Wang, Sheng Lu , ⁎ Xiaoqiang Chen State Key Laboratory of Materials-Oriented Chemical Engineering, College of Chemical Engineering, Nanjing Tech University, Nanjing 211816, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Chemiluminescent probe Fluoride ion 1,2-Dioxetane Toothpaste Living mice In vivo imaging
Fluoride plays an important role in our daily life as it associates with a variety of biological and pathological processes. Hence, an analytic method that can detect fluoride ions under physiological conditions with high sensitivity and specificity, and fast response, is needed. Herein, we introduced a dioxetane-based chemiluminescent probe, CL-F, specifically designed for the detection of fluoride ions in physiological environment. The probe CL-F emitted strong green chemiluminescent light within 1 min when incubating with fluoride ions specifically. The linear range for sensing fluoride ions and limit of detection were determined to be 0–30 μM and 0.91 μM, respectively. The probe CL-F was then successfully applied in quantifying fluoride ions in toothpaste and in vivo imaging in living mice, demonstrating CL-F as a promising tool for sensing fluoride ion in vitro and in vivo.
1. Introduction Owing to the advances in molecular sensing approaches, the development of sensors with high sensitivity and selectivity for anions has attracted much attention due to their vital roles in chemical and biological processes [1–6]. Fluoride is one of the anions that are most relevant to our daily life. It is added as supplements in our daily goods, such as drinking water, milk, toothpaste and mouth rinses, due to its function in preventing tooth decay and osteoporosis [7,8]. However, excessive fluoride intake also leads to fluorosis, urolithiasis, or even cancer [9,10]. According to the U.S. Public Health Service, the optimal level of fluoride consumption is 1 mg per day, while the World Health Organization (WHO) recommends fluoride levels below 1.5 ppm in drinking water [11]. Since artificial sources of fluoride are also used in large-scale industrial applications, the produced industrial wastes would result in fluoride contamination in water sources, affecting our fluoride intake. Therefore, efficient and reliable sensing methods for fluoride levels in aqueous environment are highly demanded. The analytic approaches used for fluoride ion detection involve standard willard and winter methods, ion-selective electrode, and ion chromatography [12,13]. These conventional analytic methods have intrinsic drawbacks, such as complicated procedures, high cost and low mobility, which hinder the rapidness and convenience of fluoride detection. Recently, fluorescent chemosensors have received considerable
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attention due to their high sensitivity and specificity, and ease of operating, providing an alternative tool for the detection of fluoride ions [14–17]. But the inherent drawbacks of fluorescence assays, including photobleaching, phototoxicity and interference of autofluorescence from other species in the samples, restrict their practical applications. Comparing to fluorescence-based mechanisms, bio- and chemiluminescent assays can generate optical signals without excitation from external light sources, rendering them higher reliability, sensitivity and signal-to-noise ratios [18]. Therefore, bio- and chemiluminescent probes have been widely used for in vitro and in vivo imaging of various enzymes and analytes [19–22]. To date, extensive examples of bio- and chemiluminescent probes are designed based on two major mechanisms, firefly luciferase-luciferin system and adamantylidene − dioxetane model [23–28]. Despite holding advantages of sensitivity and high signal-to-noise ratio, firefly luciferase-luciferin systems require exogenous gene expression of luciferase to trigger the luminescence, elevating the complexity in practical applications [29]. The adamantylidene − dioxetane developed by Schaap is a classic molecular model to construct chemiluminenscent probes, which do not require the activation by oxidation due to the relative stable dioxetane moiety [30–32]. This system exhibits a good capability of emitting light in organic solvents with medium polarity, but performs badly under aqueous conditions [33]. The addition of a surfactant, enhancer Emerald-II, was found to amplify the luminescence in aqueous solution by providing a
Corresponding authors. E-mail addresses: [email protected] (S. Lu), [email protected], [email protected] (X. Chen).
https://doi.org/10.1016/j.snb.2019.127111 Received 23 July 2019; Received in revised form 5 September 2019; Accepted 6 September 2019 Available online 09 September 2019 0925-4005/ © 2019 Elsevier B.V. All rights reserved.
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imaging was obtained using an Automatic Chemiluminescence Image Analysis System in Tris buffer solution, after distinct densities of F− (0, 5, 15, 45, 60, 80 μM) were mixed into a 96-well plate. 2.4. Chemiluminescence test to toothpaste Four grams of toothpaste dissolved in 40 ml water, then aqueous solution was filtered with a filter to get 38 mL clear solution. 30 μL filtered aqueous solution of toothpaste was added into 2970 μL buffer solution containing 2 μM CLF. The control group was carried out in the same manner except that the clear solution was replaced with Tris buffer. Chemiluminescent intensity was acquired 30 s latter after incubation of CLF (2 μM) with clear solution in buffer. All tests were performed in Tris buffer solution.
Scheme 1. The preparation path of CL-F.
hydrophobic environment that reduces the water-induced quenching [34]. However, the toxicity of surfactants impedes the applications in biological fields. More recently, Shabat’s group improved the luminescence quantum yield of the adamantylidene − dioxetane probe in aqueous conditions by introducing an electron withdrawing group (EWG) [35]. Inspired by this, our group designed and synthesized a novel fluoride ion specific dioxetane probe CLF (Scheme 1), for fluoride sensing and quantification under aqueous conditions. This probe was shown to emit strong green light rapidly after incubating with fluoride ions in aqueous solutions, and possessed an outstanding limit of detection (LOD) of 0.91 μM. The feasibility of quantifying fluoride ions in toothpaste and live imaging in mice was proven in subsequent studies. This work provides a promising tool and strategy for fluoride sensing in aqueous environment.
2.5. MTT assay The cytotoxicity of CL-F toward living cells was checked via MTT assay. HeLa cells were added to 96-well culture plate incubated for 12 h. Next, CL-F (2, 5 and 10 μM) was mixed in the wells and then 20 μL of MTT (5.0 mg/mL) were poured into each well after incubation of 12 h. Four hours later, the supernatant was thrown away and 150 μL DMSO was added to each well. Finally, the absorbance of formazan solution was collected at 490 nm. 2.6. Chemiluminescence assays in living mice CL-F (0.2 mM) and F− (6 mM) were readied in advance. The images of living mice were gained by using an Automatic Chemiluminescence Image Analysis System. Firstly, a mixture solution containing 20 μM F−, 2 μM CL-F, and Tris−HCl buffer solution at the total volume of 50 μL was injected into the back of mice, the chemiluminescence was collected immediately. As control, a 50 μL solution was carried out in the same manner except that the fluoride ion was replaced with Tris buffer solution. All imaging investigations used anesthetized BALB-C mice.
2. Experimental section 2.1. Materials and instruments Unless otherwise stated, the experimental drug was purchased directly from the supplier and was not repurified. Chemical products were purified and separated by column chromatography. Bruker 400 Nuclear Magnetic Resonance Spectrometer was used to gain 1H NMR and 13C NMR spectra. Electrospray ionization mass spectra (ESI–MS) were performed by Agilent 1260 HPLC-6500 Q-TOF Mass Spectrometer. Chemiluminescent spectra were recorded by RF-5301PC fluorescence spectrophotometer. MTT assay were performed by Multiskan Go (51119200-VAN). The chemiluminescent images were obtained by using an Automatic Chemiluminescence Image Analysis System.
3. Results and discussions 3.1. Fluoride ion triggered chemiluminescence of CL-F The general mechanism of fluoride ion sensing of this probe is presented in Scheme 2. Removal of the triggering substrate by fluoride ion releases the phenolate-dioxetane intermediate (product 1), product 1 decomposes through a chemically initiated electron exchange luminescence (CIEEL) process to produce the electronically excited benzoate ester (product 2) [36,37]. This excited benzoate ester relaxes to its
2.2. Synthesis The synthetic route of probe CLF is illustrated in Scheme 1. Starting material compound a was obtained in the light of previous literatures [35,36]. Then the phenol group of compound a was protected with tertbutyldimethylsilyl (TBS) choride to gain compound b. Finally, our desired product CLF was obtained by the oxidation of b. The identity of compound b and product CLF was proved via 1H NMR, 13C NMR, and ESI–MS (Figure S9–Figure S14). 2.3. Chemiluminescence study in vitro A stock DMSO solution of 0.2 mM CL-F was prepared. 2960 μL of Tris buffer (50 mM, pH = 7.4), 10 μL of different concentrations of F−, 30 μL DMSO solution of 0.2 mM CL-F were added to test glass tube. A chemiluminescent kinetic curve was acquired by using time scanning module. With the response of probe CL-F to F−, diverse concentrations of F− (0, 5, 10, 15, 20, 30, 45, 70, 100 μM) was treated with 2 μM CL-F in Tris buffer solution, then tested the chemiluminescent intensity 30 s latter. The selectivity of CL-F towards various anions was illustrated by testing the chemiluminescent light emission. The chemiluminescent
Scheme 2. Activation pathway of probe CL-F for fluoride ion. 2
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Fig. 1. (A) Time scans of the chemiluminescent intensity of CL-F (2 μM) reacted with fluoride ion (20 μM) in Tris buffer (50 mM, pH = 7.4). The inset shows emission spectra of CL-F for various concentrations of fluoride. (B) Chemiluminescent intensity acquired 30 s latter after incubation of CL-F (2 μM) with various concentrations of fluoride ion in Tris buffer (50 mM, pH = 7.4). (C) Time-lapse images of probe within 50 min.
shown in Fig. 2B, an evident enhancement in chemiluminescent signal appeals in the presence of fluoride ion, while the signals are barely seen after incubating with other anions. The pH effect and selectivity data support that probe CL-F is suitable for fluoride ion detection at physiologically relevant conditions with a good specificity.
ground state and yields a green chemiluminescent signal. The identity of final product 3 was proved by ESI-MS (Figure S15). With the probe CL-F in hand, we first studied the kinetics of the fluoride-dependent chemiluminescent emission. Upon the addition of 20 μM of fluoride ion, the intensity of chemiluminescent light emitted from CL-F buffered solution (50 mM Tris, pH 7.4) as a function of time was measured. As shown in Fig. 1A, the chemiluminescent light intensity increases sharply when CL-F reacted with fluoride ions, and maximizes within 1 min, following by a slow decay over a time course of 50 min. This profile of CL-F represents a typical chemiluminescence kinetic behavior. The chemiluminescent kinetic process was also visualized in Fig. 1C. Chemiluminescent light was barely observed without the presence of fluoride ions (Figure S2), validating CL-F as a sensor for fluoride ions. Then we investigated the responsibility of probe CL-F to fluoride ions at a series of concentrations ranging from 0 to 100 μM. As shown in Fig. 1B, the chemiluminescent intensity increases with elevated fluoride ion concentrations. The enhancement of the chemiluminescence is linearly proportional to the increment of fluoride ion concentration in the range of 0–30 μM, indicating the dynamic range of probe CL-F for fluoride quantification. The LOD of fluoride (3σ/k) was determined to be 0.91 μM (∼17.3 ppb), where σ represents the standard deviation of blank measurement, while k represents the slope of the plot for the chemiluminescent intensity versus fluoride concentrations. The LOD is advantageous to those of previously reported methods, which are mostly beyond 25 ppb [38–42]. The outstanding performance could be attributed to the high chemiluminescent efficiency of probe CL-F.
3.3. Comparison to a conventional chemiluminescent probe To demonstrate the superior activity of probe CL-F in fluoride ion sensing, XCL-F, a fluoride ion specific chemiluminescent probe based on the mechanism of Schapp’s adamantylidene-dioxetane [30], was synthesized to make a comparison with CL-F. The synthetic route of XCL-F is shown in Scheme S1. As expected, after incubating with fluoride ions, XCL-F only emitted a moderate blue light in DMSO (Fig. 3A and Figure S3), but not in aqueous solution (Fig. 3B and C). After the addition of Emerald-II enhancer (10%, v/v), XCL-F was able to sense fluoride ions by emitting a green light (Figure S4). The chemiluminescent intensity was enhanced by 106-fold when compared to the case without the enhancer (Fig. 3B and C). However, the intensity of chemiluminescent signal generated from probe CL-F without the enhancer was approximate 6-fold higher than that of probe XCL-F with the presence of enhancer (Fig. 3D and E). The comparison suggests that our probe CL-F holds superior efficiency for sensing fluoride ions in aqueous environment. 3.4. Quantification of fluoride ions in toothpaste Since toothpaste is one of the most common fluoride sources in our daily lives, we then continued to quantify the concentration of fluoride ions in Crest toothpaste using probe CLF as an example of practical application. To prepare the test samples, four grams of toothpaste were dissolved in water, following by filtration to obtain a clear solution. 30 μL of the filtered aqueous solution was added into 2970 μL buffer solution containing 2 μM of CLF. The control group was conducted by substituting the toothpaste sample with a blank buffer solution. The toothpaste sample induced a chemiluminescent intensity of 34.5 (Fig. 4). Given the calibration curve obtained in Fig. 1B, the concentration of NaF in toothpaste was calculated to be 1.02 mg/g, with a difference less than 8% when compared to the content provided by the manufacturer, which is 1.1 mg/g. These results clearly show that CLF
3.2. pH influence and selectivity investigation Next, the pH effect on the response of probe CL-F toward fluoride ions was tested. As shown in Fig. 2A, the chemiluminescent intensity increases gradually from pH 4.0 to 8.0, reaching a maximum in the pH range from 7.5 to 8.0, whereas decreases along with further basified environment. These data indicate that CL-F can detect fluoride ion at physiological condition at its highest sensitivity. To further evaluate the specificity of CL-F probe for fluoride sensing, twelve common anions (Br−, C2O42-, I−, SO32-, N3−, NO2−, NO3−, SCN−, OH−, SO42-, H2PO4−,Cl−) were added into CL-F buffer solutions individually and the chemiluminesent responses were monitored. As 3
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Fig. 2. (A) Chemiluminescent intensity acquired 30 s latter after incubating CL-F (2 μM) with fluoride ions (20 μM) in buffer solutions at different pH values. (B) Chemiluminescent intensity acquired 30 s latter after incubating CL-F (2 μM) with fluoride ions (20 μM) and other anions (100 μM) in Tris buffer (50 mM, pH = 7.4). The inset shows chemiluminescent imaging of CL-F (5 μM) with fluoride (20 μM) and other anions (100 μM) in buffer solution containing 5% DMSO. All the anions mentioned above are derived from the sodium salt: NaBr, Na2C2O4, NaI, Na2SO3, NaN3, NaNO2, NaNO3, NaSCN, NaOH, Na2SO4, NaH2PO4, NaCl, NaF.
Fig. 3. (A) Chemiluminescent imaging after incubation of CL-F (5 μM) and XCL-F (5 μM) with fluoride ion (20 μM) in Tris buffer (50 mM, pH 7.4), 5% DMSO, and DMSO respectively. (B) Chemiluminescent intensity of probe XCL-F (5 μM) with or without Emerald-II enhancer (10%, v/v) in Tris buffer (50 mM, pH 7.4) containing 5% DMSO, in the presence of fluoride ion (20 μM), and (C) Chemiluminescent emission spectrum. (D) Chemiluminescent intensity of probe XCL-F (5 μM) enhanced by Emerald-II enhancer (10%, v/v) and probe CL-F (5 μM) in Tris buffer (50 mM, pH7.4) containing 5% DMSO, in the presence of fluoride ion (20 μM), and (E) Chemiluminescent emission spectrum.
Fig. 4. (A) Chemiluminescent intensity acquired 30 s latter after incubation of CL-F (2 μM) with 30 μL clarified aqueous of toothpaste in buffer solution, and (B) Chemiluminescent emission spectrum.
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Fig. 5. (A) In vitro images of CLF chemiluminescence after adding CLF (2 μM) to 0, 5, 15, 45, 60, and 80 μM of fluoride ion in Tris buffer solution (n = 3). (B) Chemiluminescent imaging of fluoride ion in vivo. (right) The mouse was injected with 50 μL of buffer solution containing CLF (2 μM) and fluoride ion (20 μM). (left) The control group was injected with 50 μL of Tris buffer (50 mM, pH = 7.4) solution solely containing CLF (2 μM).
probe is capable of quantitatively sensing fluoride in a practical scenario with much complexity.
online version, at doi:https://doi.org/10.1016/j.snb.2019.127111. References
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Encouraged by the strong signal response of probe CLF toward fluoride ions in vitro, we continued on evaluating its potential for detecting fluoride ions in vivo. Beforehand, we firstly studied the chemiluminescent imaging of CLF triggered by fluoride ions with an Automatic Chemiluminescence Image Analysis System under physiological conditions in vitro. Fluoride ions were added into the wells of a 96-well black plate containing CLF (2 μM) buffer solution with final concentrations of 0, 5, 15, 45, 60, and 80 μM, respectively. As shown in Fig. 5A, the light output exhibits an observable intensity when the concentration of fluoride ions is above 5 μM. Moreover, the experimental data shown that probe CLF has little toxicity to living cells when its concentration is lower than 10 μM (Figure S5). Subsequently, the live imaging of fluoride ions was conducted on anesthetized BALB-C mice model. A mixture buffer solution containing CLF (2 μM) and fluoride ion (20 μM) was injected to the back of the mice at the volume of 50 μL, and the chemiluminescent signal was monitored under the Automatic Chemiluminescence Image Analysis System. As shown in Fig. 5B, a strong light signal is observed at the injection site while the signal from the control group is negligible. These results demonstrate that probe CLF is capable of monitoring the presence of fluoride ions in vivo. 4. Conclusion In summary, we designed and prepared a fluoride ion specific chemiluminescent probe (CL-F) by adopting 1,2-dioxetane as the chemiluminescent platform and Si-O moiety as the recognition unit [43]. The high performance and specificity towards fluoride ions of this probe have been demonstrated in aqueous solution with a dynamic range from 0 to 30 μM and a LOD of 0.91 μM. We further demonstrated the potentials of CL-F in practical applications, including quantification of fluoride ions in toothpaste and imaging fluoride ions in living mice. The features reported here strongly suggest that CL-F is a promising tool for the detection of fluoride ion in vitro and in vivo. Acknowledgements This work was supported by the National Key Research and Development Program of China (2018YFA0902200), the National Natural Science Foundation of China (Nos. 21978131,21722605 and21878156), the Six Talent Peaks Project in Jiangsu Province (XCL034) and the Project of Priority Academic Program Development of Jiangsu Higher Education Institutions(PAPD). Appendix A. Supplementary data Supplementary material related to this article can be found, in the 5
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Bowen Gu obtained his B.S. Degree in Chemistry from Yancheng Normal University (China) in 2017. Subsequently, he joined Prof. Xiaoqiang Chen’s group at Nanjing Tech University (China) as a master student. His research interests focus on developing fluorescent chemosensors. Chao Dong obtained his B.S. Degree in Chemistry from Anhui Polytechnic University. Subsequently, he joined Prof. Ruwei Shen’s group at Nanjing Tech University (China) as a master student. His research interests focus on synthetic organic chemistry and catalysis. Ruwei Shen obtained his Ph.D. in chemistry from Zhejiang University in 2009, and then moved to National Institute of Advanced Industrial Science and Technology (AIST, Japan), where he worked as a postdoctoral fellow with Prof. Li-Biao Han. After a short stay in Prof. Nobuaki Kambe’ group as a visiting scholar at Osaka University (Japan), he joined the faculty of the college of chemical engineering at Nanjing Tech University (China) in 2012. He is currently an associate professor working in the fields of synthetic organic chemistry and catalysis. Jian Qiang obtained his B.S. Degree in Chemistry from Huainan Normal University. Subsequently, he joined Prof. Xiaoqiang Chen’s group at Nanjing Tech University (China) as a master student. His research interests focus on developing fluorescent chemosensors. Tingwen Wei obtained his B.S. degree from Nantong University (China) in 2013. Subsequently, he joined Prof. Xiaoqiang Chen’s group at Nanjing Tech University (China) as a Ph.D. student. His research interests focus on developing fluorescent chemosensors. Fang Wang obtained her B.S. degree from Yan Bian University (China) in 2005 and her master degree from the College of Pharmacy, Kyung Hee University (Korea)in 2008. Subsequently, she joined Prof. Juyoung Yoon’s group at Ewha Womans University (Korea) as a Ph.D. student and received her Ph.D. in 2012. Currently, she is working at the College of Chemical Engineering, Nanjing Tech University (China). Her research interests mainly focus on developing fluorescent chemosensors. Sheng Lu received his Ph.D. in the department of Chemical Engineering from University of Waterloo (Canada) in 2015. In 2017, he started his postdoctoral fellowship in Waterloo Institute of Nanotechnology (WIN) at University of Waterloo, and continued his academic career as an assistant professor at College of Chemical Engineering, Nanjing Tech University (China) from 2019. His current research interests mainly focus on self-assembly and chemosensors. Xiaoqiang Chen received his Ph.D. in Applied Chemistry from the Dalian University of Technology (China) in 2007. In 2008, he worked as a postdoctoral fellow at Ewha Womans University (Korea). In March 2010, he moved to the Nanjing Tech University (China), where he is currently a professor at the College of Chemical Engineering. His current research interests mainly focus on fluorescent chemosensors.
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