- Email: [email protected]
A reversible fluorescent probe for Zn2+ and ATP in living cells and in vivo
Sensors and Actuators B 261 (2018) 127–134
Contents lists available at ScienceDirect
Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
A reversible fluorescent probe for Zn2+ and ATP in living cells and in vivo Xilang Jin a , Xianglong Wu b , Bo Wang c , Pu Xie a , Yaolong He a , Hongwei Zhou a,∗ , Bo Yan a , Jingjing Yang a , Weixing Chen a , Xianghan Zhang c,∗ a
School of Materials and Chemical Engineering, Xi’an Technological University, Xi’an, 710032, Shaanxi, China Key Laboratory for Space Bioscience and Biotechnology, School of Life Sciences, Northwestern Polytechnical University, Xi’an, Shaanxi, 710072, China c School of Life Sciences and Technology, Xidian University, Xi’an, 710071, China b
a r t i c l e
i n f o
Article history: Received 29 August 2017 Received in revised form 10 January 2018 Accepted 11 January 2018 Keywords: Fluorescein Zinc ion ATP Visualizing Fluorescence
a b s t r a c t A reversible fluorescein-based fluorescent probe which exhibits high sensitivity and selectivity for Zn2+ and ATP, has been designed and synthesized. The sensing process was completed via fluorescence variation induced by an opening and closing of the spiro-ring of fluorescein. Zn2+ /ATP-induced fluorescent intensity shows a good linear relationship with the concentration of Zn2+ /ATP in the range of 0–10 M with a detection limit of 0.1 M/0.5 M. Moreover, the probe was further applied to visualize and detect Zn2+ and ATP in vitro and in vivo. © 2018 Elsevier B.V. All rights reserved.
1. Introduction Zinc ion is recognized as one of the most abundant transition metals ions in the environmental and biological systems [1]. It plays indispensable roles in various physiological and pathological processes, such as genetic expression, cell apoptosis, enzyme regulation and neural transmission [2–4]. In addition, the disorder of zinc ion in living organisms may associate with Pakinson’s diseases, Alzheimer’s diseases and immune dysfunction [5–7]. On the other hand, adenosine-5 -triphosphate (ATP) is an essential biomolecules in cell biology due to its energy production and storage for many cellular events [8,9]. ATP is crucial in energy metabolism, DNA replication and transcription, and other fundamental activities [10]. Therefore, it is of great importance to develop a compelling method to quantitatively monitor the concentration of Zn2+ and ATP in both the environmental and biological systems. Compared with the traditional methods, fluorescence sensing shows obviously specific advantages in monitoring in vitro and in vivo biologically relevant species, such as metal ions, anions and active molecules due to its convenient operation and high sensitivity [11,12]. To date, numerous excellent fluorescein dyes have been
developed to detect various target objects, e.g. Cu2+ [13,14], Hg2+ [15,16], Zn2+ [17,18], Fe3+ [19,20], NO [21,22], H2 S [23,24], HOCl [25,26] and pH [27,28]. Unfortunately, there is few ATP probes have been applied in vitro and in vivo in recent decades [29,30]. Consequently, it remains a great challenge to explore highly selective and sensitive probes for monitoring the concentration of Zn2+ and ATP in vitro and in vivo. Recently, our group has designed and synthesized a series of fluorescein-based fluorescent probes for biologically relevant metal ions [31] and reactive oxygen [32,33] and sulfur species [34]. As a continuation of previous work, we report herein a fluoresceincontaining fluorescent turn-on probe with demonstrated specific selectivity to Zn2+ via formation of zinc complexes. Moreover, we developed the zinc complexes as a fluorescent probe which displayed a turn-off fluorescence change for ATP detection. We believe that this probe provides a potential and powerful approach for monitoring and visualizing Zn2+ and ATP in living cells and in vivo Scheme 1.
2. Experimental 2.1. Materials and measurements
∗ Corresponding authors. E-mail addresses: jinxilang [email protected] (X. Jin), [email protected] (H. Zhou), [email protected] (X. Zhang). https://doi.org/10.1016/j.snb.2018.01.116 0925-4005/© 2018 Elsevier B.V. All rights reserved.
All reagents were obtained from J&K Scientific, Co., Ltd. (Shanghai China) and used directly without further purification
128
X. Jin et al. / Sensors and Actuators B 261 (2018) 127–134
Scheme 1. Synthesis of probes 1.
throughout the whole experiment. Fluorescence spectra measurements were performed on a Hitachi F-4500 fluorescence spectrophotometer equipped with a xenon discharge lamp, in a 1 cm quartz cell. UV–vis spectra were recorded with a Shimadzu UV-1700 spectrphotometer. Mass spectra were measured using a Brukermicro TOF-QII ESI-Q-TOF LC/MS/MS Spectrometer by means of the electronic spray ionization (ESI). NMR spectra were recorded on a Varian INOVA-400 MHz spectrometer (at 400 MHz for 1 H NMR) using tetramethylsilane (TMS) as internal standards. Fluorescent images were performed using a Leica SPE confocal laser scanning microscope with an excitation wavelength of 488 nm. In vivo fluorescence imaging analysis was carried out in an IVIS Kinetic imaging system. Scanningelectron microscope (SEM) images were obtained on a FEI Quanta 400F scanning electron microscope system at an acceleration of 20 kV. The samples were sputter-coated with gold in vacuum before observation.
2.4. Synthesis of probes 1 Fluorescein hydrazide was synthesized in a high yield according to the procedures reported in literature [32]. Fluorescein hydrazide (10 mmol, 3.46 g) was mixed with compound 3 (11 mmol, 1.70 g) in 30 mL of ethanol with 1 drops of CH3 COOH. The reaction mixture was refluxed for 24 h. After cooling to room temperature, the white crude product was filter off from the reaction mixture and purified by column chromatography on silica gel using MeOH: CH2 Cl2 = 1: 30 as the eluent, to give 3.82 g pink power, yield 75.49%. 1 H NMR(DMSO-d6 , 400 MHz): ␦ (ppm) 1.21 (s, 9H), 6.49 (m, 4H), 6.65 (d, J = 2.0 Hz, 2H), 6.71 (d, J = 8.6 Hz, 1H), 7.19 (m, 1H), 7.26 (dd, J = 8.6, 2.5 Hz, 1H), 7.37 (d, J = 2.5 Hz, 1H), 7.65 (ddd, J = 10.5, 7.3, 1.3 Hz, 2H), 7.94 (m, 1H), 9.26 (s, 1H), 9.92 (s, 2H), 10.02 (s, 1H). MS (ESI) m/z = 529.1876 [M+ Na]+ , calc. for C28 H20 N2 O6 Na = 529.1734.
2.2. General procedure 3. Results and discussion Probes 1 stock solution (1 mM) was prepared in acetonitrile. The solutions of various testing species stock solutions (1 mM) were prepared in distilled water. During the titration experiments, different amounts of Zn2+ and 0.10 mL of 1000 M probes were mixed and filled up with PBS to 10 mL in volumetric tubes. During the interference experiments, 30 M Zn2+ , 0.10 mL of 1000 M probe 1 and 1 mL of 1000 M testing species were mixed and filled up with PBS to 10 mL in volumetric tubes. During the titration experiments of ATP, 0.10 mL of 1000 M probes, 0.1 mL of 1000 M Zn2+ and different amounts of ATP was mixed and filled up with PBS to 10 mL in volumetric tubes. 1 mL aliquots were pipetted into a 1 cm cuvette for spectral measurements. 5 nm bandpasses were used for both excitation and emission wavelengths. An excitation wavelength of 420 nm was used for the acquisition of emission spectra. 2.3. Synthesis of intermediate 3 4-tert-Butylphenol (5.14 g, 34.2 mmol) and hexamethylenetetramine (9.60 g, 68.5 mmol) were dissolved in anhydrous trifluoroacetic acid (60 mL) under N2 , and the reaction mixture was refluxed for 24 h. The mixture was poured into 4 M HCl (200 mL) and stirred for 10 min, after which it was extracted with CH2 Cl2 (2 × 150 mL). The combined organic extracts were washed with 4 M HCl(2 × 200 mL), water (200 mL), saturate brine (200 mL), then dried with Na2 SO4 and concentrated in vacuum. The crude product was purified by silica gel column chromatography to give 4.58 g yellow viscous liquid, yield 64.88%.
3.1. Optical response towardsZn2+ First of all, the time-dependent fluorescence response of the probe was investigated in the presence of 3.0 equiv. Zn2+ in CH3 CNPBS (1/99, v/v, pH 7.4) solution. As demonstrated in Fig. S2, the maximal fluorescence signal at 527 nm reached the maximum within 5 min at room temperature, indicating probe 1 is adaptive for real-time detection of Zn2+ and the reaction time of 5 min was applied for the subsequent experiments. The fluorescence titrations experiments of probe 1 with Zn2+ were then performed. As expected in Fig. 1a, the free probe (10 M) exhibited almost no emission band ( = 0.0012) due to the closed fluorescein-spirolactam form. Upon the introduction of 0.0-4.0 equiv. of Zn2+ , a dramatic enhancement of the fluorescence intensity at 527 nm was observed, with the fluorescence quantum yield of f = 0.45 (using rhodamine 6G in ethanol). This phenomenon was ascribed to Zn2+ ions induced structural transformation from the colorless spirolactam ring to the colored ring-opening delocalized form of fluorescein. As shown in Fig. 1b, the fluorescence titration curve revealed that the fluorescence intensity at 527 nm showed a good linear relationship against the concentration of Zn2+ in the range from 0.0 to 10.0 M. The regression equation was y = 23.80 x–3.323 (R2 = 0.997) and the detection limit (S/N = 3) of probe 1 for Zn2+ was found to be 0.1 M. These results demonstrated that probe 1could be potentially applicable for qualitative and quantitative analysis of Zn2+ .
X. Jin et al. / Sensors and Actuators B 261 (2018) 127–134
129
Fig. 1. (a) Fluorescence intensity (ex = 420 nm) changes of probe 1 (10 M) upon addition of Zn2+ (0.0–4.0 equiv) in CH3 CN-PBS (1/99, v/v, pH = 7.4) solution.The fluorescence intensity was measured at 527 nm. (b) Calibration curve of fluorescence intensity (ex = 420 nm) in dependence of Zn2+ concentrations. Data were acquired in CH3 CN-PBS (1/99, v/v, pH = 7.4). The fluorescence intensity was measured at 527 nm.
experiments further revealed that probe 1 was a potential candidate for the quantitative detection of Zn2+ with high selectivity and specificity. To investigate a suitable pH range for Zn2+ sensing, the pH effect on the fluorescence performance of probe 1 in the absence and presence of Zn2+ was further tested. As shown in Fig. S3, the experimental results indicated that the probe could effectively detect Zn2+ in the pH range between 6.8 and 9.0, which implies its potential for application in the physiological pH range.
3.2. Optical response towards ATP
Fig. 2. Selectivity of probe 1 (10 M) for Zn2+ in CH3 CN-PBS (1/99, v/v, pH = 7.4) solution. The pillars in the front row represent the value in the presence of representative metal ions (100 M). The pillars in the back row indicate the change in the emission intensity upon subsequent addition of Zn2+ (30 M) to the solution containing probe 1(10 M) and the respective ion of interest (100 M). For all measurements, the fluorescence intensity was measured at 527 nm.
Fig. 1 High-level selectivity and sensitivity is one of the most important criteria for the new sensing systems. As shown in Fig. 2, nineteen representative metal ions were conducted, by monitoring the fluorescence response in the presence of Li+ , Na+ , K+ , Ba2+ , Ca2+ , Cd2+ , Ag+ , Mg2+ , Co2+ , Mn2+ , Sn4+ , Pb2+ , Hg2+ , Ni2+ , Cr3+ , Cu2+ , Fe2+ , Fe3+ , Zn2+ under the identical additions. As envisioned, no obvious fluorescence response was observed in the presence of the other tested species. Only the addition of Zn2+ (30 M) caused a noticeable enhancement of the fluorescence intensity at 527 nm, displaying efficient turn-on responses. Fig. 2 To further evaluate the anti-interference properties of probe 1 towards Zn2+ , the competitive experiments were carried out in the presence of both Zn2+ (30 M) and coexisting metal ions (100 M). As shown in Fig. 3, the fluorescence intensity induced by the mixture of Zn2+ with competitive ions was similar to that caused by Zn2+ alone, which indicated that the competitive ions exerted no significant interfere during the sensing of Zn2+ .These
As shown in Fig. 3, upon the progressive addition of ATP (0.0-1.0 equiv.) to the solution of complex 1-Zn2+ resulted in about 4.59 fold decrease in the fluorescence intensity at 527 nm. The spectral phenomenon may be attributed to the strong binding ability between ATP and Zn2+ , which results in the removal of Zn2+ from the complex and structural recovery of the compound.The observed fluorescence intensity at 527 nm was nearly proportional to the concentration of ATP ranging from 0.0 to 10.0 M. The regression equation was y = − 16.94x + 220.8 (R2 = 0.994). And the detection limits (LOD) of complex1- Zn2+ to ATP is calculated to be 0.50 M based on S/N = 3. Upon the further addition of Zn2+ , the fluorescence intensity was recovered again, suggesting the reversibility of probe 1 (1+ Zn2+ ) binding to Zn2+ (ATP). This reversible process could be repeated several times with less fluorescent efficiency (Fig. 4). These results reveal that the binding of probe 1 and Zn2+ occurred via a reversible spirolactam ring open-close mechanism. To further verify the selectivity, the fluorescence emission of complex 1-Zn2+ was conducted with some representative anions, containing F− , Cl− , Br− , I− , CO3 2− , HS− , HSO3 − , Ac− , S2 O3 2− , SO4 2− , NO2 − , NO3 − , H2 PO4 − , PO4 3− , HPO4 2− , PPi, AMP, ADP. As displayed in Fig. 5, other competitive anions did not cause any notably emission quenching of complex 1-Zn2+ even at a concentration of 100 M anion under the same condition. Only ATP could remarkably quench the fluorescence intensity of the solution of complex 1-Zn2+ . Addition of ATP into a solution of 1-Zn2+ with other competitive anions together induced only slight decrease or a mildly change. Complex 1-Zn2+ also exhibited satisfactory selectivity toward ATP in a mixture of all competitive anions, which manifested the highly selectivity and sensitivity towards ATP.
130
X. Jin et al. / Sensors and Actuators B 261 (2018) 127–134
Fig. 3. (a) Fluorescence intensity (ex = 420 nm) changes of probe 1-Zn2+ (10 M) upon addition of ATP(0.0–1.0 equiv) in CH3 CN-PBS (1/99, v/v, pH 7.4) solution.The fluorescence intensity was measured at 527 nm. (b) Calibration curve of fluorescence intensity (ex = 420 nm) in dependence of ATP concentrations. Data were acquired in CH3 CN-PBS (1/99, v/v, pH 7.4). The fluorescence intensity was measured at 527 nm.
Fig. 4. Stepwise complexation/decomplexation cycles carried in CH3 CN-PBS (1/99, v/v, pH = 7.4) solution. Data were acquired in CH3 CN-PBS (1/99, v/v, pH = 7.4). The fluorescence intensity was measured at 527 nm.
3.3. Sensing mechanism study of probe 1 with Zn2+ and ATP To find out the binding stoichiometric ratio of probe 1 towards Zn2+ , Job’s plot between the change of fluorescence intensity and the mole fraction of probe 1 and Zn2+ was carried out (Fig. S4). A maximum fluorescence value was observed when the molar fraction of the probe [1] versus [1] + [Zn2+ ] was 0.66, which is indicative of a 2:1 stoichiometry for probe 1 and Zn2+ . According to the fluorescence spectra, the formation delocalized xanthane of fluorescein moiety was inferred to take place as depicted in Scheme 2. To further confirm the sensing mechanism, we performed high-resolution mass spectroscopy (HRMS) of probe 1 treated with Zn2+ and complex 1-Zn2+ treated with ATP. As depicted in Fig. S8–10, a new peak was obtained at m/z 1075.2853 (1-Zn2+ + H+ ) after the addition of excess Zn2+ , coinciding exactly with that for the transformation from probe 1 to compound 1Zn2+ + H+ (calcd: m/z 1075.2880) induced by Zn2+ .When complex 1-Zn2+ was treated with ATP, a new peak at m/z 529.1876 (probe 1, calcd: m/z 529.1734) was obtained. That is to say that this complex exhibits response to ATP based on the zinc complex ensemble displacement mechanism.
Fig. 5. Selectivity of complex 1-Zn2+ (10 M) for ATP in CH3 CN-PBS (1/99, v/v, pH = 7.4) solution. The pillars in the back row represent the value in the presence of representative anions (100 M). The pillars in the front row indicate the change in the emission intensity upon subsequent addition of ATP (10 M) to the solution containing complex 1-Zn2+ (10 M) and the respective ion of interest (100 M). For all measurements, the fluorescence intensity was measured at 527 nm.
Furthermore, IR spectra of probe 1 and complex 1-Zn2+ were also checked respectively in KBr disks, and the results were shown in Fig. S11. In the IR spectra, the peak at 1684 cm−1 of the probe 1, which corresponds to the characteristic amid carbonyl absorption, was shifted to 1654 cm−1 for the complex 1-Zn2+ , suggesting that a strong binding of the carbonyl group occurs with zinc ion. As shown in Fig. 6, the morphological changes of probe 1 and complex 1+ Zn2+ were studied by scanning electron microscope(SEM) analysis. Upon the addition of Zn2+ , the crystallographic morphology of complex became agglomerated due to the formation of 1+ Zn2+ complex. Therefore, all these data indicated that the reaction most likely followed the proposed mechanism shown in Scheme 2.
X. Jin et al. / Sensors and Actuators B 261 (2018) 127–134
131
Scheme 2. The Zn2+ /ATP-sensing mechanism of probe 1.
Fig. 6. SEM images of (a) probe 1 and (b) complex 1 + ZnCl2 .
Table 1 Detection results of Zn2+ in water samples by probe 1. Sample
Zn2+ spiked (mol/L)
Zn2+ recovered meana ± SDb (mol/L)
Recovery (%)
pool water
0 5 25 0 5 25
Not detected 4.9 ± 0.03 24.6 ± 0.02 Not detected 4.8 ± 0.03 24.4 ± 0.02
– 98.0 98.4 – 96.0 97.6
tap water
a b
Mean of three determination. SD: standard deviation.
132
X. Jin et al. / Sensors and Actuators B 261 (2018) 127–134
Fig. 7. (a–c) Confocal fluorescence images of T-cells incubated with probe 1 (50 M) for 30 min at 37 ◦ C; (d-f) The cells were stained with probe 1 (50 M) for 30 min at 37 ◦ C and then treated with ZnCl2 (50 M) for 30 min at 37 ◦ C; (g–i) The cells were stained with probe 1 (50 M) for 30 min at 37 ◦ C and then treated with ZnCl2 (50 M) followed by treatment with ATP(50 M) for 30 min at 37 ◦ C.
3.4. Applications of probe 1 in real water samples In order to understand the practicability of probe 1, we examined the concentration of Zn2+ in tap water and pool water samples by our above proposed fluorescent probe under the identical conditions, and the results were summarized in Table 1. The detecting data revealed a good agreement between the added and the found concentrations of Zn2+ . Thus, our proposed probe could be potentially used for monitoring Zn2+ in real water samples.
that probe 1 was good cell permeable and can be employed for monitoring Zn2+ and ATP within living cells. Fig. 7 Based on the desirable results in vitro, we investigated probe 1 to visualize Zn2+ and ATP in living animal. As shown in Fig. 8, the living mice without any treatment showed no fluorescence. And the mice which were given a skin-pop injection of probe 1 for 10 min also exhibited no fluorescence (Fig. 8b). When the living mice were further injected with Zn2+ (1.0 equiv.), a significant enhancement was observed observably with time (Fig. 8c–d). Upon the addition of ATP (1.0 equiv.), a significant decrease was observed. The results indicate that probe 1 could serve as a fluorescent agent for Zn2+ and ATP imaging in living organisms.
3.5. Visualization of Zn2+ in vitro and vivo 4. Conclusions In order to evaluate the capability of probe 1 for the visualization of Zn2+ in living cells, fluorescence imaging experiments were performed and shown in Fig. 7. The T-cells were first incubated in the presence of probe 1 (50 M) for 30 min at 37 ◦ C, and then treated with Zn2+ (100 M) for another 30 min. It was found that no fluorescence was observed when the T-cells were treated with the probe only. After the introduction with Zn2+ , a strong green fluorescence was observed. In a further experiment, it was found that the cells displayed weak fluorescence when the cells were further incubated with ATP for 30 min. Therefore, these results demonstrated
In summary, we demonstrated a novel fluorescein-based fluorescent probe which sequential identification for Zn2+ and ATP based on a turn on/turn off process. The probe exhibited high sensitivity and selectivity for Zn2+ and ATP with a low detection limit of 0.1 M/0.5 M, respectively. Moreover, confocal fluorescence microscopy experiments have established the feasibility of probe 1 in monitoring Zn2+ and ATP in vitro and in vivo, indicating its potential application for the detection of Zn2+ and ATP in environmental and biological systems.
X. Jin et al. / Sensors and Actuators B 261 (2018) 127–134
133
Fig. 8. Bioluminescence imaging in Kunming Mice. (a) Control mouse without any treatment; (b) The mouse was treated with 1 mM probe 1; (c) The mouse was treated with 1 mM probe 1 and then treated with ZnCl2 (1.0 equiv.) for 1 min; (d) The mouse was treated with 1 mM probe 1 and then treated with ZnCl2 (1.0 equiv.) for 10 min;(e) The mouse was treated with probe 1and then treated with ZnCl2 (1.0 equiv.) for 10 min followed by treatment with ATP(1.0 equiv.) for 30 min.
Acknowledgements The work was supported by the National Natural Science Foundation of China (No. 21202130, 51603164), the Fundamental Research Funds for the Central Universities (No.3102014JCQ15005), the Natural Science Foundation Research Project of Shaanxi Province (No.2015JM2067), the president Foundation of Xi’an Technological University (No. XAGDXJJ16010). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.snb.2018.01.116. References [1] J.M. Berg, Y. Shi, The galvanization of biology: a growing appreciation for the roles of zinc, Science 271 (1996) 1081–1085. [2] M. Warthon-Medina, V.H. Moran, A.L. Stammers, S. Dillon, P. Qualter, M. Nissensohn, L. Serra-Majem, N.M. Lowe, Zinc intake, status and indices of cognitive function in adults and children: a systematic review and meta-analysis, Eur. J. Clin. Nutr. 69 (2015) 649–661. [3] S.L. Sensi, P. Paoletti, A.I. Bush, I. Sekler, Zinc in the physiology and pathology of the CNS, Nat. Rev. Neurosci. 10 (2009) 780–791. [4] A.R. Kay, K. Tóth, Is zinc a neuromodulator? Sci. Signaling 1 (2008) re1–re3. [5] I.J. Griffin, S.C. Kim, P.D. Hicks, L.K. Liang, S.A. Abrams, Zinc metabolism in adolescents with crohn’s disease, Pediatr. Res. 56 (2004) 235–239. [6] C.J. Frederickson, J.-Y. Koh, A.I. Bush, The neurobiology of zinc in health and disease, Nat. Rev. Neurosci. 6 (2005) 449–462. [7] A. Takeda, H. Tamano, Insight into zinc signaling from dietary zinc deficiency, Brain Res. Rev. 62 (2009) 33–44. [8] H. Kioka, H. Kato, M. Fujikawa, O. Tsukamoto, T. Suzuki, H. Imamura, A. Nakano, S. Higo, S. Yamazaki, T. Matsuzaki, K. Takafuji, H. Asanuma, M. Asakura, T. Minamino, Y. Shintani, M. Yoshida, H. Noji, M. Kitakaze, I. Komuro, Y. Asano, S. Takashima, Evaluation of intramitochondrial ATP levels identifies G0/G1 switch gene 2 as a positive regulator of oxidative phosphorylation, Proc. Natl. Acad. Sci. 111 (2014) 273–278. [9] H. Imamura, K.P. Huynh Nhat, H. Togawa, K. Saito, R. Iino, Y. Kato-Yamada, T. Nagai, H. Noji, Visualization of ATP levels inside single living cells with fluorescence resonance energy transfer-based genetically encoded indicators, Proc. Natl. Acad. Sci. 106 (2009) 15651–15656. [10] D. Davalos, J. Grutzendler, G. Yang, J.V. Kim, Y. Zuo, S. Jung, D.R. Littman, M.L. Dustin, W.-B. Gan, ATP mediates rapid microglial response to local brain injury in vivo, Nat. Neurosci. 8 (2005) 752–758.
[11] Z. Yang, A. Sharma, J. Qi, X. Peng, D.Y. Lee, R. Hu, D. Lin, J. Qu, J.S. Kim, Super-resolution fluorescent materials: an insight into design and bioimaging applications, Chem. Soc. Rev. 45 (2016) 4651–4667. [12] Y.-R. Zhang, Y. Liu, X. Feng, B.-X. Zhao, Recent progress in the development of fluorescent probes for the detection of hypochlorous acid, Sens. Actuators B 240 (2017) 18–36. [13] H. Zhang, L. Feng, Y. Jiang, Y.-T. Wong, Y. He, G. Zheng, J. He, Y. Tan, H. Sun, D. Ho, A reaction-based near-infrared fluorescent sensor for Cu2+ detection in aqueous buffer and its application in living cells and tissues imaging, Biosens. Bioelectron. 94 (2017) 24–29. [14] Y. Ge, X. Zheng, R. Ji, S. Shen, X. Cao, A new pyrido[1,2-a]benzimidazole-rhodamine FRET system as an efficient ratiometric fluorescent probe for Cu2+ in living cells, Anal. Chim. Acta 965 (2017) 103–110. [15] L. Zong, Y. Xie, Q. Li, Z. Li, A new red fluorescent probe for Hg2+ based on naphthalene diimide and its application in living cells, reversibility on strip papers, Sens. Actuators B 238 (2017) 735–743. [16] Y. Qi, F.-R. Xiu, G. Yu, L. Huang, B. Li, Simple and rapid chemiluminescence aptasensor for Hg2+ in contaminated samples: a new signal amplification mechanism, Biosens. Bioelectron. 87 (2017) 439–446. [17] S. Erdemir, M. Yuksekogul, S. Karakurt, O. Kocyigit, Dual-channel fluorescent probe based on bisphenol A-rhodamine for Zn2+ and Hg2+ through different signaling mechanisms and its bioimaging studies, Sens. Actuators B 241 (2017) 230–238. [18] P. Ning, J. Jiang, L. Li, S. Wang, H. Yu, Y. Feng, M. Zhu, B. Zhang, H. Yin, Q. Guo, X. Meng, A mitochondria-targeted ratiometric two-photon fluorescent probe for biological zinc ions detection, Biosens. Bioelectron. 77 (2016) 921–927. [19] X. Yang, X. Liu, Y. Li, F. Wu, J. Mao, Y. Yuan, Y. Cui, G. Sun, G. Zhang, A differentially selective probe based on diketopyrrolopyrrole with fluorescence turn-on response to Fe3+ , and dual-mode turn-on and ratiometric response to Au3+ and its application in living cell imaging, Biosens. Bioelectron. 80 (2016) 288–293. [20] A.T. Aron, M.O. Loehr, J. Bogena, C.J. Chang, An endoperoxide reactivity-based FRET probe for ratiometric fluorescence imaging of labile iron pools in living cells, J. Am. Chem. Soc. 138 (2016) 14338–14346. [21] J. Miao, Y. Huo, X. Lv, Z. Li, H. Cao, H. Shi, Y. Shi, W. Guo, Fast-response and highly selective fluorescent probes for biological signaling molecule NO based on N-nitrosation of electron-rich aromatic secondary amines, Biomaterials 78 (2016) 11–19. [22] Z. Mao, W. Feng, Z. Li, L. Zeng, W. Lv, Z. Liu, NIR in, far-red out: developing a two-photon fluorescent probe for tracking nitric oxide in deep tissue, Chem. Sci. 7 (2016) 5230–5235. [23] P. Wang, J. Wu, C. Di, R. Zhou, H. Zhang, P. Su, C. Xu, P. Zhou, Y. Ge, D. Liu, W. Liu, Y. Tang, A novel peptide-based fluorescence chemosensor for selective imaging of hydrogen sulfide both in living cells and zebrafish, Biosens. Bioelectron. 92 (2017) 602–609. [24] R. Kawagoe, I. Takashima, S. Uchinomiya, A. Ojida, Reversible ratiometric detection of highly reactive hydropersulfides using a FRET-based dual emission fluorescent probe, Chem. Sci. 8 (2017) 1134–1140.
134
X. Jin et al. / Sensors and Actuators B 261 (2018) 127–134
[25] L. Wu, I.C. Wu, C.C. DuFort, M.A. Carlson, X. Wu, L. Chen, C.-T. Kuo, Y. Qin, J. Yu, S.R. Hingorani, D.T. Chiu, Photostable ratiometric pdot probe for in vitro and in vivo imaging of hypochlorous acid, J. Am. Chem. Soc. 139 (2017) 6911–6918. [26] K. Li, J.-T. Hou, J. Yang, X.-Q. Yu, A tumor-specific and mitochondria-targeted fluorescent probe for real-time sensing of hypochlorite in living cells, Chem. Commun. 53 (2017) 5539–5541. [27] J. Zhang, Z. Liu, P. Lian, J. Qian, X. Li, L. Wang, W. Fu, L. Chen, X. Wei, C. Li, Selective imaging and cancer cell death via pH switchable near-infrared fluorescence and photothermal effects, Chem. Sci. 7 (2016) 5995–6005. [28] A. Wallabregue, D. Moreau, P. Sherin, P. Moneva Lorente, Z. Jarolímová, E. Bakker, E. Vauthey, J. Gruenberg, J. Lacour, Selective imaging of late endosomes with a pH-Sensitive diazaoxatriangulene fluorescent probe, J. Am. Chem. Soc. 138 (2016) 1752–1755. [29] L. Zhao, S. Xie, X. Song, J. Wei, Z. Zhang, X. Li, Ratiometric fluorescent response of electrospun fibrous strips for real-time sensing of alkaline phosphatase in serum, Biosens. Bioelectron. 91 (2017) 217–224. [30] J. Deng, K. Wang, M. Wang, P. Yu, L. Mao, Mitochondria targeted nanoscale zeolitic imidazole framework-90 for ATP imaging in live cells, J. Am. Chem. Soc. 139 (2017) 5877–5882. [31] X. Jin, L. Hao, M. She, M. Obst, A. Kappler, B. Yin, P. Liu, J. Li, L. Wang, Z. Shi, Visualizing tributyltin (TBT) in bacterial aggregates by specific rhodamine-based fluorescent probes, Anal. Chim. Acta 853 (2015) 514–520. [32] X. Jin, L. Hao, Y. Hu, M. She, Y. Shi, M. Obst, J. Li, Z. Shi, Two novel fluorescein-based fluorescent probes for hypochlorite and its real applications in tap water and biological imaging, Sens. Actuators B 186 (2013) 56–60. [33] X. Jin, Y. Jia, W. Chen, P. Chui, Z. Yang, A reaction-based fluorescent probe for rapid detection of hypochlorite in tap water, serum, and living cells, Sens. Actuators B 232 (2016) 300–305. [34] X. Jin, S. Wu, M. She, Y. Jia, L. Hao, B. Yin, L. Wang, M. Obst, Y. Shen, Y. Zhang, J. Li, Novel fluorescein-based fluorescent probe for detecting H2 S and its real applications in blood plasma and biological imaging, Anal. Chem. 88 (2016) 11253–11260.
Biographies Xilang Jin received his Ph.D. in organic chemistry from Northwest University in 2014. Presently he is a University Lecturer in School of Materials and Chemical Engineering at Xi’an Technological University. His current research interests include synthesis and research methods for organic substance, fluorescent molecular devices, chemical and biological sensors and molecular recognition. Xianglong Wu received his Ph.D. in organic chemistry from Northwest University in 2010. Presently he is an associate professor in School of Life Sciences at Northwestern Polytechnical University. His main research interests include synthesis and biology application of fluorescent probes.
Bo Wang is currently a master candidate in School of Life Science and Technology, Xidian University. Her research interests focus on developing fluorescent probes and chemosensors. Xie Pu obtained a bachelor’s degree in polymer materials science and engineering from Beijing university of chemical technology in 2016.In 2017, She was studying for a master degree in materials at Xi ’an university of technology. Her current research interests include synthesis of fluorescent molecular. Yaolong He entered his BS course in Xi’an Technological University in 2010. He majors in chemistry. Hongwei Zhou received his Ph.D. degree in polymer chemistry and physics from Chengdu Institute of Organic Chemistry, Chinese Academy of Sciences and Max Planck Institute for Polymer Research (Mainz) in 2014.He is currently an associate professor at School of Materials and Chemical Engineering in Xi’an Technological University.His research interests focus on luminescent hydrogels, conductive hydrogels and high strength hydrogels. Bo Yan graduated from School of Materials and Chemical Engineering, Xi’an Technological University in June 2016.He is now a Postgraduate of Xi’an University of Technology, and now the main research direction is self-oscillating hydrogel and high performance hydrogel research. Jingjing Yang received her Ph D. in polymer chemistry and physics from Institute of Chemistry Chinese Academy of Sciences in 2013. Presently, she is a University Lecturer in School of Materials and Chemical Engineering at Xi’an Technological University. Her research interests include synthesis and characterization of functional polymers, solid state polymer electrolyte and research on the condensed structure of polymer composites. Weixing Chen received his Ph.D. in materials science from Northwestern Polytechnical University in 2010. Presently he is a professor in School of Materials and Chemical Engineering at Xi’an Technological University. His current research interests include synthesis and characterization for conjugate polymersand liquid-crystal polymers, photovoltage devices, etc. Xianghan Zhang received her Ph.D. in organic chemistry from Northwest University in 2010. She is currently an associate professor in School of Life Science and Technology, Xidian University. Her current interests include fluorescent molecular probes, near-infrared (NIR) fluorescence optical imaging, chemical and biological sensors.
Contents lists available at ScienceDirect
Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
A reversible fluorescent probe for Zn2+ and ATP in living cells and in vivo Xilang Jin a , Xianglong Wu b , Bo Wang c , Pu Xie a , Yaolong He a , Hongwei Zhou a,∗ , Bo Yan a , Jingjing Yang a , Weixing Chen a , Xianghan Zhang c,∗ a
School of Materials and Chemical Engineering, Xi’an Technological University, Xi’an, 710032, Shaanxi, China Key Laboratory for Space Bioscience and Biotechnology, School of Life Sciences, Northwestern Polytechnical University, Xi’an, Shaanxi, 710072, China c School of Life Sciences and Technology, Xidian University, Xi’an, 710071, China b
a r t i c l e
i n f o
Article history: Received 29 August 2017 Received in revised form 10 January 2018 Accepted 11 January 2018 Keywords: Fluorescein Zinc ion ATP Visualizing Fluorescence
a b s t r a c t A reversible fluorescein-based fluorescent probe which exhibits high sensitivity and selectivity for Zn2+ and ATP, has been designed and synthesized. The sensing process was completed via fluorescence variation induced by an opening and closing of the spiro-ring of fluorescein. Zn2+ /ATP-induced fluorescent intensity shows a good linear relationship with the concentration of Zn2+ /ATP in the range of 0–10 M with a detection limit of 0.1 M/0.5 M. Moreover, the probe was further applied to visualize and detect Zn2+ and ATP in vitro and in vivo. © 2018 Elsevier B.V. All rights reserved.
1. Introduction Zinc ion is recognized as one of the most abundant transition metals ions in the environmental and biological systems [1]. It plays indispensable roles in various physiological and pathological processes, such as genetic expression, cell apoptosis, enzyme regulation and neural transmission [2–4]. In addition, the disorder of zinc ion in living organisms may associate with Pakinson’s diseases, Alzheimer’s diseases and immune dysfunction [5–7]. On the other hand, adenosine-5 -triphosphate (ATP) is an essential biomolecules in cell biology due to its energy production and storage for many cellular events [8,9]. ATP is crucial in energy metabolism, DNA replication and transcription, and other fundamental activities [10]. Therefore, it is of great importance to develop a compelling method to quantitatively monitor the concentration of Zn2+ and ATP in both the environmental and biological systems. Compared with the traditional methods, fluorescence sensing shows obviously specific advantages in monitoring in vitro and in vivo biologically relevant species, such as metal ions, anions and active molecules due to its convenient operation and high sensitivity [11,12]. To date, numerous excellent fluorescein dyes have been
developed to detect various target objects, e.g. Cu2+ [13,14], Hg2+ [15,16], Zn2+ [17,18], Fe3+ [19,20], NO [21,22], H2 S [23,24], HOCl [25,26] and pH [27,28]. Unfortunately, there is few ATP probes have been applied in vitro and in vivo in recent decades [29,30]. Consequently, it remains a great challenge to explore highly selective and sensitive probes for monitoring the concentration of Zn2+ and ATP in vitro and in vivo. Recently, our group has designed and synthesized a series of fluorescein-based fluorescent probes for biologically relevant metal ions [31] and reactive oxygen [32,33] and sulfur species [34]. As a continuation of previous work, we report herein a fluoresceincontaining fluorescent turn-on probe with demonstrated specific selectivity to Zn2+ via formation of zinc complexes. Moreover, we developed the zinc complexes as a fluorescent probe which displayed a turn-off fluorescence change for ATP detection. We believe that this probe provides a potential and powerful approach for monitoring and visualizing Zn2+ and ATP in living cells and in vivo Scheme 1.
2. Experimental 2.1. Materials and measurements
∗ Corresponding authors. E-mail addresses: jinxilang [email protected] (X. Jin), [email protected] (H. Zhou), [email protected] (X. Zhang). https://doi.org/10.1016/j.snb.2018.01.116 0925-4005/© 2018 Elsevier B.V. All rights reserved.
All reagents were obtained from J&K Scientific, Co., Ltd. (Shanghai China) and used directly without further purification
128
X. Jin et al. / Sensors and Actuators B 261 (2018) 127–134
Scheme 1. Synthesis of probes 1.
throughout the whole experiment. Fluorescence spectra measurements were performed on a Hitachi F-4500 fluorescence spectrophotometer equipped with a xenon discharge lamp, in a 1 cm quartz cell. UV–vis spectra were recorded with a Shimadzu UV-1700 spectrphotometer. Mass spectra were measured using a Brukermicro TOF-QII ESI-Q-TOF LC/MS/MS Spectrometer by means of the electronic spray ionization (ESI). NMR spectra were recorded on a Varian INOVA-400 MHz spectrometer (at 400 MHz for 1 H NMR) using tetramethylsilane (TMS) as internal standards. Fluorescent images were performed using a Leica SPE confocal laser scanning microscope with an excitation wavelength of 488 nm. In vivo fluorescence imaging analysis was carried out in an IVIS Kinetic imaging system. Scanningelectron microscope (SEM) images were obtained on a FEI Quanta 400F scanning electron microscope system at an acceleration of 20 kV. The samples were sputter-coated with gold in vacuum before observation.
2.4. Synthesis of probes 1 Fluorescein hydrazide was synthesized in a high yield according to the procedures reported in literature [32]. Fluorescein hydrazide (10 mmol, 3.46 g) was mixed with compound 3 (11 mmol, 1.70 g) in 30 mL of ethanol with 1 drops of CH3 COOH. The reaction mixture was refluxed for 24 h. After cooling to room temperature, the white crude product was filter off from the reaction mixture and purified by column chromatography on silica gel using MeOH: CH2 Cl2 = 1: 30 as the eluent, to give 3.82 g pink power, yield 75.49%. 1 H NMR(DMSO-d6 , 400 MHz): ␦ (ppm) 1.21 (s, 9H), 6.49 (m, 4H), 6.65 (d, J = 2.0 Hz, 2H), 6.71 (d, J = 8.6 Hz, 1H), 7.19 (m, 1H), 7.26 (dd, J = 8.6, 2.5 Hz, 1H), 7.37 (d, J = 2.5 Hz, 1H), 7.65 (ddd, J = 10.5, 7.3, 1.3 Hz, 2H), 7.94 (m, 1H), 9.26 (s, 1H), 9.92 (s, 2H), 10.02 (s, 1H). MS (ESI) m/z = 529.1876 [M+ Na]+ , calc. for C28 H20 N2 O6 Na = 529.1734.
2.2. General procedure 3. Results and discussion Probes 1 stock solution (1 mM) was prepared in acetonitrile. The solutions of various testing species stock solutions (1 mM) were prepared in distilled water. During the titration experiments, different amounts of Zn2+ and 0.10 mL of 1000 M probes were mixed and filled up with PBS to 10 mL in volumetric tubes. During the interference experiments, 30 M Zn2+ , 0.10 mL of 1000 M probe 1 and 1 mL of 1000 M testing species were mixed and filled up with PBS to 10 mL in volumetric tubes. During the titration experiments of ATP, 0.10 mL of 1000 M probes, 0.1 mL of 1000 M Zn2+ and different amounts of ATP was mixed and filled up with PBS to 10 mL in volumetric tubes. 1 mL aliquots were pipetted into a 1 cm cuvette for spectral measurements. 5 nm bandpasses were used for both excitation and emission wavelengths. An excitation wavelength of 420 nm was used for the acquisition of emission spectra. 2.3. Synthesis of intermediate 3 4-tert-Butylphenol (5.14 g, 34.2 mmol) and hexamethylenetetramine (9.60 g, 68.5 mmol) were dissolved in anhydrous trifluoroacetic acid (60 mL) under N2 , and the reaction mixture was refluxed for 24 h. The mixture was poured into 4 M HCl (200 mL) and stirred for 10 min, after which it was extracted with CH2 Cl2 (2 × 150 mL). The combined organic extracts were washed with 4 M HCl(2 × 200 mL), water (200 mL), saturate brine (200 mL), then dried with Na2 SO4 and concentrated in vacuum. The crude product was purified by silica gel column chromatography to give 4.58 g yellow viscous liquid, yield 64.88%.
3.1. Optical response towardsZn2+ First of all, the time-dependent fluorescence response of the probe was investigated in the presence of 3.0 equiv. Zn2+ in CH3 CNPBS (1/99, v/v, pH 7.4) solution. As demonstrated in Fig. S2, the maximal fluorescence signal at 527 nm reached the maximum within 5 min at room temperature, indicating probe 1 is adaptive for real-time detection of Zn2+ and the reaction time of 5 min was applied for the subsequent experiments. The fluorescence titrations experiments of probe 1 with Zn2+ were then performed. As expected in Fig. 1a, the free probe (10 M) exhibited almost no emission band ( = 0.0012) due to the closed fluorescein-spirolactam form. Upon the introduction of 0.0-4.0 equiv. of Zn2+ , a dramatic enhancement of the fluorescence intensity at 527 nm was observed, with the fluorescence quantum yield of f = 0.45 (using rhodamine 6G in ethanol). This phenomenon was ascribed to Zn2+ ions induced structural transformation from the colorless spirolactam ring to the colored ring-opening delocalized form of fluorescein. As shown in Fig. 1b, the fluorescence titration curve revealed that the fluorescence intensity at 527 nm showed a good linear relationship against the concentration of Zn2+ in the range from 0.0 to 10.0 M. The regression equation was y = 23.80 x–3.323 (R2 = 0.997) and the detection limit (S/N = 3) of probe 1 for Zn2+ was found to be 0.1 M. These results demonstrated that probe 1could be potentially applicable for qualitative and quantitative analysis of Zn2+ .
X. Jin et al. / Sensors and Actuators B 261 (2018) 127–134
129
Fig. 1. (a) Fluorescence intensity (ex = 420 nm) changes of probe 1 (10 M) upon addition of Zn2+ (0.0–4.0 equiv) in CH3 CN-PBS (1/99, v/v, pH = 7.4) solution.The fluorescence intensity was measured at 527 nm. (b) Calibration curve of fluorescence intensity (ex = 420 nm) in dependence of Zn2+ concentrations. Data were acquired in CH3 CN-PBS (1/99, v/v, pH = 7.4). The fluorescence intensity was measured at 527 nm.
experiments further revealed that probe 1 was a potential candidate for the quantitative detection of Zn2+ with high selectivity and specificity. To investigate a suitable pH range for Zn2+ sensing, the pH effect on the fluorescence performance of probe 1 in the absence and presence of Zn2+ was further tested. As shown in Fig. S3, the experimental results indicated that the probe could effectively detect Zn2+ in the pH range between 6.8 and 9.0, which implies its potential for application in the physiological pH range.
3.2. Optical response towards ATP
Fig. 2. Selectivity of probe 1 (10 M) for Zn2+ in CH3 CN-PBS (1/99, v/v, pH = 7.4) solution. The pillars in the front row represent the value in the presence of representative metal ions (100 M). The pillars in the back row indicate the change in the emission intensity upon subsequent addition of Zn2+ (30 M) to the solution containing probe 1(10 M) and the respective ion of interest (100 M). For all measurements, the fluorescence intensity was measured at 527 nm.
Fig. 1 High-level selectivity and sensitivity is one of the most important criteria for the new sensing systems. As shown in Fig. 2, nineteen representative metal ions were conducted, by monitoring the fluorescence response in the presence of Li+ , Na+ , K+ , Ba2+ , Ca2+ , Cd2+ , Ag+ , Mg2+ , Co2+ , Mn2+ , Sn4+ , Pb2+ , Hg2+ , Ni2+ , Cr3+ , Cu2+ , Fe2+ , Fe3+ , Zn2+ under the identical additions. As envisioned, no obvious fluorescence response was observed in the presence of the other tested species. Only the addition of Zn2+ (30 M) caused a noticeable enhancement of the fluorescence intensity at 527 nm, displaying efficient turn-on responses. Fig. 2 To further evaluate the anti-interference properties of probe 1 towards Zn2+ , the competitive experiments were carried out in the presence of both Zn2+ (30 M) and coexisting metal ions (100 M). As shown in Fig. 3, the fluorescence intensity induced by the mixture of Zn2+ with competitive ions was similar to that caused by Zn2+ alone, which indicated that the competitive ions exerted no significant interfere during the sensing of Zn2+ .These
As shown in Fig. 3, upon the progressive addition of ATP (0.0-1.0 equiv.) to the solution of complex 1-Zn2+ resulted in about 4.59 fold decrease in the fluorescence intensity at 527 nm. The spectral phenomenon may be attributed to the strong binding ability between ATP and Zn2+ , which results in the removal of Zn2+ from the complex and structural recovery of the compound.The observed fluorescence intensity at 527 nm was nearly proportional to the concentration of ATP ranging from 0.0 to 10.0 M. The regression equation was y = − 16.94x + 220.8 (R2 = 0.994). And the detection limits (LOD) of complex1- Zn2+ to ATP is calculated to be 0.50 M based on S/N = 3. Upon the further addition of Zn2+ , the fluorescence intensity was recovered again, suggesting the reversibility of probe 1 (1+ Zn2+ ) binding to Zn2+ (ATP). This reversible process could be repeated several times with less fluorescent efficiency (Fig. 4). These results reveal that the binding of probe 1 and Zn2+ occurred via a reversible spirolactam ring open-close mechanism. To further verify the selectivity, the fluorescence emission of complex 1-Zn2+ was conducted with some representative anions, containing F− , Cl− , Br− , I− , CO3 2− , HS− , HSO3 − , Ac− , S2 O3 2− , SO4 2− , NO2 − , NO3 − , H2 PO4 − , PO4 3− , HPO4 2− , PPi, AMP, ADP. As displayed in Fig. 5, other competitive anions did not cause any notably emission quenching of complex 1-Zn2+ even at a concentration of 100 M anion under the same condition. Only ATP could remarkably quench the fluorescence intensity of the solution of complex 1-Zn2+ . Addition of ATP into a solution of 1-Zn2+ with other competitive anions together induced only slight decrease or a mildly change. Complex 1-Zn2+ also exhibited satisfactory selectivity toward ATP in a mixture of all competitive anions, which manifested the highly selectivity and sensitivity towards ATP.
130
X. Jin et al. / Sensors and Actuators B 261 (2018) 127–134
Fig. 3. (a) Fluorescence intensity (ex = 420 nm) changes of probe 1-Zn2+ (10 M) upon addition of ATP(0.0–1.0 equiv) in CH3 CN-PBS (1/99, v/v, pH 7.4) solution.The fluorescence intensity was measured at 527 nm. (b) Calibration curve of fluorescence intensity (ex = 420 nm) in dependence of ATP concentrations. Data were acquired in CH3 CN-PBS (1/99, v/v, pH 7.4). The fluorescence intensity was measured at 527 nm.
Fig. 4. Stepwise complexation/decomplexation cycles carried in CH3 CN-PBS (1/99, v/v, pH = 7.4) solution. Data were acquired in CH3 CN-PBS (1/99, v/v, pH = 7.4). The fluorescence intensity was measured at 527 nm.
3.3. Sensing mechanism study of probe 1 with Zn2+ and ATP To find out the binding stoichiometric ratio of probe 1 towards Zn2+ , Job’s plot between the change of fluorescence intensity and the mole fraction of probe 1 and Zn2+ was carried out (Fig. S4). A maximum fluorescence value was observed when the molar fraction of the probe [1] versus [1] + [Zn2+ ] was 0.66, which is indicative of a 2:1 stoichiometry for probe 1 and Zn2+ . According to the fluorescence spectra, the formation delocalized xanthane of fluorescein moiety was inferred to take place as depicted in Scheme 2. To further confirm the sensing mechanism, we performed high-resolution mass spectroscopy (HRMS) of probe 1 treated with Zn2+ and complex 1-Zn2+ treated with ATP. As depicted in Fig. S8–10, a new peak was obtained at m/z 1075.2853 (1-Zn2+ + H+ ) after the addition of excess Zn2+ , coinciding exactly with that for the transformation from probe 1 to compound 1Zn2+ + H+ (calcd: m/z 1075.2880) induced by Zn2+ .When complex 1-Zn2+ was treated with ATP, a new peak at m/z 529.1876 (probe 1, calcd: m/z 529.1734) was obtained. That is to say that this complex exhibits response to ATP based on the zinc complex ensemble displacement mechanism.
Fig. 5. Selectivity of complex 1-Zn2+ (10 M) for ATP in CH3 CN-PBS (1/99, v/v, pH = 7.4) solution. The pillars in the back row represent the value in the presence of representative anions (100 M). The pillars in the front row indicate the change in the emission intensity upon subsequent addition of ATP (10 M) to the solution containing complex 1-Zn2+ (10 M) and the respective ion of interest (100 M). For all measurements, the fluorescence intensity was measured at 527 nm.
Furthermore, IR spectra of probe 1 and complex 1-Zn2+ were also checked respectively in KBr disks, and the results were shown in Fig. S11. In the IR spectra, the peak at 1684 cm−1 of the probe 1, which corresponds to the characteristic amid carbonyl absorption, was shifted to 1654 cm−1 for the complex 1-Zn2+ , suggesting that a strong binding of the carbonyl group occurs with zinc ion. As shown in Fig. 6, the morphological changes of probe 1 and complex 1+ Zn2+ were studied by scanning electron microscope(SEM) analysis. Upon the addition of Zn2+ , the crystallographic morphology of complex became agglomerated due to the formation of 1+ Zn2+ complex. Therefore, all these data indicated that the reaction most likely followed the proposed mechanism shown in Scheme 2.
X. Jin et al. / Sensors and Actuators B 261 (2018) 127–134
131
Scheme 2. The Zn2+ /ATP-sensing mechanism of probe 1.
Fig. 6. SEM images of (a) probe 1 and (b) complex 1 + ZnCl2 .
Table 1 Detection results of Zn2+ in water samples by probe 1. Sample
Zn2+ spiked (mol/L)
Zn2+ recovered meana ± SDb (mol/L)
Recovery (%)
pool water
0 5 25 0 5 25
Not detected 4.9 ± 0.03 24.6 ± 0.02 Not detected 4.8 ± 0.03 24.4 ± 0.02
– 98.0 98.4 – 96.0 97.6
tap water
a b
Mean of three determination. SD: standard deviation.
132
X. Jin et al. / Sensors and Actuators B 261 (2018) 127–134
Fig. 7. (a–c) Confocal fluorescence images of T-cells incubated with probe 1 (50 M) for 30 min at 37 ◦ C; (d-f) The cells were stained with probe 1 (50 M) for 30 min at 37 ◦ C and then treated with ZnCl2 (50 M) for 30 min at 37 ◦ C; (g–i) The cells were stained with probe 1 (50 M) for 30 min at 37 ◦ C and then treated with ZnCl2 (50 M) followed by treatment with ATP(50 M) for 30 min at 37 ◦ C.
3.4. Applications of probe 1 in real water samples In order to understand the practicability of probe 1, we examined the concentration of Zn2+ in tap water and pool water samples by our above proposed fluorescent probe under the identical conditions, and the results were summarized in Table 1. The detecting data revealed a good agreement between the added and the found concentrations of Zn2+ . Thus, our proposed probe could be potentially used for monitoring Zn2+ in real water samples.
that probe 1 was good cell permeable and can be employed for monitoring Zn2+ and ATP within living cells. Fig. 7 Based on the desirable results in vitro, we investigated probe 1 to visualize Zn2+ and ATP in living animal. As shown in Fig. 8, the living mice without any treatment showed no fluorescence. And the mice which were given a skin-pop injection of probe 1 for 10 min also exhibited no fluorescence (Fig. 8b). When the living mice were further injected with Zn2+ (1.0 equiv.), a significant enhancement was observed observably with time (Fig. 8c–d). Upon the addition of ATP (1.0 equiv.), a significant decrease was observed. The results indicate that probe 1 could serve as a fluorescent agent for Zn2+ and ATP imaging in living organisms.
3.5. Visualization of Zn2+ in vitro and vivo 4. Conclusions In order to evaluate the capability of probe 1 for the visualization of Zn2+ in living cells, fluorescence imaging experiments were performed and shown in Fig. 7. The T-cells were first incubated in the presence of probe 1 (50 M) for 30 min at 37 ◦ C, and then treated with Zn2+ (100 M) for another 30 min. It was found that no fluorescence was observed when the T-cells were treated with the probe only. After the introduction with Zn2+ , a strong green fluorescence was observed. In a further experiment, it was found that the cells displayed weak fluorescence when the cells were further incubated with ATP for 30 min. Therefore, these results demonstrated
In summary, we demonstrated a novel fluorescein-based fluorescent probe which sequential identification for Zn2+ and ATP based on a turn on/turn off process. The probe exhibited high sensitivity and selectivity for Zn2+ and ATP with a low detection limit of 0.1 M/0.5 M, respectively. Moreover, confocal fluorescence microscopy experiments have established the feasibility of probe 1 in monitoring Zn2+ and ATP in vitro and in vivo, indicating its potential application for the detection of Zn2+ and ATP in environmental and biological systems.
X. Jin et al. / Sensors and Actuators B 261 (2018) 127–134
133
Fig. 8. Bioluminescence imaging in Kunming Mice. (a) Control mouse without any treatment; (b) The mouse was treated with 1 mM probe 1; (c) The mouse was treated with 1 mM probe 1 and then treated with ZnCl2 (1.0 equiv.) for 1 min; (d) The mouse was treated with 1 mM probe 1 and then treated with ZnCl2 (1.0 equiv.) for 10 min;(e) The mouse was treated with probe 1and then treated with ZnCl2 (1.0 equiv.) for 10 min followed by treatment with ATP(1.0 equiv.) for 30 min.
Acknowledgements The work was supported by the National Natural Science Foundation of China (No. 21202130, 51603164), the Fundamental Research Funds for the Central Universities (No.3102014JCQ15005), the Natural Science Foundation Research Project of Shaanxi Province (No.2015JM2067), the president Foundation of Xi’an Technological University (No. XAGDXJJ16010). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.snb.2018.01.116. References [1] J.M. Berg, Y. Shi, The galvanization of biology: a growing appreciation for the roles of zinc, Science 271 (1996) 1081–1085. [2] M. Warthon-Medina, V.H. Moran, A.L. Stammers, S. Dillon, P. Qualter, M. Nissensohn, L. Serra-Majem, N.M. Lowe, Zinc intake, status and indices of cognitive function in adults and children: a systematic review and meta-analysis, Eur. J. Clin. Nutr. 69 (2015) 649–661. [3] S.L. Sensi, P. Paoletti, A.I. Bush, I. Sekler, Zinc in the physiology and pathology of the CNS, Nat. Rev. Neurosci. 10 (2009) 780–791. [4] A.R. Kay, K. Tóth, Is zinc a neuromodulator? Sci. Signaling 1 (2008) re1–re3. [5] I.J. Griffin, S.C. Kim, P.D. Hicks, L.K. Liang, S.A. Abrams, Zinc metabolism in adolescents with crohn’s disease, Pediatr. Res. 56 (2004) 235–239. [6] C.J. Frederickson, J.-Y. Koh, A.I. Bush, The neurobiology of zinc in health and disease, Nat. Rev. Neurosci. 6 (2005) 449–462. [7] A. Takeda, H. Tamano, Insight into zinc signaling from dietary zinc deficiency, Brain Res. Rev. 62 (2009) 33–44. [8] H. Kioka, H. Kato, M. Fujikawa, O. Tsukamoto, T. Suzuki, H. Imamura, A. Nakano, S. Higo, S. Yamazaki, T. Matsuzaki, K. Takafuji, H. Asanuma, M. Asakura, T. Minamino, Y. Shintani, M. Yoshida, H. Noji, M. Kitakaze, I. Komuro, Y. Asano, S. Takashima, Evaluation of intramitochondrial ATP levels identifies G0/G1 switch gene 2 as a positive regulator of oxidative phosphorylation, Proc. Natl. Acad. Sci. 111 (2014) 273–278. [9] H. Imamura, K.P. Huynh Nhat, H. Togawa, K. Saito, R. Iino, Y. Kato-Yamada, T. Nagai, H. Noji, Visualization of ATP levels inside single living cells with fluorescence resonance energy transfer-based genetically encoded indicators, Proc. Natl. Acad. Sci. 106 (2009) 15651–15656. [10] D. Davalos, J. Grutzendler, G. Yang, J.V. Kim, Y. Zuo, S. Jung, D.R. Littman, M.L. Dustin, W.-B. Gan, ATP mediates rapid microglial response to local brain injury in vivo, Nat. Neurosci. 8 (2005) 752–758.
[11] Z. Yang, A. Sharma, J. Qi, X. Peng, D.Y. Lee, R. Hu, D. Lin, J. Qu, J.S. Kim, Super-resolution fluorescent materials: an insight into design and bioimaging applications, Chem. Soc. Rev. 45 (2016) 4651–4667. [12] Y.-R. Zhang, Y. Liu, X. Feng, B.-X. Zhao, Recent progress in the development of fluorescent probes for the detection of hypochlorous acid, Sens. Actuators B 240 (2017) 18–36. [13] H. Zhang, L. Feng, Y. Jiang, Y.-T. Wong, Y. He, G. Zheng, J. He, Y. Tan, H. Sun, D. Ho, A reaction-based near-infrared fluorescent sensor for Cu2+ detection in aqueous buffer and its application in living cells and tissues imaging, Biosens. Bioelectron. 94 (2017) 24–29. [14] Y. Ge, X. Zheng, R. Ji, S. Shen, X. Cao, A new pyrido[1,2-a]benzimidazole-rhodamine FRET system as an efficient ratiometric fluorescent probe for Cu2+ in living cells, Anal. Chim. Acta 965 (2017) 103–110. [15] L. Zong, Y. Xie, Q. Li, Z. Li, A new red fluorescent probe for Hg2+ based on naphthalene diimide and its application in living cells, reversibility on strip papers, Sens. Actuators B 238 (2017) 735–743. [16] Y. Qi, F.-R. Xiu, G. Yu, L. Huang, B. Li, Simple and rapid chemiluminescence aptasensor for Hg2+ in contaminated samples: a new signal amplification mechanism, Biosens. Bioelectron. 87 (2017) 439–446. [17] S. Erdemir, M. Yuksekogul, S. Karakurt, O. Kocyigit, Dual-channel fluorescent probe based on bisphenol A-rhodamine for Zn2+ and Hg2+ through different signaling mechanisms and its bioimaging studies, Sens. Actuators B 241 (2017) 230–238. [18] P. Ning, J. Jiang, L. Li, S. Wang, H. Yu, Y. Feng, M. Zhu, B. Zhang, H. Yin, Q. Guo, X. Meng, A mitochondria-targeted ratiometric two-photon fluorescent probe for biological zinc ions detection, Biosens. Bioelectron. 77 (2016) 921–927. [19] X. Yang, X. Liu, Y. Li, F. Wu, J. Mao, Y. Yuan, Y. Cui, G. Sun, G. Zhang, A differentially selective probe based on diketopyrrolopyrrole with fluorescence turn-on response to Fe3+ , and dual-mode turn-on and ratiometric response to Au3+ and its application in living cell imaging, Biosens. Bioelectron. 80 (2016) 288–293. [20] A.T. Aron, M.O. Loehr, J. Bogena, C.J. Chang, An endoperoxide reactivity-based FRET probe for ratiometric fluorescence imaging of labile iron pools in living cells, J. Am. Chem. Soc. 138 (2016) 14338–14346. [21] J. Miao, Y. Huo, X. Lv, Z. Li, H. Cao, H. Shi, Y. Shi, W. Guo, Fast-response and highly selective fluorescent probes for biological signaling molecule NO based on N-nitrosation of electron-rich aromatic secondary amines, Biomaterials 78 (2016) 11–19. [22] Z. Mao, W. Feng, Z. Li, L. Zeng, W. Lv, Z. Liu, NIR in, far-red out: developing a two-photon fluorescent probe for tracking nitric oxide in deep tissue, Chem. Sci. 7 (2016) 5230–5235. [23] P. Wang, J. Wu, C. Di, R. Zhou, H. Zhang, P. Su, C. Xu, P. Zhou, Y. Ge, D. Liu, W. Liu, Y. Tang, A novel peptide-based fluorescence chemosensor for selective imaging of hydrogen sulfide both in living cells and zebrafish, Biosens. Bioelectron. 92 (2017) 602–609. [24] R. Kawagoe, I. Takashima, S. Uchinomiya, A. Ojida, Reversible ratiometric detection of highly reactive hydropersulfides using a FRET-based dual emission fluorescent probe, Chem. Sci. 8 (2017) 1134–1140.
134
X. Jin et al. / Sensors and Actuators B 261 (2018) 127–134
[25] L. Wu, I.C. Wu, C.C. DuFort, M.A. Carlson, X. Wu, L. Chen, C.-T. Kuo, Y. Qin, J. Yu, S.R. Hingorani, D.T. Chiu, Photostable ratiometric pdot probe for in vitro and in vivo imaging of hypochlorous acid, J. Am. Chem. Soc. 139 (2017) 6911–6918. [26] K. Li, J.-T. Hou, J. Yang, X.-Q. Yu, A tumor-specific and mitochondria-targeted fluorescent probe for real-time sensing of hypochlorite in living cells, Chem. Commun. 53 (2017) 5539–5541. [27] J. Zhang, Z. Liu, P. Lian, J. Qian, X. Li, L. Wang, W. Fu, L. Chen, X. Wei, C. Li, Selective imaging and cancer cell death via pH switchable near-infrared fluorescence and photothermal effects, Chem. Sci. 7 (2016) 5995–6005. [28] A. Wallabregue, D. Moreau, P. Sherin, P. Moneva Lorente, Z. Jarolímová, E. Bakker, E. Vauthey, J. Gruenberg, J. Lacour, Selective imaging of late endosomes with a pH-Sensitive diazaoxatriangulene fluorescent probe, J. Am. Chem. Soc. 138 (2016) 1752–1755. [29] L. Zhao, S. Xie, X. Song, J. Wei, Z. Zhang, X. Li, Ratiometric fluorescent response of electrospun fibrous strips for real-time sensing of alkaline phosphatase in serum, Biosens. Bioelectron. 91 (2017) 217–224. [30] J. Deng, K. Wang, M. Wang, P. Yu, L. Mao, Mitochondria targeted nanoscale zeolitic imidazole framework-90 for ATP imaging in live cells, J. Am. Chem. Soc. 139 (2017) 5877–5882. [31] X. Jin, L. Hao, M. She, M. Obst, A. Kappler, B. Yin, P. Liu, J. Li, L. Wang, Z. Shi, Visualizing tributyltin (TBT) in bacterial aggregates by specific rhodamine-based fluorescent probes, Anal. Chim. Acta 853 (2015) 514–520. [32] X. Jin, L. Hao, Y. Hu, M. She, Y. Shi, M. Obst, J. Li, Z. Shi, Two novel fluorescein-based fluorescent probes for hypochlorite and its real applications in tap water and biological imaging, Sens. Actuators B 186 (2013) 56–60. [33] X. Jin, Y. Jia, W. Chen, P. Chui, Z. Yang, A reaction-based fluorescent probe for rapid detection of hypochlorite in tap water, serum, and living cells, Sens. Actuators B 232 (2016) 300–305. [34] X. Jin, S. Wu, M. She, Y. Jia, L. Hao, B. Yin, L. Wang, M. Obst, Y. Shen, Y. Zhang, J. Li, Novel fluorescein-based fluorescent probe for detecting H2 S and its real applications in blood plasma and biological imaging, Anal. Chem. 88 (2016) 11253–11260.
Biographies Xilang Jin received his Ph.D. in organic chemistry from Northwest University in 2014. Presently he is a University Lecturer in School of Materials and Chemical Engineering at Xi’an Technological University. His current research interests include synthesis and research methods for organic substance, fluorescent molecular devices, chemical and biological sensors and molecular recognition. Xianglong Wu received his Ph.D. in organic chemistry from Northwest University in 2010. Presently he is an associate professor in School of Life Sciences at Northwestern Polytechnical University. His main research interests include synthesis and biology application of fluorescent probes.
Bo Wang is currently a master candidate in School of Life Science and Technology, Xidian University. Her research interests focus on developing fluorescent probes and chemosensors. Xie Pu obtained a bachelor’s degree in polymer materials science and engineering from Beijing university of chemical technology in 2016.In 2017, She was studying for a master degree in materials at Xi ’an university of technology. Her current research interests include synthesis of fluorescent molecular. Yaolong He entered his BS course in Xi’an Technological University in 2010. He majors in chemistry. Hongwei Zhou received his Ph.D. degree in polymer chemistry and physics from Chengdu Institute of Organic Chemistry, Chinese Academy of Sciences and Max Planck Institute for Polymer Research (Mainz) in 2014.He is currently an associate professor at School of Materials and Chemical Engineering in Xi’an Technological University.His research interests focus on luminescent hydrogels, conductive hydrogels and high strength hydrogels. Bo Yan graduated from School of Materials and Chemical Engineering, Xi’an Technological University in June 2016.He is now a Postgraduate of Xi’an University of Technology, and now the main research direction is self-oscillating hydrogel and high performance hydrogel research. Jingjing Yang received her Ph D. in polymer chemistry and physics from Institute of Chemistry Chinese Academy of Sciences in 2013. Presently, she is a University Lecturer in School of Materials and Chemical Engineering at Xi’an Technological University. Her research interests include synthesis and characterization of functional polymers, solid state polymer electrolyte and research on the condensed structure of polymer composites. Weixing Chen received his Ph.D. in materials science from Northwestern Polytechnical University in 2010. Presently he is a professor in School of Materials and Chemical Engineering at Xi’an Technological University. His current research interests include synthesis and characterization for conjugate polymersand liquid-crystal polymers, photovoltage devices, etc. Xianghan Zhang received her Ph.D. in organic chemistry from Northwest University in 2010. She is currently an associate professor in School of Life Science and Technology, Xidian University. Her current interests include fluorescent molecular probes, near-infrared (NIR) fluorescence optical imaging, chemical and biological sensors.