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An ultrasensitive fluorescent probe for hydrazine detection and its application in water samples and living cells
Accepted Manuscript An ultrasensitive fluorescent probe for hydrazine detection and its application in water samples and living cells Shao-Hua Guo, Zhi-Qian Guo, Cheng-Yun Wang, Yongjia Shen, Wei-Hong Zhu PII:
S0040-4020(19)30297-2
DOI:
https://doi.org/10.1016/j.tet.2019.03.022
Reference:
TET 30208
To appear in:
Tetrahedron
Received Date: 29 January 2019 Revised Date:
8 March 2019
Accepted Date: 11 March 2019
Please cite this article as: Guo S-H, Guo Z-Q, Wang C-Y, Shen Y, Zhu W-H, An ultrasensitive fluorescent probe for hydrazine detection and its application in water samples and living cells, Tetrahedron (2019), doi: https://doi.org/10.1016/j.tet.2019.03.022. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Graphical Abstract
An ultrasensitive fluorescent probe for hydrazine detection and its application in water samples and living cells
A coumarin-based fluorescent probe for ultrasensitive hydrazine sensing has been developed. The practical utilities of probe have been successfully proved through quantitative N2H4 detection in environmental water samples and bioimaging of N2H4 in living cells.
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Shao-Hua Guo, Zhi-Qian Guo, Cheng-Yun Wang*, Yongjia Shen and Wei-Hong Zhu*
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Shanghai Key Laboratory of Functional Materials Chemistry, Key Laboratory for Advanced Materials and Institute of Fine Chemicals, School of Chemistry and Molecular Engineering, East China University of Science & Technology, Shanghai 200237, P. R. China.
ACCEPTED MANUSCRIPT Graphical Abstract A coumarin-based fluorescent probe for ultrasensitive hydrazine sensing has been developed. The practical utilities of probe have been successfully proved through quantitative N2H4 detection in environmental water samples and bioimaging of N2H4 in living cells.
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An ultrasensitive fluorescent probe for hydrazine detection and its application in water samples and living cells
Shao-Hua Guo, Zhi-Qian Guo, Cheng-Yun Wang*, Yongjia Shen and Wei-Hong Zhu*
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Shanghai Key Laboratory of Functional Materials Chemistry, Key Laboratory for Advanced Materials and Institute of Fine Chemicals, School of Chemistry and Molecular Engineering, East China University of Science & Technology, Shanghai 200237, P. R. China.
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Tetrahedron journal homepage: www.elsevier.com
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An ultrasensitive fluorescent probe for hydrazine detection and its application in water samples and living cells Shao-Hua Guo, Zhi-Qian Guo, Cheng-Yun Wang*, Yongjia Shen and Wei-Hong Zhu ∗
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Shanghai Key Laboratory of Functional Materials Chemistry, Key Laboratory for Advanced Materials and Institute of Fine Chemicals, School of Chemistry and Molecular Engineering, East China University of Science & Technology, Shanghai 200237, P. R. China.
ABSTRACT
Article history: Received Received in revised form Accepted Available online
Hydrazine, as a strong reducing agent, has been extensively used in many industrial manufactures. However, it is a potential human carcinogen and an environmental contaminant due to its high toxicity. Therefore, developing an ultrasensitive method for determining hydrazine in real water and biosystems is of great significance. Herein, based on coumarin dye, a turn-on fluorescent probe Cou-1-N2H4, which contains an acetyl group as the trigger unit and the fluorescence quencher, is developed. The probe can achieve a rapid (3min) and colorimetric sensing detection for hydrazine with an extremely low limit detection (11.9 nM or 0.38 ppb). More importantly, the practical utilities of probe have been successfully proved through quantitative N2H4 detection in environmental water samples and bioimaging of N2H4 in living cells. 2019 Elsevier Ltd. All rights reserved.
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ARTICLE INFO
Keywords: Ultrasensitive Hydrazine Real water Bioimaging
1. Introduction
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Hydrazine (N2H4) has been wildly applied to many chemical industries.[1] As one of the most essential strong reducing agent or reactive bases, hydrazine plays a key role in the preparation of emulsifiers, pharmaceuticals, paints and pesticides.[2,3] In addition, hydrazine can be also used as fuel for missile and rocket propulsion systems due to its high enthalpy.[4] However, hydrazine is poisonous, excess exposure to it may lead to serious organ damage, including kidney, liver, lung, and especially the central nervous system.[5, 6] Thus, the governmental regulations have been issued to control the residual hydrazine content in the final products for the prevention of health problems, for instance, its threshold in drinking water is 10 ppb.[7] In view of this, it is of great significance to develop a highly effective method for hydrazine sensing in the fields of chemistry, biology and environment. Up to now, a variety of techniques, such as chemiluminescence,[8] surface enhanced Raman spectroscopy,[9] chromatography,[10] electrochemistry,[11-14] have been developed to detect hydrazine. However, they often need time-consuming procedures, complicated instruments and are inappropriate to intracellular detection. The fluorescence spectra analysis has attracted a lot of attention because of its good compatibility to biological samples, convenience, simplicity and excellent selectivity. Until now, many kinds of fluorescent chemosensors for hydrazine detection have been reported based on three main mechanisms, which includes specific deprotection of acetyl groups or levulinoyl ester,[15-22] specific reaction with
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arylidenemalononitrile,[23-26] and condensation with arylaldehydes.[27,28] Xu’s group[29] initially synthesized a trioutput probe for specific detection of hydrazine, the probe could achieve a rapid detection (1 min) of hydrazine with a low detection (0.1 µM). Liu and co-workers[30] developed a ratiometric fluorescent probe for hydrazine based on a chemoselective hydrazine-induced chalcone cyclization. The probe exhibits a low limit of detection 0.3 µM and the detection is complete within 60 min. By selecting coumarin derivative as fluorophore and acetyl group as a recognizing group, Yin’s group[31] design a fluorescent probe based on the cyclization mechanism for hydrazine. This probe shows a limit of detection 31 µM and a rapid detection progress (7 min). Despite the great progress has been made, there are still some drawbacks. For instance, some of fluorescent chemosensors had relatively long synthetic route, high limit detection or long detection time, which limited their application. In view of this, the development of simple and ultrasensitive fluorescent probes for hydrazine sensing with a fast detection process is still urgent. Herein, a new coumarin-based colorimetric and fluorescent two-output probe has been prepared for ultrasensitive hydrazine sensing. In the design, Cou-1 (3-benzothiazolyl-7hydroxycoumarin, Scheme 1) is employed as a fluorophore due to its excellent optical properties such as high fluorescence quantum yields (ΦF=0.56, see Table S1) and large absorption extinction coefficients, and acetyl group as an excellent recognition unit and the fluorescence quencher. Cou-1-N2H4 probe (Scheme 1) not only shows excellent colorimetric (colorless to green yellow) and rapid response toward hydrazine
∗ Corresponding author. Tel./fax: +86-021-64252967, e-mail: [email protected] (C. -Y. Wang), [email protected] (W. -H. Zhu)
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To further evaluate the sensing properties of Cou-1-N2H4 (3 min), but also has extremely low detection limit (11.9 nM). MANUSCRIPT ACCEPTED probe, the reaction time of Cou-1-N2H4 probe with N2H4 was More importantly, the probe can be used to detect hydrazine in investigated by carrying out time-dependent fluorescence environmental water samples quantitatively and bioimage experiments. As it can be seen in Fig. S1, after addition of 10 hydrazine in living Hela cells. equiv. of N2H4, fluorescence intensity (at 496 nm) of probe (10 µM) was gradually increased and appeared a fluorescent maximum after 3 min. Therefore, the reaction time needed to produce stable fluorescence intensity was 3 min.
2.1. Absorption and fluorescent detection of hydrazine
Fig.1 The linearity of fluorescence intensity (at 496 nm) versus N2H4 concentrations.
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Initially, we investigated the response of Cou-1-N2H4 toward N2H4 in PBS-DMSO (pH 7.4, 20 mM, v/v = 1/1). As it can be seen in Fig. 1A, the probe solution showed an absorption band at 376 nm. But after gradual addition of hydrazine, we could find the absorption peak at 376 nm evidently decreased along with the appearance of a new absorption peak around 462 nm (isosbestic point at 410 nm), and there was a clear color change from colorless to green yellow under natural light during the detection. This result indicated that the probe was able to serve as a “nakedeye” colorimetric indicator for N2H4.
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2. Results and discussion
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Scheme 1 Synthesis of Cou-1-N2H4
2.3. Selectivity and effect of pH
Besides highly sensitive, a satisfactory N2H4 probe should also have high selectivity. In order to investigate the selectivity of the probe for N2H4, a wide variety of analytes were added to the probe solution. As it can be seen in Fig. 3, the fluorescence spectra displayed almost no changes after addition of various other analytes, including Cu2+, Fe3+, Al3+, Na+, Fe2+, Ba2+, Zn2+, K+, HS-, Asn, Asp, Ala, Arg, Gln, GSH, Hcy, Cys, Glu, Gly, Phe, Pro, Trp, His, Leu, Thr and Val. However, a significant fluorescence enhancement was observed upon addition of N2H4, indicating the probe had good selectivity toward N2H4 over these analytes. In addition, competing experiment showed that other analytes could hardly interfere with the detection process (Fig. 4).
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As shown in Fig. 1B, the probe shows an extremely low fluorescence intensity at 496 nm. However, within 3 minutes, a green remarkable fluorescence enhancement was clearly observed at 496 nm after addition of N2H4. These results implied that the probe could play the part of a promising turn-on fluorescent indicator.
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Fig.1 (A) Absorption spectra properties of Cou-1-N2H4 upon treatment with 15 equiv. of N2H4, (B) Fluorescent spectra changes of different concentration of N2H4 (0–15equiv.) in a mixed PBS-DMSO solution (pH 7.4, 20 mM, v/v = 1/1). Insert: Emission color and color change of Cou1-N2H4 before and after addition of N2H4. λex = 465 nm. Slit width:10 nm/10 nm.
2.2. The sensitivity
To investigate the sensitivity of the probe, the probe solution was handled with different concentrations of N2H4. As shown in Fig. 2, the fluorescence intensity of the probe solution increased upon gradual addition of N2H4. The relative fluorescence intensity at 496 nm showed a satisfactory linearity (R2 = 0.9978) with the N2H4 concentration ranging from 0 to 60 µM. Based on the definition by IUPAC (CDL = 3σ / S),[32-35] the detection limit was calculated to be about 11.9 nM (0.38 ppb) in PBS-DMSO (20 mM, pH 7.4, v/v = 1/1), which was much lower than 10 ppb suggested by EPA. These results indicated that the probe was ultrasensitive toward N2H4.
Fig.3 Fluorescent spectra changes of Cou-1-N2H4 (10 µM) for various analytes (10 equiv.) in a mixed solution PBS-DMSO (20 mM, pH 7.4, v/v = 1/1). Analytes are: Cu2+, Fe3+, Al3+, Na+, Fe2+, Ba2+, Zn2+, K+, HS-, Asn, Asp, Ala, Arg, Gln, GSH, Hcy, Cys, Glu, Gly, Phe, Pro, Trp, His, Leu, Thr, Val. λex = 465 nm. Slit width:10 nm/10 nm.
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N2H4 spiked (M)
N2H4 recovered (M)
0
Not detected
Tap water
Fig.4 The selectivity of Cou-1-N2H4 (10 µM). The black bars represent the emission intensity of Cou-1-N2H4 in the presence of other analytes (100 µM). The red bars represent the fluorescence intensity (at 496 nm) that occurs upon addition of (100 µM) N2H4 to the above solution. 1: Cu2+, 2: Fe3+, 3: Al3+, 4: Na+, 5: Fe2+, 6: Ba2+, 7: Zn2+, 8: K+, 9: NaHS, 10: Asn, 11: Asp, 12: Ala, 13: Arg, 14: Gln, 15: GSH, 16: Hcy, 17: Cys, 18: Glu, 19: Gly, 20: Phe, 21: Pro, 22: Trp, 23: His, 24: Leu, 25: Thr, 26: Val
Huangpu River
3×10
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(3.10±0.09)×10
3×10-5
(2.94±0.13)×10
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0
Not detected
3×10
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3×10
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3×10
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0
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As a suspected carcinogen, hydrazine has been extensively applied to various industrial manufactures. Even worse, owing to the perfect water solubility of N2H4, biological systems can absorb N2H4 easily through inhalation or skin contact. Thus, it is of great necessity to determine hydrazine in water samples. Standard addition method[36-38] was used to deal with the real water samples from the tap water, Qingchun River (ECUST) and Huangpu River in Shanghai, then the samples were used in the test. As shown in Table 1, the recovery rates were in the range of 97%-105%. The result indicated that Cou-1-N2H4 could be qualified to determine hydrazine in environmental water samples quantitatively. 2.5. Proposed mechanism
3×10-5
103.3 98
(2.95±0.12)×10-7
98.3
(2.97±0.16)×10
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99
(3.08±0.14)×10
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102.6 —
(3.15±0.13)×10
-7
105
(3.06±0.08)×10
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102
(3.11±0.12)×10-5
103.7
Note: the results shown in the table are reported as the mean ± standard deviation of triplicate experiments.
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2.4. Determination of hydrazine in water samples
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97
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Not detected
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3×10
In order to investigate the applicability, the effect of pH on the fluorescence intensity of Cou-1-N2H4 in the presence or absence of N2H4 was studied in wide pH range 5.0-12.0. As shown in Fig. S2, the appropriate working pH was from 7.0 to 10.0. The result implied that the probe could work over a relatively wide pH range, especially in normal physiological condition.
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Qingchun River
3×10
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recovery (%)
To extend the application of Cou-1-N2H4 into biological systems, the CCK8 assay was used to estimate the cytotoxicity of Cou-1-N2H4 (0, 10, 20 and 30 µM) in HeLa cells.[39] As it can be seen in Fig. S7, the cell viability still remained more than 85 % even cells co-cultured with Cou-1-N2H4 (30 µM) for 12 h. This result indicated that Cou-1-N2H4 has low cytotoxicity.
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The highly specific N2H4-triggered deprotection of acetyl group could explain the excellent selectivity of the Cou-1-N2H4 for N2H4. In order to confirm this mechanism, TLC and mass analysis were conducted. TLC analysis (Fig. S3) showed that when reacted with N2H4 for 3 min, Cou-1-N2H4 disappeared. Meanwhile, a new fluorescent product formed, which had the same retention factor (Rf) value as Cou-1. This result indicated that the reaction product was Cou-1. When Cou-1-N2H4 reacted with N2H4 for 3 min, the reaction mixture of Cou-1-N2H4 and N2H4 was isolated by a silica gel column (product A). Then product A was subjected to mass analysis. The peak was found at m/z 296.0375 correponding to Cou-1 (Fig. S4). In addition, the absorption and emission spectra of reaction mixture of Cou1-N2H4 with N2H4 were almost the same as Cou-1 (Fig. S5, Fig. S6 ). These results indicated that the mechanism of Cou-1-N2H4 for N2H4 detection might be as follows: Cou-1-N2H4 probe reacted with hydrazine due to the specific deprotection of acetyl group of the probe, to deliver Cou-1, and eventually led to the appearance of fluorogenic signaling (Scheme 2).
Scheme 2 The proposed sensing mechanism of Cou-1-N2H4 for N2H4
2.6. Cellular imaging
Fig.5 Confocal fluorescence images Hela cells. a1, a2 and a3: HeLa cells were cultured only with Cou-1-N2H4 (10 µM) for 30 min. b1, b2 and b3: HeLa cells were incubated with N2H4 (10 equiv.) for 30 min, and then treated with Cou-1-N2H4 (10 µM) for another 30 min. (a1 and b1) Bright-field images, (a2 and b2) Green channel, (a3 and b3) Merged images.
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triethylamine was added and stirring for 2 h. Then, the reaction Then, Cou-1-N2H4 (10 µM) was used ACCEPTED to carry out the MANUSCRIPT mixture was dried under vacuum. In the end, the combined bioimaging experiments. As shown in Fig. 5, we found that residue was subjected to silica gel chromatography to generate almost no intracellular fluorescence was detected after HeLa cells Cou-1-N2H4 as green floc with yield of 78%. 1H NMR (400 MHz, were co-cultured only with Cou-1-N2H4 (a2). By contrast, when cells incubated with N2H4 (100 µM) for first 30 min, and then DMSO-d6) δ (ppm) δ 9.28 (s 1H) 8.22 (d J = 8.0 Hz 1 H) treated with Cou-1-N2H4 for another 30 min, a green fluorescence 8.16 (d J = 8.6 Hz 1 H) 8.10 (d J = 8.3 Hz 1 H) 7.60 enhancement could be observed (b2). All of these results (m J = 7.9 Hz 1 H) 7.50 (m J = 6.9 Hz 1 H) 7.46 (d J demonstrated that Cou-1-N2H4 could determine intracellular = 1.8 Hz 1 H) 7.29 (dd J = 8.5 Hz 1 H) 2.34 (s, 3 H) hydrazine in living cells. HRMS (ESI): Calc. [M+Na]+ 360.0306, found 360.0307 (Fig. S9, Fig. S11). 3. Conclusion 4.4. Determination of hydrazine in real water samples In summary, a coumarin-based fluorescent probe for The real water samples were from the tap water, Qingchun ultrasensitive hydrazine sensing has been developed. The probe River (ECUST) and Huangpu River in Shanghai. We used a shows a dramatic green fluorescence enhancement toward N2H4. microfiltration membrane to filter the real water samples before In addition, the fluorescence intensity at 496 nm exhibits a being used in the test. PBS solution was used to adjust the pH of satisfactory linearity with the concentration of N2H4 in the range water samples. Four different concentrations of N2H4 (0, 0.3, 3 of 0-60 µM. Moreover, the probe is also able to selectively detect and 30 µM) were spiked in each sample. Unless clearly stated, all N2H4 in minutes against other interfering analytes and the of the fluorescence tests were conducted after mixing the samples detection limit is as low as 11.9 nM (0.38 ppb). More importantly, for 3 min. Eventually, Cou-1-N2H4 was used for determining the we have successfully proved the practical utility of Cou-1-N2H4 concentration of N2H4 through the calibration curve. through quantitative N2H4 detection in water samples and bioimaging of hydrazine in living HeLa cells. 4.5. Cell imaging 4. Materials and methods
4.2. Absorption and fluorescence spectra analysis
Acknowlegements
4.1. Material and instruments
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All chemical reagents and solvents were analytical grade and commercially available. Bruker AM 400 spectrometer was used to obtain 13C NMR and 1H NMR spectra. We used Waters LCT Premier XE spectrometer to collect HRMS data. Fluorescence emission spectra and UV-vis absorption spectra were performed on F97pro fluorospectrophotometer and Varian Cary 500 spectrophotometer respectively.
First, Cou-1-N2H4 (10 µM) co-cultured with Hela cells for 30 min at 37 oC, and then bioimaging after the Hela cells being washed three times with PBS (pH 7.4, 20 mM). Next, the cells were incubated with N2H4 (100 µM) for 30 min and were treated by Cou-1-N2H4 (10 µM) for another 30 min at 37 oC. The Hela cells were washed by PBS (pH 7.4, 20 mM) for three times before conducting bioimaging experiments. All the measurements of fluorescence spectra were performed on the confocal laser scanning microscopy (Nikon A1R). The emissions were recorded within the range of 475 − 575 nm.
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Fluorescence and absorption spectra analysis were conducted in PBS-DMSO (pH 7.4, 20 mM, v/v = 1/1). Test solutions were adjusted to a suitable concentration by PBS-DMSO (pH 7.4, 20 mM, v/v = 1/1). The final samples mixed with N2H4 were stirred for 3 min, and then fluorescence and UV test were carried out, 465 nm was selected as the excitation wavelength in the fluorescence test. 4.3. Synthesis
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4.3.1 Synthesis of Cou-1. Cou-1 was synthesized according to the published procedure.[40, 41] Briefly, 2, 4-Dihydroxybenzaldehyde (1.05 g, 7.5 mmol) and 2-(1, 3-benzothiazol-2-yl)-acetonitrile (1.32 g, 7.85 mmol) were dissolved in 15 mL ethanol, and 0.5 ml piperidine were then added. The mixture was stirred at room temperature overnight. After filtration, the yellow solid was treated with 10% hydrochloric acid. The suspended solution was stirred at 100 °C overnight. Then the resulting yellow residue was collected by filtration, and then purified by recrystallization using N, Ndimethylformamide to afford the target compound. 1H NMR (400 MHz, DMSO-d6) δ (ppm) δ 11.09 (s 1H) 9.17 (s 1H) 8.17 (d J = 8.0 Hz 1 H) 8.06 (d J = 8.0 Hz 1 H) 7.94 (d J = 8.6 Hz 1 H) 7.56 (m J = 7.1 Hz 1 H) 7.46 (m J = 7.1 Hz 1 H) 6.92 (dd J = 8.6 Hz 1 H) 6.86 (d J = 2.1 Hz 1 H) HRMS (ESI): Calc. [M+H]+ 296.0381, found 296.0375 (Fig. S8, Fig. S10). 4.3.2. Synthesis of Cou-1-N2H4. Acetyl bromide (492 mg, 4 mmol) and compound Cou-1 (176 mg, 1 mmol) were dissolved in 30 mL of CH2Cl2. Subsequently,
The authors are grateful to financial support from the Natural Science Foundation of Shanghai (16ZR1408000) and the Fundamental Research Funds for the Central Universities (WJ1814055). Supplementary data Additional data related to this article can be found in ESI References and notes 1.
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ACCEPTED MANUSCRIPT
Highlights
Colorimetric and fluorescent two-output probe with dramatic responses
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to N2H4. An extremely low detection limit for N2H4 determination.
Fluorescence bioimaging application in living HeLa cells.
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Quantitative N2H4 detection in water samples.
S0040-4020(19)30297-2
DOI:
https://doi.org/10.1016/j.tet.2019.03.022
Reference:
TET 30208
To appear in:
Tetrahedron
Received Date: 29 January 2019 Revised Date:
8 March 2019
Accepted Date: 11 March 2019
Please cite this article as: Guo S-H, Guo Z-Q, Wang C-Y, Shen Y, Zhu W-H, An ultrasensitive fluorescent probe for hydrazine detection and its application in water samples and living cells, Tetrahedron (2019), doi: https://doi.org/10.1016/j.tet.2019.03.022. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Graphical Abstract
An ultrasensitive fluorescent probe for hydrazine detection and its application in water samples and living cells
A coumarin-based fluorescent probe for ultrasensitive hydrazine sensing has been developed. The practical utilities of probe have been successfully proved through quantitative N2H4 detection in environmental water samples and bioimaging of N2H4 in living cells.
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Shao-Hua Guo, Zhi-Qian Guo, Cheng-Yun Wang*, Yongjia Shen and Wei-Hong Zhu*
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Shanghai Key Laboratory of Functional Materials Chemistry, Key Laboratory for Advanced Materials and Institute of Fine Chemicals, School of Chemistry and Molecular Engineering, East China University of Science & Technology, Shanghai 200237, P. R. China.
ACCEPTED MANUSCRIPT Graphical Abstract A coumarin-based fluorescent probe for ultrasensitive hydrazine sensing has been developed. The practical utilities of probe have been successfully proved through quantitative N2H4 detection in environmental water samples and bioimaging of N2H4 in living cells.
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An ultrasensitive fluorescent probe for hydrazine detection and its application in water samples and living cells
Shao-Hua Guo, Zhi-Qian Guo, Cheng-Yun Wang*, Yongjia Shen and Wei-Hong Zhu*
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Shanghai Key Laboratory of Functional Materials Chemistry, Key Laboratory for Advanced Materials and Institute of Fine Chemicals, School of Chemistry and Molecular Engineering, East China University of Science & Technology, Shanghai 200237, P. R. China.
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Tetrahedron journal homepage: www.elsevier.com
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An ultrasensitive fluorescent probe for hydrazine detection and its application in water samples and living cells Shao-Hua Guo, Zhi-Qian Guo, Cheng-Yun Wang*, Yongjia Shen and Wei-Hong Zhu ∗
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Shanghai Key Laboratory of Functional Materials Chemistry, Key Laboratory for Advanced Materials and Institute of Fine Chemicals, School of Chemistry and Molecular Engineering, East China University of Science & Technology, Shanghai 200237, P. R. China.
ABSTRACT
Article history: Received Received in revised form Accepted Available online
Hydrazine, as a strong reducing agent, has been extensively used in many industrial manufactures. However, it is a potential human carcinogen and an environmental contaminant due to its high toxicity. Therefore, developing an ultrasensitive method for determining hydrazine in real water and biosystems is of great significance. Herein, based on coumarin dye, a turn-on fluorescent probe Cou-1-N2H4, which contains an acetyl group as the trigger unit and the fluorescence quencher, is developed. The probe can achieve a rapid (3min) and colorimetric sensing detection for hydrazine with an extremely low limit detection (11.9 nM or 0.38 ppb). More importantly, the practical utilities of probe have been successfully proved through quantitative N2H4 detection in environmental water samples and bioimaging of N2H4 in living cells. 2019 Elsevier Ltd. All rights reserved.
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ARTICLE INFO
Keywords: Ultrasensitive Hydrazine Real water Bioimaging
1. Introduction
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Hydrazine (N2H4) has been wildly applied to many chemical industries.[1] As one of the most essential strong reducing agent or reactive bases, hydrazine plays a key role in the preparation of emulsifiers, pharmaceuticals, paints and pesticides.[2,3] In addition, hydrazine can be also used as fuel for missile and rocket propulsion systems due to its high enthalpy.[4] However, hydrazine is poisonous, excess exposure to it may lead to serious organ damage, including kidney, liver, lung, and especially the central nervous system.[5, 6] Thus, the governmental regulations have been issued to control the residual hydrazine content in the final products for the prevention of health problems, for instance, its threshold in drinking water is 10 ppb.[7] In view of this, it is of great significance to develop a highly effective method for hydrazine sensing in the fields of chemistry, biology and environment. Up to now, a variety of techniques, such as chemiluminescence,[8] surface enhanced Raman spectroscopy,[9] chromatography,[10] electrochemistry,[11-14] have been developed to detect hydrazine. However, they often need time-consuming procedures, complicated instruments and are inappropriate to intracellular detection. The fluorescence spectra analysis has attracted a lot of attention because of its good compatibility to biological samples, convenience, simplicity and excellent selectivity. Until now, many kinds of fluorescent chemosensors for hydrazine detection have been reported based on three main mechanisms, which includes specific deprotection of acetyl groups or levulinoyl ester,[15-22] specific reaction with
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arylidenemalononitrile,[23-26] and condensation with arylaldehydes.[27,28] Xu’s group[29] initially synthesized a trioutput probe for specific detection of hydrazine, the probe could achieve a rapid detection (1 min) of hydrazine with a low detection (0.1 µM). Liu and co-workers[30] developed a ratiometric fluorescent probe for hydrazine based on a chemoselective hydrazine-induced chalcone cyclization. The probe exhibits a low limit of detection 0.3 µM and the detection is complete within 60 min. By selecting coumarin derivative as fluorophore and acetyl group as a recognizing group, Yin’s group[31] design a fluorescent probe based on the cyclization mechanism for hydrazine. This probe shows a limit of detection 31 µM and a rapid detection progress (7 min). Despite the great progress has been made, there are still some drawbacks. For instance, some of fluorescent chemosensors had relatively long synthetic route, high limit detection or long detection time, which limited their application. In view of this, the development of simple and ultrasensitive fluorescent probes for hydrazine sensing with a fast detection process is still urgent. Herein, a new coumarin-based colorimetric and fluorescent two-output probe has been prepared for ultrasensitive hydrazine sensing. In the design, Cou-1 (3-benzothiazolyl-7hydroxycoumarin, Scheme 1) is employed as a fluorophore due to its excellent optical properties such as high fluorescence quantum yields (ΦF=0.56, see Table S1) and large absorption extinction coefficients, and acetyl group as an excellent recognition unit and the fluorescence quencher. Cou-1-N2H4 probe (Scheme 1) not only shows excellent colorimetric (colorless to green yellow) and rapid response toward hydrazine
∗ Corresponding author. Tel./fax: +86-021-64252967, e-mail: [email protected] (C. -Y. Wang), [email protected] (W. -H. Zhu)
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To further evaluate the sensing properties of Cou-1-N2H4 (3 min), but also has extremely low detection limit (11.9 nM). MANUSCRIPT ACCEPTED probe, the reaction time of Cou-1-N2H4 probe with N2H4 was More importantly, the probe can be used to detect hydrazine in investigated by carrying out time-dependent fluorescence environmental water samples quantitatively and bioimage experiments. As it can be seen in Fig. S1, after addition of 10 hydrazine in living Hela cells. equiv. of N2H4, fluorescence intensity (at 496 nm) of probe (10 µM) was gradually increased and appeared a fluorescent maximum after 3 min. Therefore, the reaction time needed to produce stable fluorescence intensity was 3 min.
2.1. Absorption and fluorescent detection of hydrazine
Fig.1 The linearity of fluorescence intensity (at 496 nm) versus N2H4 concentrations.
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Initially, we investigated the response of Cou-1-N2H4 toward N2H4 in PBS-DMSO (pH 7.4, 20 mM, v/v = 1/1). As it can be seen in Fig. 1A, the probe solution showed an absorption band at 376 nm. But after gradual addition of hydrazine, we could find the absorption peak at 376 nm evidently decreased along with the appearance of a new absorption peak around 462 nm (isosbestic point at 410 nm), and there was a clear color change from colorless to green yellow under natural light during the detection. This result indicated that the probe was able to serve as a “nakedeye” colorimetric indicator for N2H4.
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2. Results and discussion
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Scheme 1 Synthesis of Cou-1-N2H4
2.3. Selectivity and effect of pH
Besides highly sensitive, a satisfactory N2H4 probe should also have high selectivity. In order to investigate the selectivity of the probe for N2H4, a wide variety of analytes were added to the probe solution. As it can be seen in Fig. 3, the fluorescence spectra displayed almost no changes after addition of various other analytes, including Cu2+, Fe3+, Al3+, Na+, Fe2+, Ba2+, Zn2+, K+, HS-, Asn, Asp, Ala, Arg, Gln, GSH, Hcy, Cys, Glu, Gly, Phe, Pro, Trp, His, Leu, Thr and Val. However, a significant fluorescence enhancement was observed upon addition of N2H4, indicating the probe had good selectivity toward N2H4 over these analytes. In addition, competing experiment showed that other analytes could hardly interfere with the detection process (Fig. 4).
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As shown in Fig. 1B, the probe shows an extremely low fluorescence intensity at 496 nm. However, within 3 minutes, a green remarkable fluorescence enhancement was clearly observed at 496 nm after addition of N2H4. These results implied that the probe could play the part of a promising turn-on fluorescent indicator.
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Fig.1 (A) Absorption spectra properties of Cou-1-N2H4 upon treatment with 15 equiv. of N2H4, (B) Fluorescent spectra changes of different concentration of N2H4 (0–15equiv.) in a mixed PBS-DMSO solution (pH 7.4, 20 mM, v/v = 1/1). Insert: Emission color and color change of Cou1-N2H4 before and after addition of N2H4. λex = 465 nm. Slit width:10 nm/10 nm.
2.2. The sensitivity
To investigate the sensitivity of the probe, the probe solution was handled with different concentrations of N2H4. As shown in Fig. 2, the fluorescence intensity of the probe solution increased upon gradual addition of N2H4. The relative fluorescence intensity at 496 nm showed a satisfactory linearity (R2 = 0.9978) with the N2H4 concentration ranging from 0 to 60 µM. Based on the definition by IUPAC (CDL = 3σ / S),[32-35] the detection limit was calculated to be about 11.9 nM (0.38 ppb) in PBS-DMSO (20 mM, pH 7.4, v/v = 1/1), which was much lower than 10 ppb suggested by EPA. These results indicated that the probe was ultrasensitive toward N2H4.
Fig.3 Fluorescent spectra changes of Cou-1-N2H4 (10 µM) for various analytes (10 equiv.) in a mixed solution PBS-DMSO (20 mM, pH 7.4, v/v = 1/1). Analytes are: Cu2+, Fe3+, Al3+, Na+, Fe2+, Ba2+, Zn2+, K+, HS-, Asn, Asp, Ala, Arg, Gln, GSH, Hcy, Cys, Glu, Gly, Phe, Pro, Trp, His, Leu, Thr, Val. λex = 465 nm. Slit width:10 nm/10 nm.
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N2H4 spiked (M)
N2H4 recovered (M)
0
Not detected
Tap water
Fig.4 The selectivity of Cou-1-N2H4 (10 µM). The black bars represent the emission intensity of Cou-1-N2H4 in the presence of other analytes (100 µM). The red bars represent the fluorescence intensity (at 496 nm) that occurs upon addition of (100 µM) N2H4 to the above solution. 1: Cu2+, 2: Fe3+, 3: Al3+, 4: Na+, 5: Fe2+, 6: Ba2+, 7: Zn2+, 8: K+, 9: NaHS, 10: Asn, 11: Asp, 12: Ala, 13: Arg, 14: Gln, 15: GSH, 16: Hcy, 17: Cys, 18: Glu, 19: Gly, 20: Phe, 21: Pro, 22: Trp, 23: His, 24: Leu, 25: Thr, 26: Val
Huangpu River
3×10
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(3.10±0.09)×10
3×10-5
(2.94±0.13)×10
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0
Not detected
3×10
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3×10
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3×10
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As a suspected carcinogen, hydrazine has been extensively applied to various industrial manufactures. Even worse, owing to the perfect water solubility of N2H4, biological systems can absorb N2H4 easily through inhalation or skin contact. Thus, it is of great necessity to determine hydrazine in water samples. Standard addition method[36-38] was used to deal with the real water samples from the tap water, Qingchun River (ECUST) and Huangpu River in Shanghai, then the samples were used in the test. As shown in Table 1, the recovery rates were in the range of 97%-105%. The result indicated that Cou-1-N2H4 could be qualified to determine hydrazine in environmental water samples quantitatively. 2.5. Proposed mechanism
3×10-5
103.3 98
(2.95±0.12)×10-7
98.3
(2.97±0.16)×10
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99
(3.08±0.14)×10
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102.6 —
(3.15±0.13)×10
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105
(3.06±0.08)×10
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102
(3.11±0.12)×10-5
103.7
Note: the results shown in the table are reported as the mean ± standard deviation of triplicate experiments.
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2.4. Determination of hydrazine in water samples
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97
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Not detected
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In order to investigate the applicability, the effect of pH on the fluorescence intensity of Cou-1-N2H4 in the presence or absence of N2H4 was studied in wide pH range 5.0-12.0. As shown in Fig. S2, the appropriate working pH was from 7.0 to 10.0. The result implied that the probe could work over a relatively wide pH range, especially in normal physiological condition.
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3×10
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recovery (%)
To extend the application of Cou-1-N2H4 into biological systems, the CCK8 assay was used to estimate the cytotoxicity of Cou-1-N2H4 (0, 10, 20 and 30 µM) in HeLa cells.[39] As it can be seen in Fig. S7, the cell viability still remained more than 85 % even cells co-cultured with Cou-1-N2H4 (30 µM) for 12 h. This result indicated that Cou-1-N2H4 has low cytotoxicity.
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The highly specific N2H4-triggered deprotection of acetyl group could explain the excellent selectivity of the Cou-1-N2H4 for N2H4. In order to confirm this mechanism, TLC and mass analysis were conducted. TLC analysis (Fig. S3) showed that when reacted with N2H4 for 3 min, Cou-1-N2H4 disappeared. Meanwhile, a new fluorescent product formed, which had the same retention factor (Rf) value as Cou-1. This result indicated that the reaction product was Cou-1. When Cou-1-N2H4 reacted with N2H4 for 3 min, the reaction mixture of Cou-1-N2H4 and N2H4 was isolated by a silica gel column (product A). Then product A was subjected to mass analysis. The peak was found at m/z 296.0375 correponding to Cou-1 (Fig. S4). In addition, the absorption and emission spectra of reaction mixture of Cou1-N2H4 with N2H4 were almost the same as Cou-1 (Fig. S5, Fig. S6 ). These results indicated that the mechanism of Cou-1-N2H4 for N2H4 detection might be as follows: Cou-1-N2H4 probe reacted with hydrazine due to the specific deprotection of acetyl group of the probe, to deliver Cou-1, and eventually led to the appearance of fluorogenic signaling (Scheme 2).
Scheme 2 The proposed sensing mechanism of Cou-1-N2H4 for N2H4
2.6. Cellular imaging
Fig.5 Confocal fluorescence images Hela cells. a1, a2 and a3: HeLa cells were cultured only with Cou-1-N2H4 (10 µM) for 30 min. b1, b2 and b3: HeLa cells were incubated with N2H4 (10 equiv.) for 30 min, and then treated with Cou-1-N2H4 (10 µM) for another 30 min. (a1 and b1) Bright-field images, (a2 and b2) Green channel, (a3 and b3) Merged images.
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triethylamine was added and stirring for 2 h. Then, the reaction Then, Cou-1-N2H4 (10 µM) was used ACCEPTED to carry out the MANUSCRIPT mixture was dried under vacuum. In the end, the combined bioimaging experiments. As shown in Fig. 5, we found that residue was subjected to silica gel chromatography to generate almost no intracellular fluorescence was detected after HeLa cells Cou-1-N2H4 as green floc with yield of 78%. 1H NMR (400 MHz, were co-cultured only with Cou-1-N2H4 (a2). By contrast, when cells incubated with N2H4 (100 µM) for first 30 min, and then DMSO-d6) δ (ppm) δ 9.28 (s 1H) 8.22 (d J = 8.0 Hz 1 H) treated with Cou-1-N2H4 for another 30 min, a green fluorescence 8.16 (d J = 8.6 Hz 1 H) 8.10 (d J = 8.3 Hz 1 H) 7.60 enhancement could be observed (b2). All of these results (m J = 7.9 Hz 1 H) 7.50 (m J = 6.9 Hz 1 H) 7.46 (d J demonstrated that Cou-1-N2H4 could determine intracellular = 1.8 Hz 1 H) 7.29 (dd J = 8.5 Hz 1 H) 2.34 (s, 3 H) hydrazine in living cells. HRMS (ESI): Calc. [M+Na]+ 360.0306, found 360.0307 (Fig. S9, Fig. S11). 3. Conclusion 4.4. Determination of hydrazine in real water samples In summary, a coumarin-based fluorescent probe for The real water samples were from the tap water, Qingchun ultrasensitive hydrazine sensing has been developed. The probe River (ECUST) and Huangpu River in Shanghai. We used a shows a dramatic green fluorescence enhancement toward N2H4. microfiltration membrane to filter the real water samples before In addition, the fluorescence intensity at 496 nm exhibits a being used in the test. PBS solution was used to adjust the pH of satisfactory linearity with the concentration of N2H4 in the range water samples. Four different concentrations of N2H4 (0, 0.3, 3 of 0-60 µM. Moreover, the probe is also able to selectively detect and 30 µM) were spiked in each sample. Unless clearly stated, all N2H4 in minutes against other interfering analytes and the of the fluorescence tests were conducted after mixing the samples detection limit is as low as 11.9 nM (0.38 ppb). More importantly, for 3 min. Eventually, Cou-1-N2H4 was used for determining the we have successfully proved the practical utility of Cou-1-N2H4 concentration of N2H4 through the calibration curve. through quantitative N2H4 detection in water samples and bioimaging of hydrazine in living HeLa cells. 4.5. Cell imaging 4. Materials and methods
4.2. Absorption and fluorescence spectra analysis
Acknowlegements
4.1. Material and instruments
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All chemical reagents and solvents were analytical grade and commercially available. Bruker AM 400 spectrometer was used to obtain 13C NMR and 1H NMR spectra. We used Waters LCT Premier XE spectrometer to collect HRMS data. Fluorescence emission spectra and UV-vis absorption spectra were performed on F97pro fluorospectrophotometer and Varian Cary 500 spectrophotometer respectively.
First, Cou-1-N2H4 (10 µM) co-cultured with Hela cells for 30 min at 37 oC, and then bioimaging after the Hela cells being washed three times with PBS (pH 7.4, 20 mM). Next, the cells were incubated with N2H4 (100 µM) for 30 min and were treated by Cou-1-N2H4 (10 µM) for another 30 min at 37 oC. The Hela cells were washed by PBS (pH 7.4, 20 mM) for three times before conducting bioimaging experiments. All the measurements of fluorescence spectra were performed on the confocal laser scanning microscopy (Nikon A1R). The emissions were recorded within the range of 475 − 575 nm.
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Fluorescence and absorption spectra analysis were conducted in PBS-DMSO (pH 7.4, 20 mM, v/v = 1/1). Test solutions were adjusted to a suitable concentration by PBS-DMSO (pH 7.4, 20 mM, v/v = 1/1). The final samples mixed with N2H4 were stirred for 3 min, and then fluorescence and UV test were carried out, 465 nm was selected as the excitation wavelength in the fluorescence test. 4.3. Synthesis
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4.3.1 Synthesis of Cou-1. Cou-1 was synthesized according to the published procedure.[40, 41] Briefly, 2, 4-Dihydroxybenzaldehyde (1.05 g, 7.5 mmol) and 2-(1, 3-benzothiazol-2-yl)-acetonitrile (1.32 g, 7.85 mmol) were dissolved in 15 mL ethanol, and 0.5 ml piperidine were then added. The mixture was stirred at room temperature overnight. After filtration, the yellow solid was treated with 10% hydrochloric acid. The suspended solution was stirred at 100 °C overnight. Then the resulting yellow residue was collected by filtration, and then purified by recrystallization using N, Ndimethylformamide to afford the target compound. 1H NMR (400 MHz, DMSO-d6) δ (ppm) δ 11.09 (s 1H) 9.17 (s 1H) 8.17 (d J = 8.0 Hz 1 H) 8.06 (d J = 8.0 Hz 1 H) 7.94 (d J = 8.6 Hz 1 H) 7.56 (m J = 7.1 Hz 1 H) 7.46 (m J = 7.1 Hz 1 H) 6.92 (dd J = 8.6 Hz 1 H) 6.86 (d J = 2.1 Hz 1 H) HRMS (ESI): Calc. [M+H]+ 296.0381, found 296.0375 (Fig. S8, Fig. S10). 4.3.2. Synthesis of Cou-1-N2H4. Acetyl bromide (492 mg, 4 mmol) and compound Cou-1 (176 mg, 1 mmol) were dissolved in 30 mL of CH2Cl2. Subsequently,
The authors are grateful to financial support from the Natural Science Foundation of Shanghai (16ZR1408000) and the Fundamental Research Funds for the Central Universities (WJ1814055). Supplementary data Additional data related to this article can be found in ESI References and notes 1.
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Highlights
Colorimetric and fluorescent two-output probe with dramatic responses
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to N2H4. An extremely low detection limit for N2H4 determination.
Fluorescence bioimaging application in living HeLa cells.
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Quantitative N2H4 detection in water samples.