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A smart reaction-based fluorescence probe for ratio detection of hydrazine and its application in living cells
Microchemical Journal 156 (2020) 104809
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A smart reaction-based fluorescence probe for ratio detection of hydrazine and its application in living cells
T
Zhaodi Liu , Zhouhua Yang, Sizhe Chen, Yue Liu, Liangquan Sheng, Zhimei Tian, Deqian Huang, ⁎ Huajie Xu ⁎
School of Chemistry and Materials Engineering, Fuyang Normal University, Fuyang, Anhui 236037, PR China
ARTICLE INFO
ABSTRACT
Keywords: Triphenylamine Thiophene Ratiometric Fluorescent probe Hydrazine
Hydrazine (N2H4) has been widely used in many areas of the chemical industry, but it can cause potential harm to human health and environmental pollution. Herein, a novel fluorescent probe (L) benefit from smart cyclization mechanism has been constructed for ratio detection of hydrazine. Due to the destruction of the conjugate system by cyclic reaction, the intramolecular charge transfer (ICT) process was blocked, and a large hypsochromic shift could be observed both in the absorption and fluorescence spectra with excellent ratio response. The sensing mechanism was also studied by NMR spectra and DFT calculation. This ratiometric probe was highly selective and sensitive for detecting hydrazine with a low detection limit of 0.23 μM. Moreover, that probe was also effective in monitoring hydrazine in live cells.
1. Introduction The high reactivity of hydrazine (N2H4) makes it widely used in pharmacy, military, agriculture, industry, aerospace and other fields [1–4]. However, hydrazine is highly toxic and has a strong irritant to the eyes and skin. Especially, it can cause acute poisoning or cancer. Its widespread usage leads to the release of hydrazine, causing serious environmental problems [5–9]. The U.S. Environmental Protection Agency (EPA) suggested a safe limit for hydrazine of 10 ppb [10–12]. Therefore, it was important to develop a reliable and sensitive method for the detection of hydrazine. Fluorescent probes have attracted much attention for their simplicity, real-time detection, high selectivity and sensitivity [13–20]. Further, ratiometric fluorescent probes have some advantages evaluating the analyte concentration via self-calibrating two output signals, and provide a built-in correction for environmental effects [21–23], such as photo bleaching, molecule concentration, stability under illumination, and the environmental factors (pH, polarity, temperature, etc.) [24–27]. Up to now, there are only few reports of N2H4 probes even fewer of N2H4 ratiometric fluorescent probes based on its unique nucleophilic characteristics including 1,3-diketones, acetonate, levulinate, and phthalimide derivatives [28–32]. In spite of the reported probes have made distinct progress, some drawbacks were still unavoidable, such as poor water solubility, high pH dependence, long response time or unsatisfactory emission shift, which limited their further biological
⁎
applications [33–36]. As a result, the design of fluorescent probes for N2H4 excellent ratiometric measurement remains a challenge. Based on these above insights, we designed and synthesized a probe (E)-3-(5-(4-(diphenylamino)phenyl)thiophen-2-yl)-1-(2-hydroxyphenyl)prop-2-en-1-onefor (L) detecting N2H4 (Scheme 1). In the presence of hydrazine, a novel cyclization reaction was generated involving the conjugation addition of an amine and the condensation of ketone with another amine of hydrazine, which destroy the large conjugate system resulting in a blocked intramolecular charge transfer (ICT) processes with a large degree of spectral blue shift. All these induced the probe to achieve high sensitivity and selectivity of ratio detection for hydrazine, and the sensing performance and mechanism have been studied in detail by experiments and theory calculations. Moreover, the probe demonstrated excellent capability to monitor hydrazine in living cells. 2. Experiments 2.1. General NMR spectra were recorded on a Varian INOVA-400 MHz spectrometer with tetramethylsilane (TMS) as internal standard. Absorbance spectra were measured on a Purkinje general UV-1901 spectrophotometer. IR spectra were recorded on NEXUS 870 (Nicolet) spectrophotometer. Fluorescence spectra were performed on a Cary Eclipse
Corresponding authors. E-mail addresses: [email protected] (Z. Liu), [email protected] (H. Xu).
https://doi.org/10.1016/j.microc.2020.104809 Received 20 February 2020; Received in revised form 8 March 2020; Accepted 8 March 2020 Available online 10 March 2020 0026-265X/ Published by Elsevier B.V.
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Scheme. 1. Synthesis of the probe L and its reaction with hydrazine to form L’.
fluorescence spectrophotometer. X-ray data were collected on Bruker Smart APEX II CCD diffractometer. The structure was solved by direct methods and refined by full matrix least-square on F2 using the SHELXTL-97 program. All calculations were performed with a Gaussian 16 program using the PBE38 functional with a 6-31+G (d) basis set [37–39]. Excited states in DMSO were calculated by a TD-DFT approach using the polarizable continuum model (PCM). The electron and hole distribution of the S1 state is analyzed using the Multiwfn program (version 3.5) [40]. All reagents were of analytical grade and were not further purified for use. Double distilled water was used throughout the experiment. Anions and metal ions were prepared from their sodium salts and chloride salts. Hydrazine, ethanediamine, hydroxylamine, phenyl hydrazine, ammoniumhydroxide, triethylamine, urea, hexamethylenetetramine hydrate and some amino acids were tested their sensing behaviors in deionized water.
δ 12.51 (s, 1H), 8.13 (d, J = 8.2 Hz, 1H), 7.99 (d, J = 15.1 Hz, 1H), 7.72 (d, J = 3.6 Hz, 1H), 7.68–7.59 (m, 3H), 7.58–7.45 (m, 2H), 7.35 (t, J = 7.7 Hz, 4H), 7.11 (dd, J = 16.0, 7.8 Hz, 6H), 6.99 (t, J = 6.2 Hz, 4H); 13C NMR (100 MHz, d6-DMSO) δ 192.88, 162.08, 148.55, 148.32, 147.04, 138.29, 137.86, 136.57, 136.10, 130.97, 130.23, 127.42, 126.74, 125.25, 124.69, 124.33, 122.67, 121.41, 119.90, 119.64, 118.12; MALDI-TOF MS (M + 1 = 474.14); IR (KBr, cm−1) selected bands: 3449 (w), 1631 (s), 1584 (m), 1562 (s), 1487 (s), 1466 (m), 1275 (s), 1153 (m), 803 (w), 758 (m), 693 (m). 2.3. General UV–vis and Fluorescence Spectra Measurements A stock solution of L (1 × 10−3 M) was prepared in DMSO. The UV–vis and fluorescence measurements of the probe L were fixed at 10 μM in buffered DMSO/HEPES (10 mM, pH = 7.40, 9/1, v/v) solution at room temperature, and then the fluorescence and UV absorption spectra were recorded.
2.2. Preparation and characterization of the probe
2.4. Imaging of HeLa cells
5-[4-(Diphenylamino)phenyl]thiophene-2-carbaldehyde (1). To a solution of 4-bromotriphenylamine (1.44 g, 4.5 mmol) and PdCl2(dppf) (0.35 g, 0.3 mmol) in dry toluene (30 mL) was added a solution of 5-formylthiophene-2-yl-2-boronic acid (0.47 g, 3 mmol) and K2CO3 (4.1 g, 30 mmol) in methanol (30 mL). The mixture lasted for 10 h at 70 °C, then adding 30 mL of water to quench the reaction, and extracting with DCM (3 × 50 mL). The combined organic extract was dried over anhydrous MgSO4 and filtered. Solvent removal by rotary vaporation followed by column chromatography over silica gel with ethyl acetate/petroleum ether (1:5) yielded a yellow solid. (0.91 g, 85.1 %). 1H NMR (400 MHz, Acetone) δ: 9.91 (s, 1H), 7.94 (d, J = 4.0 Hz, 1H), 7.69 (d, J = 8.7 Hz, 2H), 7.56 (d, J = 3.9 = Hz, 1H), 7.37 (t, J = 7.9 Hz, 4H), 7.20–7.10 (m, 6H), 7.05 (d, J = 8.7 Hz, 2H); 13C NMR (100 MHz, DMSO) δ: 184.21, 153.38, 149.02, 146.88, 141.33, 140.00, 130.26, 127.90, 125.98, 125.46, 124.58, 124.46, 122.15; MALDI-TOF MS (M+1 = 356.11); IR (KBr,cm−1) selected bands: 2359(w), 1662(s), 1591(s), 1489(s), 1448(s), 1329(m), 1284(m), 1228(m), 1057(m), 808(m), 752(m), 698(s), 492(w). (E)-3-(5-(4-(diphenylamino)phenyl)thiophen-2-yl)-1-(2-hydroxyphenyl)prop-2-en-1-one (L) To a solution of 5-[4-(Diphenylamino)phenyl]thiophene-2-carbaldehyde (1) (0.71 g, 2 mmol) and 2-hydroxyacetophenone (0.27 g, 2.0 mmol) in ethanol (50 mL) was added 20% NaOH solvents. The mixture was refluxed for 8 h. A precipitate was produced with addition of ethanol, a tangerine solid was prepared after filtered, washed with ethanol and drying. Yield: 0.39 g, 40.7%. 1H NMR (400 MHz, d6-DMSO)
HeLa cells were cultured in DMEM medium (10% fetal bovine serum, FBS) with a humidified atmosphere containing CO2 5% (v/v) at 37 °C. The conventional MTT assays were first operated to evaluate the cytotoxicity of L (0–20 μM, 1% DMSO). Then, the first group was imaged as cell blank. The second group of the HeLa cells incubated with 10 μM of the probe L for 60 min at 37 °C,and the third group of the cells were incubated with the probe (10 μM) for 60 min and then incubated with N2H4 for another 60 min. In all above operations, cells are washed 3 times with PBS after each incubating, then confocal laser scanning microscope was used to image cell. 3. Results and discussion 3.1. Crystal structure description The X-ray diffraction measurements for the probe made on a Rigaku Oxford Diffraction area detector diffractometer with graphite monochromated Mo-Kα radiation (λ = 0.71073 Å) at 293 K. For data collection, block-shaped red crystals with dimensions of 0.2 × 0.2 × 0.1 mm were used. It belongs to the monoclinic system with space group P21/c. Its unit cell dimensions are: a = 19.432 (5) Å b = 12.305 (2) Åc = 10.389 (2) Å,β = 76.416 (2)°, with Z = 4. Table S1 shows the experimental conditions for the single crystal diffraction data collection and structure refinement. The selected bond lengths and angles have been provided in Tables S2. The structure of L 2
Microchemical Journal 156 (2020) 104809
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Fig. 1. The absorption (a) and fluorescent spectra (b, λex = 380 nm; c, λex = 446 nm) of L (10 μM) to N2H4 (200 μM) and other amine species (500 μM). (d) The fluorescence intensity ratios (I433 nm/I628 nm) and color of L in the presence of N2H4 (200 μM) and other amine species. Inset in (a,b): the color changes of L with hydrazine. Inset in (d): The emission color of L with amine species. Abbreviation: 1, ethanediamine; 2, hydroxylamine; 3, phenyl hydrazine; 4, ammoniumhydroxide; 5, triethylamine; 6, urea; 7, hexamethylenetetramine; 8, probe L; 9, hydrazine.
(Fig. S1) shows s-cis geometry of the enone as expected, with less torsion angle 9.3 (9)° through the enone torsion (O]CeC]C), and the olefinic in the trans-conformations. There is an intramolecular hydrogen bond between the enone carbonyl and ortho-hydroxy group on the benzene ring (Tables S3). The dihedral angles between thiophene ring and benzene ring (C13-C18, C27-C32) are 16.8 (1)° and 26.5(2)°, respectively. So the molecular backbone is twisted significantly.
were also investigated. As seen in Fig. S3, while the fluorescence changes caused by other analytes were negligible during the same experimental operation. Remarkably, due to the specific reaction with hydrazine, a large emission band hyposochromic shift from 628 nm to 433 nm (~200 nm) could be observed, which allowed the visible monitoring of N2H4 with an ultraviolet lamp. The emission color also changed from red to blue (inside Fig. 1d), so the presence of hydrazine could be easily observed.
3.2. Selectivity of L to hydrazine and other analytes
3.3. Spectra of probe L titrated with N2H4
In order to verify the design idea of probe L, the absorption and fluorescence spectrum were investigated in buffered DMSO/HEPES (10 mM, pH = 7.40, 9/1, v/v) to evaluated the selectivity of the probe L in various cations, anions, and amines solution. As shown in Fig. 1a and Fig. S2, in the absence of N2H4, the solution containing probe L was yellow. With the addition of N2H4, the probes exhibited a new blue shift absorption peak at 353 nm from 463 nm. The color change from yellow to colorless enables one to distinguish N2H4 by the naked eye (inside Fig. 1a). The fluorescence response of L was also measured towards amine species. As shown in Fig. 1b and Fig. 1c, upon excitation at 380 nm and 446 nm respectively, only the addition of hydrazine significantly enhanced the fluorescence of L at 433 nm and gradually decreased it at 628 nm. Other amines had no significant effect on the fluorescence of L, and their fluorescence ratio (I433 nm/I628 nm) also indicated that L was highly selective to hydrazine (Fig. 1d). Various cations, anions and small biomolecules which often exist in environmental and biological samples were selected and their effects on fluorescence spectra of L
The absorption and fluorescence titration of probe L were performed in detail with hydrazine in a solution of DMSO/HEPES (10 mM, pH = 7.40, 9/1, v/v). The probe L exhibited a strong absorption peak at 463 nm as observed in Fig. S4. When the hydrazine from 0 to 200 μM was added, the absorption peak at 353 nm gradually increased, while the absorption peak at 463 nm gradually decreased, accompanied by a well-defined isosbestic point at 380 nm, and there was also a significant color change from yellow to colorless. As shown in Fig. S5a,b, when the probe L was excited at 380 nm and displayed a weak emission peak at 433 nm. Interestingly, with the addition of N2H4, which induced significantly increase in fluorescence intensity at 433 nm. In Fig. S5c,d, we used excitation at 446 nm to observe the change of fluorescence spectra, and the fluorescence intensity at 628 nm, significant quenching with a large hypsochromic shift from 628 nm to 571 nm upon the addition of N2H4 (0-200 μM). The fluorescence ratio of the two emission intensities (I433 nm/I628 nm) increased about 360-fold and showed a good linear relationship with the concentration of N2H4 from 0 to 80 3
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μM (R2 = 0.99869) as shown in Fig. S6a. The detection limit for N2H4 was calculated to be about 0.23 μM in terms of the equation of LOD = 3 Sb/m, and indicated that the probe possessed excellent analytical performance and comparable to the performance of the most reported probes (Table S4).
program (version 3.5). The electron and hole distribution of the S1 state for L and L’ molecules are shown in Fig. 3 and table S5. For L, the electrons mainly locate at the thiophene and enone group, but holes are distributed at the triphenylamine and thiophene group. These indicated electrons transfer from the triphenylamine to enone groups showing typical ICT excitation. However, for L’, electrons mainly locate at the central phenyl and thiophene, and holes are still distributed at the triphenylamine and thiophene group, which means electrons transfer from the triphenylamine to the central phenyl and thiophene, showing ICT excitation, too. As can be seen from Table S5, the S/D values of L (0.13 Å−1) is smaller than that of L’ (0.35 Å−1), so L shows more remarkable CT modes [41]. That is to say, with addition of hydrazine to L, the enone was broken to form L’ which hinder the electron transitions.
3.4. pH-dependence and time-dependence for hydrazine Furthermore, the effect of pH on the emission ratio (I433 nm/I628 nm) of the probe had also been investigated in the absence and presence of N2H4. As shown in Fig. S6b, without adding N2H4, the fluorescence intensity ratio (I433 nm/I628 nm) of the probe L hardly changed when the pH value was less than 8.5, but the emission ratio significantly increased with pH ≧ 8.5, indicating that L was stable under neutral conditions and easy to inhibit the electron transfer process under alkaline condition. Upon the addition of N2H4, the fluorescence emission ratio (I433 nm/I628 nm) gradually increased from pH 6.0 to 8.0, implying that probe L enable a selective detection of N2H4 in a wide pH range including physiological conditions. The time-dependence of probe L towards N2H4 was also measured in DMSO/HEPES (10 mM, pH = 7.40, 9/1, v/v). As shown in Fig. S7, after treatment with N2H4 (200 μM), the absorption peak at 336 nm redshifted slightly to 353 nm and another absorption peak at 463 nm gradually reduced with time. The main absorption peak strength decrease at 463 nm was due to the conjugation addition of N2H4 to the unsaturated carbonyl configuration. Time course studies showed that the absorbance reached a plateau at about 30 min. Fluorescence intensity changed with time after the same amount of N2H4 treatment (Fig. S8) was also consistent with the absorption result, and the fluorescence emission ratio (I433 nm/I628 nm) of probe L gradually increased and remained constant within 30 min. In conclusion, the probe L was sensitive to hydrazine, and enabling to detect N2H4 as a practical method.
3.6. Bio-imaging of hydrazine Cellular imaging studies were performed to examine the sensing behavior of probe L in living Hela cells. The cytotoxicity of probe L was first evaluated using the MTT assay (Fig. S9), which revealed that probe L had highly biocompatible to Hela cells. As such, we attempted to explore the potential applications of probe L in the living systems. When probe L loaded HeLa cells at 37 °C for 30 min, and strong red and negligible blue fluorescent emissions can be observed (Fig. S10a-c), such stable photophysical properties indicated that the probe can enter the cell smoothly. Compared to another controlled experiment, after HeLa cells loaded with probes were further incubated with N2H4 for another 30 min, the fluorescence intensity in the blue channel increased significantly and the red fluorescence emissions faded simultaneously (Fig. S10e-g). Moreover, the merged image (Fig. S10d,h) overlaid very well in the two channels, and these cell experiments clearly demonstrated excellent performance tracing N2H4 in living cells for probe L. 4. Conclusions In summary, a ratiometric fluorescent probe based on a novel cyclization platform was set up for the efficient detection of N2H4. The synthesized probe L was equipped with chalcone and thiophene groups and to ensure active reaction sites and aqueous solubility. As expected, probe L show highly sensitive and selective to hydrazine for its cyclization reaction with chalcone leading to a ratiometric response. The excellent anti-interference ability for cations, amino acids and pH has been studied, and the sensing mechanism was also explored by NMR spectra and DFT calculation. When N2H4 was added to L, the enone configuration was broken to form L’, which hinder the electron transitions with spectral hypochromatic shift. Moreover, the developed new chemosignaling system based on cyclization principles will be very helpful for the development of other probes for N2H4.
3.5. Mechanism of probe L responding to hydrazine On the basis of results achieved above, the sensing mechanism of probe L toward hydrazine was proposed to proceed a cyclization reaction forming pyrazoline ring by 1H NMR spectroscopic. Upon addition of 1 equiv. of hydrazine into the assay system, the yellow liquid turned to colorless immediately. The 1H NMR spectra recorded the changes in turn (Fig. 2). The nuclear magnetic resonance shows that the outcome is 2-(5-(5-(4-(diphenylamino)phenyl)thiophen-2-yl)-4,5-dihydro-1H-pyrazol-3-yl)phenol (L’) which is quite pure, so the 1H NMR and 13C NMR datas were easily obtained: 1H NMR (400 MHz, d6-DMSO) δ: 11.54-10.47 (s, 1H), 8.01 (d, J = 3.5 Hz, 1H), 7.52 (d, J = 8.7 Hz, 2H), 7.38–7.27 (m, 5H), 7.28–7.19 (m, 2H), 7.14–7.00 (m, 7H), 6.99–6.82 (m, 4H), 5.11 (td, J = 10.3, 3.5 Hz, 1H), 3.75-3.58 (m, 1H), 3.15-3.01 (m, 1H). 13C NMR (100 MHz, d6-DMSO) δ: 157.19, 153.53, 147.33, 147.10, 145.04, 142.54, 130.45, 130.09, 128.41, 128.36, 126.78, 126.69, 124.67, 123.82, 123.60, 122.72, 119.69, 117.06, 116.29, 58.50, 41.91. Compared with L, the conjugation extent in L’ is lower than it in L, and the chemical shifts in low field was reduced to some extent. Concurrently, three proton signals appeared at 3.75–3.58 ppm (m, 1H, -CH2-), 3.15–3.01 ppm (m, 1H, -CH2-) and 5.1 ppm (td, 1H,-CH-) in pyrazoline ring. The results clearly supported that the probe L can recognize hydrazine by a very efficient cyclization to form L’. To further identify the proposed mechanism, the electronic property of L and L’ at excited states were also studied by TD-DFT method, which was good agreement with experimental results. We only consider the first low-lying excited-states (S1) based on the experimental data. Compares with L, L’ possess the relatively higher electron excitation energies, which lead to a hypochromatic shift of the max absorption. In order to discover the changes of electron transition between the L and L’, the electron-hole distributions were analyzed using the Multiwfn
Novelty statement In this manuscript, we develop a novel fluorescent probe (L) benefit from smart cyclization mechanism has been constructed for ratio detection of hydrazine. And we believe this work provide a new strategy for future constructing more high performance ratiometric fluorescent probes for hydrazine. Declaration of Competing Interest The authors declared that they have no conflicts of interest to this work. Acknowledgments This work was supported by the National Natural Science Foundation of China (No. 21701025), Natural Science Foundation of Anhui Province (1908085MB44) and Natural Science Foundation of 4
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Fig. 2. The 1H NMR spectras of L and L + N2H4.
Fig. 3. Electron and hole distributions of the S1 state and the experimental absorbtion for L and L’ molecule.
Higher Education Institutions in Anhui province (KJ2019A0524), the Horizontal Cooperation Project of Fuyang Municipal Government and Fuyang Normal University (No. XDHX2016030, XDHX201728 and XDHX201701), Excellent Young Talents Fund Program of Higher Education Institutions of Anhui Province (No. gxyq2017039), the Training Programs of Innovation and Entrepreneurship for Undergraduates (201810371039, S201910371043) and the modern
analysis technology innovation team (kytd201701). The authors are also thankful to Engineering Research Centre of Biomass Conversion and Pollution Prevention Control of Anhui Provincial Department of Education, and Anhui Province Key Laboratory of Environmental Hormone and Reproduction.
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Supplementary materials
[20] B. Liu, Q. Liu, M. Shah, J. Wang, G. Zhang, Y. Pang, Fluorescence monitor of hydrazine in vivo by selective deprotection of flavonoid, Sens. Actuators B 202 (2014) 194–200. [21] M. Xing, K. Wang, X. Wu, S. Ma, D. Cao, R. Guan, Z. Liu, A coumarin chalcone ratiometric fluorescent probe for hydrazine based on deprotection, addition and subsequent cyclization mechanism, Chem. Commun. (Camb.) 55 (99) (2019) 14980–14983. [22] W. Zhang, F. Huo, T. Liu, C. Yin, Ratiometric fluorescence probe for hydrazine vapor detection and biological imaging, J. Mater. Chem. B 6 (48) (2018) 8085–8089. [23] Z. Luo, B. Liu, T. Qin, K. Zhu, C. Zhao, C. Pan, L. Wang, Cyclization of chalcone enables ratiometric fluorescence determination of hydrazine with a high selectivity, Sens. Actuators B 263 (2018) 229–236. [24] Q. Han, Z. Dong, X. Tang, L. Wang, Z. Ju, W. Liu, A ratiometric nanoprobe consisting of up-conversion nanoparticles functionalized with cobalt oxyhydroxide for detecting and imaging ascorbic acid, J. Mater. Chem. B 5 (1) (2017) 167–172. [25] P. Su, Z. Zhu, J. Wang, B. Cheng, W. Wu, K. Iqbal, Y. Tang, A biomolecule-based fluorescence chemosensor for sequential detection of Ag+ and H2S in 100% aqueous solution and living cells, Sens. Actuators B 273 (2018) 93–100. [26] K. Luan, R. Meng, C. Shan, J. Cao, J. Jia, W. Liu, Y. Tang, Terbium functionalized micelle nanoprobe for ratiometric fluorescence detection of anthrax spore biomarker, Anal. Chem. 90 (5) (2018) 3600–3607. [27] D. Liu, S. Liu, X. Zhang, Q. Zhang, J. Yu, M. Yang, X. Yang, Y. Tian, X. Zhu, H Zhou, A water-soluble benzoxazole-based probe: Real-time monitoring PPi via situ reaction by two-photon cells imaging, Talanta 195 (2019) 158–164. [28] X. Shi, F. Huo, J. Chao, C. Yin, A ratiometric fluorescent probe for hydrazine based on novel cyclization mechanism and its application in living cells, Sens. Actuators B 260 (2018) 609–616. [29] X. Xia, F. Zeng, P. Zhang, J. Lyu, Y. Huang, S. Wu, An ICT-based ratiometric fluorescent probe for hydrazine detection and its application in living cells and in vivo, Sens. Actuators B 227 (2016) 411–418. [30] Q. Wu, J. Zheng, W. Zhang, J. Wang, W. Liang, F.J. Stadler, A new quinoline-derived highly-sensitive fluorescent probe for the detection of hydrazine with excellent large-emission-shift ratiometric response, Talanta 195 (2019) 857–864. [31] X.-Y. Qiu, S.-J. Liu, Y.-Q. Hao, J.-W. Sun, S. Chen, Phenothiazine-based fluorescence probe for ratiometric imaging of hydrazine in living cells with remarkable Stokes shift, Spectrochim. Acta Part A 227 (2020) 117675. [32] Y. Sun, D. Zhao, S. Fan, L. Duan, A 4-hydroxynaphthalimide-derived ratiometric fluorescent probe for hydrazine and its in vivo applications, Sens. Actuators B 208 (2015) 512–517. [33] K. Vijay, C. Nandi, S.D. Samant, Synthesis of a dihydroquinoline based merocyanine as a ‘naked eye’ and ‘fluorogenic’ sensor for hydrazine hydrate in aqueous medium and hydrazine gas, RSC Adv. 4 (58) (2014) 30712–30717. [34] W.-N. Wu, H. Wu, Y. Wang, X.-J. Mao, X.-L. Zhao, Z.-Q. Xu, Y.-C. Fan, Z.-H. Xu, A highly sensitive and selective off-on fluorescent chemosensor for hydrazine based on coumarin β-diketone, Spectrochim. Acta Part A 188 (2018) 80–84. [35] H. Xu, Z. Huang, Y. Li, B. Gu, Z. Zhou, R. Xie, X. Pang, H. Li, Y. Zhang, A highly sensitive naked-eye fluorescent probe for trace hydrazine based on ‘C-CN’ bond cleavage, Analyst 143 (18) (2018) 4354–4358. [36] L. Wang, F.-y Liu, H.-y Liu, Y.-s Dong, T.-q Liu, J.-f Liu, Y.-w Yao, X.-j Wan, A novel pyrazoline-based fluorescent probe for detection of hydrazine in aqueous solution and gas state and its imaging in living cells, Sens. Actuators B 229 (2016) 441–452. [37] M. Frisch, G. Trucks, H. Schlegel, G. Scuseria, M. Robb, J. Cheeseman, et al., Gaussian 16, Revision A. 03, Gaussian Inc, Wallingford CT, 2016. [38] S. Grimme, J. Antony, S. Ehrlich, H. Krieg, A consistent and accurate ab initio parametrization of density functional dispersion correction (DFT-D) for the 94 elements H-Pu, J. Chem. Phys. 132 (15) (2010) 154104. [39] C. Lee, W. Yang, R.G. Parr, Development of the Colle-Salvetti correlation-energyformula into a functional of the electron density, Phys. Rev. B 37 (1988) 785. [40] T. Lu, F. Chen, Multiwfn: A multifunctional wavefunction analyzer, J. Comput. Chem. 33 (5) (2012) 580–592. [41] J. Yuan, Y. Yuan, X. Tian, Y. Liu, J. Sun, Insights into the photobehavior of fluorescent oxazinone, quinazoline, and difluoroboron derivatives: molecular design based on the structure-property relationships, J. Phys. Chem. 121 (14) (2017) 8091–8108.
Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.microc.2020.104809. References [1] U. Ragnarsson, Synthetic methodology for alkyl substituted hydrazines, Chem. Soc. Rev 30 (4) (2001) 205–213. [2] K.H. Nguyen, Y. Hao, W. Chen, Y. Zhang, M. Xu, M. Yang, Y.-N. Liu, Recent progress in the development of fluorescent probes for hydrazine, Luminescence 33 (5) (2018) 816–836. [3] J. Li, Y. Cui, C. Bi, S. Feng, F. Yu, E. Yuan, S. Xu, Z. Hu, Q. Sun, D. Wei, J. Yoon, Oligo(ethylene glycol)-functionalized ratiometric fluorescent probe for the detection of hydrazine in vitro and in vivo, Anal. Chem. 91 (11) (2019) 7360–7365. [4] A.D. Smolenkov, O.A. Shpigun, Direct liquid chromatographic determination of hydrazines: A review, Talanta 102 (2012) 93–100. [5] J. Fan, W. Sun, M. Hu, J. Cao, G. Cheng, H. Dong, K. Song, Y. Liu, S. Sun, X. Peng, An ICT-based ratiometric probe for hydrazine and its application in live cells, Chem. Commun. (Camb.) 48 (65) (2012) 8117–8119. [6] S. Goswami, S. Das, K. Aich, B. Pakhira, S. Panja, S.K. Mukherjee, S. Sarkar, A chemodosimeter for the ratiometric detection of hydrazine based on return of esipt and its application in live-cell imaging, Org. Lett. 15 (21) (2013) 5412–5415. [7] Z. An, Z. Li, Y. He, B. Shi, L. Wei, M. Yu, Ratiometric luminescence detection of hydrazine with a carbon dots–hemicyanine nanohybrid system, RSC Adv. 7 (18) (2017) 10875–10880. [8] J. Zhang, L. Ning, J. Liu, J. Wang, B. Yu, X. Liu, X. Yao, Z. Zhang, H. Zhang, Nakedeye and near-infrared fluorescence probe for hydrazine and its applications in in vitro and in vivo bioimaging, Anal. Chem. 87 (17) (2015) 9101–9107. [9] T. Li, J. Liu, L. Song, Z. Li, Q. Qi, W. Huang, A hemicyanine-based fluorescent probe for hydrazine detection in aqueous solution and its application in real time bioimaging of hydrazine as a metabolite in mice, J. Mater. Chem. B 7 (20) (2019) 3197–3200. [10] P. Zhang, Y. Gong, Q. Zhang, X. Guo, C. Ding, In situ generated chromophore as the indicator for background-free sensing strategy of hydrazine with high sensitivity with in vitro and in vivo applications, J. Mater. Chem. B 7 (34) (2019) 5182–5189. [11] Y. Jung, I.G. Ju, Y.H. Choe, Y. Kim, S. Park, Y.-M. Hyun, M.S. Oh, D. Kim, Hydrazine exposé: the next-generation fluorescent probe, ACS Sens. 4 (2) (2019) 441–449. [12] S. Nandi, A. Sahana, S. Mandal, A. Sengupta, A. Chatterjee, D.A. Safin, M.G. Babashkina, N.A. Tumanov, Y. Filinchuk, D. Das, Hydrazine selective dual signaling chemodosimetric probe in physiological conditions and its application in live cells, Anal. Chim. Acta 893 (2015) 84–90. [13] M.H. Lee, B. Yoon, J.S. Kim, J.L. Sessler, Naphthalimide trifluoroacetyl acetonate: a hydrazine-selective chemodosimetric sensor, Chem. Sci. 4 (11) (2013) 4121–4126. [14] Y. Qian, J. Lin, L. Han, L. Lin, H. Zhu, A resorufin-based colorimetric and fluorescent probe for live-cell monitoring of hydrazine, Biosens. Bioelectron 58 (2014) 282–286. [15] W. Xi, Y. Gong, B. Mei, X. Zhang, Y. Zhang, B. Chen, J. Wu, Y. Tian, H. Zhou, Schiff base derivatives based on diaminomaleonitrile: Colorimetric and fluorescent recognition of Cu(II), cell imaging application, polymorph-dependent fluorescence and aggregation-enhanced emission, Sens. Actuators B 205 (2014) 158–167. [16] Z. Feng, D. Li, M. Zhang, T. Shao, Y. Shen, X. Tian, Q. Zhang, S. Li, J. Wu, Y. Tian, Enhanced three-photon activity triggered by the AIE behaviour of a novel terpyridine-based Zn(ii) complex bearing a thiophene bridge, Chem. Sci. 10 (30) (2019) 7228–7232. [17] J. Hai, T. Li, J. Su, W. Liu, Y. Ju, B. Wang, Y. Hou, Reversible response of luminescent terbium(III)–nanocellulose hydrogels to anions for latent fingerprint detection and encryption, Angew. Chem. Int 57 (23) (2018) 6786–6790. [18] Z. Hu, T. Yang, J. Liu, Z. Zhang, G. Feng, Preparation and application of a highly sensitive conjugated polymer-copper (Ⅱ) composite fluorescent sensor for detecting hydrazine in aqueous solution, Talanta 207 (2020) 120203. [19] W. Chen, W. Liu, X.-J. Liu, Y.-Q. Kuang, R.-Q. Yu, J.-H. Jiang, A novel fluorescent probe for sensitive detection and imaging of hydrazine in living cells, Talanta 162 (2017) 225–231.
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Microchemical Journal journal homepage: www.elsevier.com/locate/microc
A smart reaction-based fluorescence probe for ratio detection of hydrazine and its application in living cells
T
Zhaodi Liu , Zhouhua Yang, Sizhe Chen, Yue Liu, Liangquan Sheng, Zhimei Tian, Deqian Huang, ⁎ Huajie Xu ⁎
School of Chemistry and Materials Engineering, Fuyang Normal University, Fuyang, Anhui 236037, PR China
ARTICLE INFO
ABSTRACT
Keywords: Triphenylamine Thiophene Ratiometric Fluorescent probe Hydrazine
Hydrazine (N2H4) has been widely used in many areas of the chemical industry, but it can cause potential harm to human health and environmental pollution. Herein, a novel fluorescent probe (L) benefit from smart cyclization mechanism has been constructed for ratio detection of hydrazine. Due to the destruction of the conjugate system by cyclic reaction, the intramolecular charge transfer (ICT) process was blocked, and a large hypsochromic shift could be observed both in the absorption and fluorescence spectra with excellent ratio response. The sensing mechanism was also studied by NMR spectra and DFT calculation. This ratiometric probe was highly selective and sensitive for detecting hydrazine with a low detection limit of 0.23 μM. Moreover, that probe was also effective in monitoring hydrazine in live cells.
1. Introduction The high reactivity of hydrazine (N2H4) makes it widely used in pharmacy, military, agriculture, industry, aerospace and other fields [1–4]. However, hydrazine is highly toxic and has a strong irritant to the eyes and skin. Especially, it can cause acute poisoning or cancer. Its widespread usage leads to the release of hydrazine, causing serious environmental problems [5–9]. The U.S. Environmental Protection Agency (EPA) suggested a safe limit for hydrazine of 10 ppb [10–12]. Therefore, it was important to develop a reliable and sensitive method for the detection of hydrazine. Fluorescent probes have attracted much attention for their simplicity, real-time detection, high selectivity and sensitivity [13–20]. Further, ratiometric fluorescent probes have some advantages evaluating the analyte concentration via self-calibrating two output signals, and provide a built-in correction for environmental effects [21–23], such as photo bleaching, molecule concentration, stability under illumination, and the environmental factors (pH, polarity, temperature, etc.) [24–27]. Up to now, there are only few reports of N2H4 probes even fewer of N2H4 ratiometric fluorescent probes based on its unique nucleophilic characteristics including 1,3-diketones, acetonate, levulinate, and phthalimide derivatives [28–32]. In spite of the reported probes have made distinct progress, some drawbacks were still unavoidable, such as poor water solubility, high pH dependence, long response time or unsatisfactory emission shift, which limited their further biological
⁎
applications [33–36]. As a result, the design of fluorescent probes for N2H4 excellent ratiometric measurement remains a challenge. Based on these above insights, we designed and synthesized a probe (E)-3-(5-(4-(diphenylamino)phenyl)thiophen-2-yl)-1-(2-hydroxyphenyl)prop-2-en-1-onefor (L) detecting N2H4 (Scheme 1). In the presence of hydrazine, a novel cyclization reaction was generated involving the conjugation addition of an amine and the condensation of ketone with another amine of hydrazine, which destroy the large conjugate system resulting in a blocked intramolecular charge transfer (ICT) processes with a large degree of spectral blue shift. All these induced the probe to achieve high sensitivity and selectivity of ratio detection for hydrazine, and the sensing performance and mechanism have been studied in detail by experiments and theory calculations. Moreover, the probe demonstrated excellent capability to monitor hydrazine in living cells. 2. Experiments 2.1. General NMR spectra were recorded on a Varian INOVA-400 MHz spectrometer with tetramethylsilane (TMS) as internal standard. Absorbance spectra were measured on a Purkinje general UV-1901 spectrophotometer. IR spectra were recorded on NEXUS 870 (Nicolet) spectrophotometer. Fluorescence spectra were performed on a Cary Eclipse
Corresponding authors. E-mail addresses: [email protected] (Z. Liu), [email protected] (H. Xu).
https://doi.org/10.1016/j.microc.2020.104809 Received 20 February 2020; Received in revised form 8 March 2020; Accepted 8 March 2020 Available online 10 March 2020 0026-265X/ Published by Elsevier B.V.
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Scheme. 1. Synthesis of the probe L and its reaction with hydrazine to form L’.
fluorescence spectrophotometer. X-ray data were collected on Bruker Smart APEX II CCD diffractometer. The structure was solved by direct methods and refined by full matrix least-square on F2 using the SHELXTL-97 program. All calculations were performed with a Gaussian 16 program using the PBE38 functional with a 6-31+G (d) basis set [37–39]. Excited states in DMSO were calculated by a TD-DFT approach using the polarizable continuum model (PCM). The electron and hole distribution of the S1 state is analyzed using the Multiwfn program (version 3.5) [40]. All reagents were of analytical grade and were not further purified for use. Double distilled water was used throughout the experiment. Anions and metal ions were prepared from their sodium salts and chloride salts. Hydrazine, ethanediamine, hydroxylamine, phenyl hydrazine, ammoniumhydroxide, triethylamine, urea, hexamethylenetetramine hydrate and some amino acids were tested their sensing behaviors in deionized water.
δ 12.51 (s, 1H), 8.13 (d, J = 8.2 Hz, 1H), 7.99 (d, J = 15.1 Hz, 1H), 7.72 (d, J = 3.6 Hz, 1H), 7.68–7.59 (m, 3H), 7.58–7.45 (m, 2H), 7.35 (t, J = 7.7 Hz, 4H), 7.11 (dd, J = 16.0, 7.8 Hz, 6H), 6.99 (t, J = 6.2 Hz, 4H); 13C NMR (100 MHz, d6-DMSO) δ 192.88, 162.08, 148.55, 148.32, 147.04, 138.29, 137.86, 136.57, 136.10, 130.97, 130.23, 127.42, 126.74, 125.25, 124.69, 124.33, 122.67, 121.41, 119.90, 119.64, 118.12; MALDI-TOF MS (M + 1 = 474.14); IR (KBr, cm−1) selected bands: 3449 (w), 1631 (s), 1584 (m), 1562 (s), 1487 (s), 1466 (m), 1275 (s), 1153 (m), 803 (w), 758 (m), 693 (m). 2.3. General UV–vis and Fluorescence Spectra Measurements A stock solution of L (1 × 10−3 M) was prepared in DMSO. The UV–vis and fluorescence measurements of the probe L were fixed at 10 μM in buffered DMSO/HEPES (10 mM, pH = 7.40, 9/1, v/v) solution at room temperature, and then the fluorescence and UV absorption spectra were recorded.
2.2. Preparation and characterization of the probe
2.4. Imaging of HeLa cells
5-[4-(Diphenylamino)phenyl]thiophene-2-carbaldehyde (1). To a solution of 4-bromotriphenylamine (1.44 g, 4.5 mmol) and PdCl2(dppf) (0.35 g, 0.3 mmol) in dry toluene (30 mL) was added a solution of 5-formylthiophene-2-yl-2-boronic acid (0.47 g, 3 mmol) and K2CO3 (4.1 g, 30 mmol) in methanol (30 mL). The mixture lasted for 10 h at 70 °C, then adding 30 mL of water to quench the reaction, and extracting with DCM (3 × 50 mL). The combined organic extract was dried over anhydrous MgSO4 and filtered. Solvent removal by rotary vaporation followed by column chromatography over silica gel with ethyl acetate/petroleum ether (1:5) yielded a yellow solid. (0.91 g, 85.1 %). 1H NMR (400 MHz, Acetone) δ: 9.91 (s, 1H), 7.94 (d, J = 4.0 Hz, 1H), 7.69 (d, J = 8.7 Hz, 2H), 7.56 (d, J = 3.9 = Hz, 1H), 7.37 (t, J = 7.9 Hz, 4H), 7.20–7.10 (m, 6H), 7.05 (d, J = 8.7 Hz, 2H); 13C NMR (100 MHz, DMSO) δ: 184.21, 153.38, 149.02, 146.88, 141.33, 140.00, 130.26, 127.90, 125.98, 125.46, 124.58, 124.46, 122.15; MALDI-TOF MS (M+1 = 356.11); IR (KBr,cm−1) selected bands: 2359(w), 1662(s), 1591(s), 1489(s), 1448(s), 1329(m), 1284(m), 1228(m), 1057(m), 808(m), 752(m), 698(s), 492(w). (E)-3-(5-(4-(diphenylamino)phenyl)thiophen-2-yl)-1-(2-hydroxyphenyl)prop-2-en-1-one (L) To a solution of 5-[4-(Diphenylamino)phenyl]thiophene-2-carbaldehyde (1) (0.71 g, 2 mmol) and 2-hydroxyacetophenone (0.27 g, 2.0 mmol) in ethanol (50 mL) was added 20% NaOH solvents. The mixture was refluxed for 8 h. A precipitate was produced with addition of ethanol, a tangerine solid was prepared after filtered, washed with ethanol and drying. Yield: 0.39 g, 40.7%. 1H NMR (400 MHz, d6-DMSO)
HeLa cells were cultured in DMEM medium (10% fetal bovine serum, FBS) with a humidified atmosphere containing CO2 5% (v/v) at 37 °C. The conventional MTT assays were first operated to evaluate the cytotoxicity of L (0–20 μM, 1% DMSO). Then, the first group was imaged as cell blank. The second group of the HeLa cells incubated with 10 μM of the probe L for 60 min at 37 °C,and the third group of the cells were incubated with the probe (10 μM) for 60 min and then incubated with N2H4 for another 60 min. In all above operations, cells are washed 3 times with PBS after each incubating, then confocal laser scanning microscope was used to image cell. 3. Results and discussion 3.1. Crystal structure description The X-ray diffraction measurements for the probe made on a Rigaku Oxford Diffraction area detector diffractometer with graphite monochromated Mo-Kα radiation (λ = 0.71073 Å) at 293 K. For data collection, block-shaped red crystals with dimensions of 0.2 × 0.2 × 0.1 mm were used. It belongs to the monoclinic system with space group P21/c. Its unit cell dimensions are: a = 19.432 (5) Å b = 12.305 (2) Åc = 10.389 (2) Å,β = 76.416 (2)°, with Z = 4. Table S1 shows the experimental conditions for the single crystal diffraction data collection and structure refinement. The selected bond lengths and angles have been provided in Tables S2. The structure of L 2
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Fig. 1. The absorption (a) and fluorescent spectra (b, λex = 380 nm; c, λex = 446 nm) of L (10 μM) to N2H4 (200 μM) and other amine species (500 μM). (d) The fluorescence intensity ratios (I433 nm/I628 nm) and color of L in the presence of N2H4 (200 μM) and other amine species. Inset in (a,b): the color changes of L with hydrazine. Inset in (d): The emission color of L with amine species. Abbreviation: 1, ethanediamine; 2, hydroxylamine; 3, phenyl hydrazine; 4, ammoniumhydroxide; 5, triethylamine; 6, urea; 7, hexamethylenetetramine; 8, probe L; 9, hydrazine.
(Fig. S1) shows s-cis geometry of the enone as expected, with less torsion angle 9.3 (9)° through the enone torsion (O]CeC]C), and the olefinic in the trans-conformations. There is an intramolecular hydrogen bond between the enone carbonyl and ortho-hydroxy group on the benzene ring (Tables S3). The dihedral angles between thiophene ring and benzene ring (C13-C18, C27-C32) are 16.8 (1)° and 26.5(2)°, respectively. So the molecular backbone is twisted significantly.
were also investigated. As seen in Fig. S3, while the fluorescence changes caused by other analytes were negligible during the same experimental operation. Remarkably, due to the specific reaction with hydrazine, a large emission band hyposochromic shift from 628 nm to 433 nm (~200 nm) could be observed, which allowed the visible monitoring of N2H4 with an ultraviolet lamp. The emission color also changed from red to blue (inside Fig. 1d), so the presence of hydrazine could be easily observed.
3.2. Selectivity of L to hydrazine and other analytes
3.3. Spectra of probe L titrated with N2H4
In order to verify the design idea of probe L, the absorption and fluorescence spectrum were investigated in buffered DMSO/HEPES (10 mM, pH = 7.40, 9/1, v/v) to evaluated the selectivity of the probe L in various cations, anions, and amines solution. As shown in Fig. 1a and Fig. S2, in the absence of N2H4, the solution containing probe L was yellow. With the addition of N2H4, the probes exhibited a new blue shift absorption peak at 353 nm from 463 nm. The color change from yellow to colorless enables one to distinguish N2H4 by the naked eye (inside Fig. 1a). The fluorescence response of L was also measured towards amine species. As shown in Fig. 1b and Fig. 1c, upon excitation at 380 nm and 446 nm respectively, only the addition of hydrazine significantly enhanced the fluorescence of L at 433 nm and gradually decreased it at 628 nm. Other amines had no significant effect on the fluorescence of L, and their fluorescence ratio (I433 nm/I628 nm) also indicated that L was highly selective to hydrazine (Fig. 1d). Various cations, anions and small biomolecules which often exist in environmental and biological samples were selected and their effects on fluorescence spectra of L
The absorption and fluorescence titration of probe L were performed in detail with hydrazine in a solution of DMSO/HEPES (10 mM, pH = 7.40, 9/1, v/v). The probe L exhibited a strong absorption peak at 463 nm as observed in Fig. S4. When the hydrazine from 0 to 200 μM was added, the absorption peak at 353 nm gradually increased, while the absorption peak at 463 nm gradually decreased, accompanied by a well-defined isosbestic point at 380 nm, and there was also a significant color change from yellow to colorless. As shown in Fig. S5a,b, when the probe L was excited at 380 nm and displayed a weak emission peak at 433 nm. Interestingly, with the addition of N2H4, which induced significantly increase in fluorescence intensity at 433 nm. In Fig. S5c,d, we used excitation at 446 nm to observe the change of fluorescence spectra, and the fluorescence intensity at 628 nm, significant quenching with a large hypsochromic shift from 628 nm to 571 nm upon the addition of N2H4 (0-200 μM). The fluorescence ratio of the two emission intensities (I433 nm/I628 nm) increased about 360-fold and showed a good linear relationship with the concentration of N2H4 from 0 to 80 3
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μM (R2 = 0.99869) as shown in Fig. S6a. The detection limit for N2H4 was calculated to be about 0.23 μM in terms of the equation of LOD = 3 Sb/m, and indicated that the probe possessed excellent analytical performance and comparable to the performance of the most reported probes (Table S4).
program (version 3.5). The electron and hole distribution of the S1 state for L and L’ molecules are shown in Fig. 3 and table S5. For L, the electrons mainly locate at the thiophene and enone group, but holes are distributed at the triphenylamine and thiophene group. These indicated electrons transfer from the triphenylamine to enone groups showing typical ICT excitation. However, for L’, electrons mainly locate at the central phenyl and thiophene, and holes are still distributed at the triphenylamine and thiophene group, which means electrons transfer from the triphenylamine to the central phenyl and thiophene, showing ICT excitation, too. As can be seen from Table S5, the S/D values of L (0.13 Å−1) is smaller than that of L’ (0.35 Å−1), so L shows more remarkable CT modes [41]. That is to say, with addition of hydrazine to L, the enone was broken to form L’ which hinder the electron transitions.
3.4. pH-dependence and time-dependence for hydrazine Furthermore, the effect of pH on the emission ratio (I433 nm/I628 nm) of the probe had also been investigated in the absence and presence of N2H4. As shown in Fig. S6b, without adding N2H4, the fluorescence intensity ratio (I433 nm/I628 nm) of the probe L hardly changed when the pH value was less than 8.5, but the emission ratio significantly increased with pH ≧ 8.5, indicating that L was stable under neutral conditions and easy to inhibit the electron transfer process under alkaline condition. Upon the addition of N2H4, the fluorescence emission ratio (I433 nm/I628 nm) gradually increased from pH 6.0 to 8.0, implying that probe L enable a selective detection of N2H4 in a wide pH range including physiological conditions. The time-dependence of probe L towards N2H4 was also measured in DMSO/HEPES (10 mM, pH = 7.40, 9/1, v/v). As shown in Fig. S7, after treatment with N2H4 (200 μM), the absorption peak at 336 nm redshifted slightly to 353 nm and another absorption peak at 463 nm gradually reduced with time. The main absorption peak strength decrease at 463 nm was due to the conjugation addition of N2H4 to the unsaturated carbonyl configuration. Time course studies showed that the absorbance reached a plateau at about 30 min. Fluorescence intensity changed with time after the same amount of N2H4 treatment (Fig. S8) was also consistent with the absorption result, and the fluorescence emission ratio (I433 nm/I628 nm) of probe L gradually increased and remained constant within 30 min. In conclusion, the probe L was sensitive to hydrazine, and enabling to detect N2H4 as a practical method.
3.6. Bio-imaging of hydrazine Cellular imaging studies were performed to examine the sensing behavior of probe L in living Hela cells. The cytotoxicity of probe L was first evaluated using the MTT assay (Fig. S9), which revealed that probe L had highly biocompatible to Hela cells. As such, we attempted to explore the potential applications of probe L in the living systems. When probe L loaded HeLa cells at 37 °C for 30 min, and strong red and negligible blue fluorescent emissions can be observed (Fig. S10a-c), such stable photophysical properties indicated that the probe can enter the cell smoothly. Compared to another controlled experiment, after HeLa cells loaded with probes were further incubated with N2H4 for another 30 min, the fluorescence intensity in the blue channel increased significantly and the red fluorescence emissions faded simultaneously (Fig. S10e-g). Moreover, the merged image (Fig. S10d,h) overlaid very well in the two channels, and these cell experiments clearly demonstrated excellent performance tracing N2H4 in living cells for probe L. 4. Conclusions In summary, a ratiometric fluorescent probe based on a novel cyclization platform was set up for the efficient detection of N2H4. The synthesized probe L was equipped with chalcone and thiophene groups and to ensure active reaction sites and aqueous solubility. As expected, probe L show highly sensitive and selective to hydrazine for its cyclization reaction with chalcone leading to a ratiometric response. The excellent anti-interference ability for cations, amino acids and pH has been studied, and the sensing mechanism was also explored by NMR spectra and DFT calculation. When N2H4 was added to L, the enone configuration was broken to form L’, which hinder the electron transitions with spectral hypochromatic shift. Moreover, the developed new chemosignaling system based on cyclization principles will be very helpful for the development of other probes for N2H4.
3.5. Mechanism of probe L responding to hydrazine On the basis of results achieved above, the sensing mechanism of probe L toward hydrazine was proposed to proceed a cyclization reaction forming pyrazoline ring by 1H NMR spectroscopic. Upon addition of 1 equiv. of hydrazine into the assay system, the yellow liquid turned to colorless immediately. The 1H NMR spectra recorded the changes in turn (Fig. 2). The nuclear magnetic resonance shows that the outcome is 2-(5-(5-(4-(diphenylamino)phenyl)thiophen-2-yl)-4,5-dihydro-1H-pyrazol-3-yl)phenol (L’) which is quite pure, so the 1H NMR and 13C NMR datas were easily obtained: 1H NMR (400 MHz, d6-DMSO) δ: 11.54-10.47 (s, 1H), 8.01 (d, J = 3.5 Hz, 1H), 7.52 (d, J = 8.7 Hz, 2H), 7.38–7.27 (m, 5H), 7.28–7.19 (m, 2H), 7.14–7.00 (m, 7H), 6.99–6.82 (m, 4H), 5.11 (td, J = 10.3, 3.5 Hz, 1H), 3.75-3.58 (m, 1H), 3.15-3.01 (m, 1H). 13C NMR (100 MHz, d6-DMSO) δ: 157.19, 153.53, 147.33, 147.10, 145.04, 142.54, 130.45, 130.09, 128.41, 128.36, 126.78, 126.69, 124.67, 123.82, 123.60, 122.72, 119.69, 117.06, 116.29, 58.50, 41.91. Compared with L, the conjugation extent in L’ is lower than it in L, and the chemical shifts in low field was reduced to some extent. Concurrently, three proton signals appeared at 3.75–3.58 ppm (m, 1H, -CH2-), 3.15–3.01 ppm (m, 1H, -CH2-) and 5.1 ppm (td, 1H,-CH-) in pyrazoline ring. The results clearly supported that the probe L can recognize hydrazine by a very efficient cyclization to form L’. To further identify the proposed mechanism, the electronic property of L and L’ at excited states were also studied by TD-DFT method, which was good agreement with experimental results. We only consider the first low-lying excited-states (S1) based on the experimental data. Compares with L, L’ possess the relatively higher electron excitation energies, which lead to a hypochromatic shift of the max absorption. In order to discover the changes of electron transition between the L and L’, the electron-hole distributions were analyzed using the Multiwfn
Novelty statement In this manuscript, we develop a novel fluorescent probe (L) benefit from smart cyclization mechanism has been constructed for ratio detection of hydrazine. And we believe this work provide a new strategy for future constructing more high performance ratiometric fluorescent probes for hydrazine. Declaration of Competing Interest The authors declared that they have no conflicts of interest to this work. Acknowledgments This work was supported by the National Natural Science Foundation of China (No. 21701025), Natural Science Foundation of Anhui Province (1908085MB44) and Natural Science Foundation of 4
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Fig. 2. The 1H NMR spectras of L and L + N2H4.
Fig. 3. Electron and hole distributions of the S1 state and the experimental absorbtion for L and L’ molecule.
Higher Education Institutions in Anhui province (KJ2019A0524), the Horizontal Cooperation Project of Fuyang Municipal Government and Fuyang Normal University (No. XDHX2016030, XDHX201728 and XDHX201701), Excellent Young Talents Fund Program of Higher Education Institutions of Anhui Province (No. gxyq2017039), the Training Programs of Innovation and Entrepreneurship for Undergraduates (201810371039, S201910371043) and the modern
analysis technology innovation team (kytd201701). The authors are also thankful to Engineering Research Centre of Biomass Conversion and Pollution Prevention Control of Anhui Provincial Department of Education, and Anhui Province Key Laboratory of Environmental Hormone and Reproduction.
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Supplementary materials
[20] B. Liu, Q. Liu, M. Shah, J. Wang, G. Zhang, Y. Pang, Fluorescence monitor of hydrazine in vivo by selective deprotection of flavonoid, Sens. Actuators B 202 (2014) 194–200. [21] M. Xing, K. Wang, X. Wu, S. Ma, D. Cao, R. Guan, Z. Liu, A coumarin chalcone ratiometric fluorescent probe for hydrazine based on deprotection, addition and subsequent cyclization mechanism, Chem. Commun. (Camb.) 55 (99) (2019) 14980–14983. [22] W. Zhang, F. Huo, T. Liu, C. Yin, Ratiometric fluorescence probe for hydrazine vapor detection and biological imaging, J. Mater. Chem. B 6 (48) (2018) 8085–8089. [23] Z. Luo, B. Liu, T. Qin, K. Zhu, C. Zhao, C. Pan, L. Wang, Cyclization of chalcone enables ratiometric fluorescence determination of hydrazine with a high selectivity, Sens. Actuators B 263 (2018) 229–236. [24] Q. Han, Z. Dong, X. Tang, L. Wang, Z. Ju, W. Liu, A ratiometric nanoprobe consisting of up-conversion nanoparticles functionalized with cobalt oxyhydroxide for detecting and imaging ascorbic acid, J. Mater. Chem. B 5 (1) (2017) 167–172. [25] P. Su, Z. Zhu, J. Wang, B. Cheng, W. Wu, K. Iqbal, Y. Tang, A biomolecule-based fluorescence chemosensor for sequential detection of Ag+ and H2S in 100% aqueous solution and living cells, Sens. Actuators B 273 (2018) 93–100. [26] K. Luan, R. Meng, C. Shan, J. Cao, J. Jia, W. Liu, Y. Tang, Terbium functionalized micelle nanoprobe for ratiometric fluorescence detection of anthrax spore biomarker, Anal. Chem. 90 (5) (2018) 3600–3607. [27] D. Liu, S. Liu, X. Zhang, Q. Zhang, J. Yu, M. Yang, X. Yang, Y. Tian, X. Zhu, H Zhou, A water-soluble benzoxazole-based probe: Real-time monitoring PPi via situ reaction by two-photon cells imaging, Talanta 195 (2019) 158–164. [28] X. Shi, F. Huo, J. Chao, C. Yin, A ratiometric fluorescent probe for hydrazine based on novel cyclization mechanism and its application in living cells, Sens. Actuators B 260 (2018) 609–616. [29] X. Xia, F. Zeng, P. Zhang, J. Lyu, Y. Huang, S. Wu, An ICT-based ratiometric fluorescent probe for hydrazine detection and its application in living cells and in vivo, Sens. Actuators B 227 (2016) 411–418. [30] Q. Wu, J. Zheng, W. Zhang, J. Wang, W. Liang, F.J. Stadler, A new quinoline-derived highly-sensitive fluorescent probe for the detection of hydrazine with excellent large-emission-shift ratiometric response, Talanta 195 (2019) 857–864. [31] X.-Y. Qiu, S.-J. Liu, Y.-Q. Hao, J.-W. Sun, S. Chen, Phenothiazine-based fluorescence probe for ratiometric imaging of hydrazine in living cells with remarkable Stokes shift, Spectrochim. Acta Part A 227 (2020) 117675. [32] Y. Sun, D. Zhao, S. Fan, L. Duan, A 4-hydroxynaphthalimide-derived ratiometric fluorescent probe for hydrazine and its in vivo applications, Sens. Actuators B 208 (2015) 512–517. [33] K. Vijay, C. Nandi, S.D. Samant, Synthesis of a dihydroquinoline based merocyanine as a ‘naked eye’ and ‘fluorogenic’ sensor for hydrazine hydrate in aqueous medium and hydrazine gas, RSC Adv. 4 (58) (2014) 30712–30717. [34] W.-N. Wu, H. Wu, Y. Wang, X.-J. Mao, X.-L. Zhao, Z.-Q. Xu, Y.-C. Fan, Z.-H. Xu, A highly sensitive and selective off-on fluorescent chemosensor for hydrazine based on coumarin β-diketone, Spectrochim. Acta Part A 188 (2018) 80–84. [35] H. Xu, Z. Huang, Y. Li, B. Gu, Z. Zhou, R. Xie, X. Pang, H. Li, Y. Zhang, A highly sensitive naked-eye fluorescent probe for trace hydrazine based on ‘C-CN’ bond cleavage, Analyst 143 (18) (2018) 4354–4358. [36] L. Wang, F.-y Liu, H.-y Liu, Y.-s Dong, T.-q Liu, J.-f Liu, Y.-w Yao, X.-j Wan, A novel pyrazoline-based fluorescent probe for detection of hydrazine in aqueous solution and gas state and its imaging in living cells, Sens. Actuators B 229 (2016) 441–452. [37] M. Frisch, G. Trucks, H. Schlegel, G. Scuseria, M. Robb, J. Cheeseman, et al., Gaussian 16, Revision A. 03, Gaussian Inc, Wallingford CT, 2016. [38] S. Grimme, J. Antony, S. Ehrlich, H. Krieg, A consistent and accurate ab initio parametrization of density functional dispersion correction (DFT-D) for the 94 elements H-Pu, J. Chem. Phys. 132 (15) (2010) 154104. [39] C. Lee, W. Yang, R.G. Parr, Development of the Colle-Salvetti correlation-energyformula into a functional of the electron density, Phys. Rev. B 37 (1988) 785. [40] T. Lu, F. Chen, Multiwfn: A multifunctional wavefunction analyzer, J. Comput. Chem. 33 (5) (2012) 580–592. [41] J. Yuan, Y. Yuan, X. Tian, Y. Liu, J. Sun, Insights into the photobehavior of fluorescent oxazinone, quinazoline, and difluoroboron derivatives: molecular design based on the structure-property relationships, J. Phys. Chem. 121 (14) (2017) 8091–8108.
Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.microc.2020.104809. References [1] U. Ragnarsson, Synthetic methodology for alkyl substituted hydrazines, Chem. Soc. Rev 30 (4) (2001) 205–213. [2] K.H. Nguyen, Y. Hao, W. Chen, Y. Zhang, M. Xu, M. Yang, Y.-N. Liu, Recent progress in the development of fluorescent probes for hydrazine, Luminescence 33 (5) (2018) 816–836. [3] J. Li, Y. Cui, C. Bi, S. Feng, F. Yu, E. Yuan, S. Xu, Z. Hu, Q. Sun, D. Wei, J. Yoon, Oligo(ethylene glycol)-functionalized ratiometric fluorescent probe for the detection of hydrazine in vitro and in vivo, Anal. Chem. 91 (11) (2019) 7360–7365. [4] A.D. Smolenkov, O.A. Shpigun, Direct liquid chromatographic determination of hydrazines: A review, Talanta 102 (2012) 93–100. [5] J. Fan, W. Sun, M. Hu, J. Cao, G. Cheng, H. Dong, K. Song, Y. Liu, S. Sun, X. Peng, An ICT-based ratiometric probe for hydrazine and its application in live cells, Chem. Commun. (Camb.) 48 (65) (2012) 8117–8119. [6] S. Goswami, S. Das, K. Aich, B. Pakhira, S. Panja, S.K. Mukherjee, S. Sarkar, A chemodosimeter for the ratiometric detection of hydrazine based on return of esipt and its application in live-cell imaging, Org. Lett. 15 (21) (2013) 5412–5415. [7] Z. An, Z. Li, Y. He, B. Shi, L. Wei, M. Yu, Ratiometric luminescence detection of hydrazine with a carbon dots–hemicyanine nanohybrid system, RSC Adv. 7 (18) (2017) 10875–10880. [8] J. Zhang, L. Ning, J. Liu, J. Wang, B. Yu, X. Liu, X. Yao, Z. Zhang, H. Zhang, Nakedeye and near-infrared fluorescence probe for hydrazine and its applications in in vitro and in vivo bioimaging, Anal. Chem. 87 (17) (2015) 9101–9107. [9] T. Li, J. Liu, L. Song, Z. Li, Q. Qi, W. Huang, A hemicyanine-based fluorescent probe for hydrazine detection in aqueous solution and its application in real time bioimaging of hydrazine as a metabolite in mice, J. Mater. Chem. B 7 (20) (2019) 3197–3200. [10] P. Zhang, Y. Gong, Q. Zhang, X. Guo, C. Ding, In situ generated chromophore as the indicator for background-free sensing strategy of hydrazine with high sensitivity with in vitro and in vivo applications, J. Mater. Chem. B 7 (34) (2019) 5182–5189. [11] Y. Jung, I.G. Ju, Y.H. Choe, Y. Kim, S. Park, Y.-M. Hyun, M.S. Oh, D. Kim, Hydrazine exposé: the next-generation fluorescent probe, ACS Sens. 4 (2) (2019) 441–449. [12] S. Nandi, A. Sahana, S. Mandal, A. Sengupta, A. Chatterjee, D.A. Safin, M.G. Babashkina, N.A. Tumanov, Y. Filinchuk, D. Das, Hydrazine selective dual signaling chemodosimetric probe in physiological conditions and its application in live cells, Anal. Chim. Acta 893 (2015) 84–90. [13] M.H. Lee, B. Yoon, J.S. Kim, J.L. Sessler, Naphthalimide trifluoroacetyl acetonate: a hydrazine-selective chemodosimetric sensor, Chem. Sci. 4 (11) (2013) 4121–4126. [14] Y. Qian, J. Lin, L. Han, L. Lin, H. Zhu, A resorufin-based colorimetric and fluorescent probe for live-cell monitoring of hydrazine, Biosens. Bioelectron 58 (2014) 282–286. [15] W. Xi, Y. Gong, B. Mei, X. Zhang, Y. Zhang, B. Chen, J. Wu, Y. Tian, H. Zhou, Schiff base derivatives based on diaminomaleonitrile: Colorimetric and fluorescent recognition of Cu(II), cell imaging application, polymorph-dependent fluorescence and aggregation-enhanced emission, Sens. Actuators B 205 (2014) 158–167. [16] Z. Feng, D. Li, M. Zhang, T. Shao, Y. Shen, X. Tian, Q. Zhang, S. Li, J. Wu, Y. Tian, Enhanced three-photon activity triggered by the AIE behaviour of a novel terpyridine-based Zn(ii) complex bearing a thiophene bridge, Chem. Sci. 10 (30) (2019) 7228–7232. [17] J. Hai, T. Li, J. Su, W. Liu, Y. Ju, B. Wang, Y. Hou, Reversible response of luminescent terbium(III)–nanocellulose hydrogels to anions for latent fingerprint detection and encryption, Angew. Chem. Int 57 (23) (2018) 6786–6790. [18] Z. Hu, T. Yang, J. Liu, Z. Zhang, G. Feng, Preparation and application of a highly sensitive conjugated polymer-copper (Ⅱ) composite fluorescent sensor for detecting hydrazine in aqueous solution, Talanta 207 (2020) 120203. [19] W. Chen, W. Liu, X.-J. Liu, Y.-Q. Kuang, R.-Q. Yu, J.-H. Jiang, A novel fluorescent probe for sensitive detection and imaging of hydrazine in living cells, Talanta 162 (2017) 225–231.
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