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A novel ratiometric and colorimetric fluorescent probe for hydrazine based on ring-opening reaction and its applications
Accepted Manuscript Title: A novel ratiometric and colorimetric fluorescent probe for hydrazine based on ring-opening reaction and its applications Authors: Xinrong Shi, Caixia Yin, Yongbin Zhang, Ying Wen, Fangjun Huo PII: DOI: Reference:
S0925-4005(19)30101-7 https://doi.org/10.1016/j.snb.2019.01.075 SNB 26001
To appear in:
Sensors and Actuators B
Received date: Revised date: Accepted date:
9 November 2018 14 January 2019 16 January 2019
Please cite this article as: Shi X, Yin C, Zhang Y, Wen Y, Huo F, A novel ratiometric and colorimetric fluorescent probe for hydrazine based on ring-opening reaction and its applications, Sensors and amp; Actuators: B. Chemical (2019), https://doi.org/10.1016/j.snb.2019.01.075 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.
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Graphical Abstract
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A novel ratiometric and colorimetric fluorescent probe for hydrazine
based
on
ring-opening
reaction
and
its
applications
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Highlights
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Xinrong Shi,a Caixia Yin,*,a Yongbin Zhang,b Ying Wen, a Fangjun Huo*,b a Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education, Key Laboratory of Materials for Energy Conversion and Storage of Shanxi Province, Institute of Molecular Science, Shanxi University, Taiyuan, 030006, China. b Research Institute of Applied Chemistry, Shanxi University, Taiyuan, 030006, China. *Corresponding author: F. J. Huo, E-mail: [email protected]； C. X. Yin, E-mail: [email protected].
The colorimetric and ratiometric probe can detect hydrazine with good sensitivity and selectivity.
The lactone ring opening of the probe is the sensing mechanism.
The probe can be used for hydrazine vapor monitoring and cell imaging experiments.
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Abstract: In recent years, with the increasing awareness of environmental protection, the detection of industrial emissions has become more and more strict. Hydrazine, as a common reactive group, the excessive emissions by the widespread use is inevitably
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harmful to the environment and human beings. To respond positively to environment protection policies at home and abroad, the development of a convenient and effective hydrazine probe has become an important subject for many research groups. In this work, a derivative containing lactone ring was synthesized based on naphthalimide.
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This probe displayed a fast colorimetric and ratiometric fluorescent response for hydrazine with high selectivity and sensitivity based on the ring-opening reaction. The detection limit of the probe for hydrazine is about 0.2032 μM (< 10 ppb). It is worth mentioning that the probe can successfully apply to detect gaseous hydrazine when
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the probe is loaded on the periopaper. Furthermore, the results of biological imaging experiment indicated that the probe can detect the hydrazine at the cellular level.
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Keywords: Lactone ring, Ratiometric and colorimetric, Ring-opening, Hydrazine, Cell
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imaging
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1. Introduction
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Hydrazine is a crucial reactive base which has been widely used in various
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industries，chemical，pharmaceutical, and agricultural [1]. It usually involves the
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preparation of catalysts, herbicides, preservatives, dyes and pharmaceuticals [2]. Due to its high enthalpy of combustion, flammable and detonable characteristics,
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hydrazine plays an important fuel in missile and rocket propulsion system [3–5]. And
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as such, the spread of hydrazine will inevitably bring harm to the environment and human beings. Medical research shows that inhalation or skin contact with hydrazine will injure organism and cytoarchitecture，even induce cancer, genetic mutations and
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neurological disorders [6]. In addition, the U.S. Environmental Protection Agency (EPA) identified hydrazine as a potential carcinogen with a threshold limit of 10 ppb [7]. Thus, it is imperative to explore a convenient and rapid sensor for detecting trace amounts of hydrazine in the environment and organisms.
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Some traditional methods have already been exploited, include chromatography [8, 9], chemiluminescence [10, 11], spectrophotometry [12, 13], electrochemistry [14–15], titrimetry [16] and surface-enhanced Raman spectroscopy [17]. But all of these techniques are time-consuming and insensitive [18]. By contrast, fluorescence
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detection has become one of the most popular detection methods because of its remarkable sensitivity and celerity [19]. The most ideal ones are turn-on probes and
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ratio probes, they are preferred due to their larger signal-to-noise ratio and less
interference from the nonspecific quenching by factors other than the analytes [20].
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Especially ratio fluorescent probes realize more effective quantitative detection
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through measuring the ratio of fluorescence intensities at two different wavelengths
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[21–23]. In consequence, a probe that can combine the visuality of visual colorimetry
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with the sensitivity and rapidity of ratio fluorescent to detect hydrazine is highly
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desirable.
Various dyes have been selected to design synthetic fluorescent probes, such as
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BODIPY [24, 25], coumarin [26–29], resorufin [30,31], benzothiazole [32–35],
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heptamethine cyanine and hemicyanine [36–38], fluorescein and rhodamine [39,40]. Beyond these fluorophore, 1, 8-naphthalimide is an ideal fluorophore for the construction of ratiometric fluorescent probes because of its desirable photophysical
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properties [41–44]. It is rarely reported that hydrazine induces the release of fluorophore based on the open-loop reaction. This work showed that hydrazine as a nucleophile can break the ester bonds to extend conjugate resulting in cyan fluorescence shifting orange
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fluorescence. Based on this specific identification, the probe successfully was applied to detect hydrazine vapor when the probe was loaded on the periopaper and hydrazine molecules at the cellular level. 2. Experimental
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2.1. Materials and instrumentations All chemicals were purchased from commercial suppliers and used without further
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purification. All solvents were of analytical grade also without further purification. Deionized water was used to prepare all aqueous solutions.
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A pH meter (Mettler Toledo, Switzerland) was used to determine the pH of buffer
spectrophotometer and
F-7000
FL fluorescence spectrophotometer
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UV-Vis
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solution. UV-Vis spectra and fluorescence spectra were measured on a Cary 50 Bio
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respectively. A PO-120 quartz cuvette (10 mm) was purchased from Shanghai 13
C NMR spectra were
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Huamei Experiment Instrument Plants, China. 1H NMR and
recorded on a Bruker AVANCE III-600 MHz and 150 MHz NMR spectrometer
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(Bruker, Billerica, MA) respectively, using tetramethylsilane as the internal standard
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for chemical shifts. ESI-MS was carried out on AB Triple TOF 5600 plus System (AB SCIEX, Framingham, USA). The ability of probe reacting to hydrazine in the living cells was evaluated by using a Zeiss LSM880 Airyscan confocal laser scanning
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microscope.
2.2. Preparation and characterization of compounds. 2.2.1. Synthesis of compounds The compound 1 and 2 was prepared according to our group's previous reports
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[45]. 2.2.2. Synthesis and Characterization of Probe Compound 2 (0.594g, 2.0 mmol) and ethyl acetoacetate (0.38 mL, 63.0 mmol) were dissoloved in dry EtOH (30 mL) with piperidine (40 µL). All of these liquid
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mixtures were heated at 85 degrees Celsius for four hours. Then cooled down to room temperature; the resulting precipitate was filtered; washed with cold ethanol and dried
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in vacuum, thus a desired light yellow solid was obtained (0.336g, 46 %). 1H NMR (600 MHz, CDCl3) δ = 8.89 (d, J = 8.4 Hz, 1H), 8.80 (d, J = 7.2 Hz, 1H), 8.77 (s, 2H),
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7.97 (t, J = 7.8 Hz, 1H), 4.21 (t, J = 7.5 Hz, 2H), 2.81 (s, 3H), 1.74 (dt, J = 14.5, 7.4
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Hz, 2H), 1.52 – 1.43 (m, 2H), 1.01 (t, J = 7.3 Hz, 3H). 13C NMR (151 MHz, CDCl3) δ
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= 147.6, 134.1, 131.3, 129.0, 128.5, 77.2, 77.0, 76.8, 40.6, 30.6, 30.2, 20.4, 13.9.
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ESI-MS m/z: [Probe + H]+ Calcd. for C21H18NO5 364.11850, Found 364.11823 (Fig.
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S6).
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2.3. Preparation of stock solutions for Probe and other analytes
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Stock solution of probe (2 mM) was dissolved in DMSO, which was further diluted with PBS solution (pH = 7.4, including 50 % DMSO) to the required concentration. Other analytes solutions (20 mM) were prepared in deionized water.
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2.4. General optical measurements For the optical measurement, probe (10 μL or 20 μL) was added in 2 mL solvent (PBS: DMSO = 1: 1, pH = 7.4) in a quartz cell for fluorescence and UV detection, respectively. The samples to be tested were placed for 15 minutes before testing. All
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optical spectra were recorded at room temperature. The fluorescence spectra were measured with the excitation at 325 nm and slit width are 5 nm/5 nm. 2.5. Detection of gaseous hydrazine The filter paper strips (5 cm × 5 mm) were tiled in a dish and soaked in a
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chloroform solution of 2 mM probe for 5 minutes, to ensure the probe can evenly distribute in every filter paper strip. After that, filter paper strips was taken out and
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dried for use. Different concentrations of hydrazine solution were prepared in glass
bottles, the above filter paper strips were remained into respective glass bottles at
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room temperature for 30 minutes.
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2.6. Cell viability assay
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HepG-2 cells were selected to verify the feasibility of the probe in biological
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system by Cell Counting Kit-8 (CCK-8). HepG-2 cells were cultured in DMEM
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medium (12% Fetal Bovine Serum and 1% antibiotics) at 37 °C and 5 % CO2 in the incubator. The cells were plated on 6-well plates and were incubated overnight..
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Different concentrations of probe (0 μM, 1 μM, 2.5 μM, 5 μM, 10 μM, 20 μM, 30 μM,
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50 μM) were then added to the wells. After incubation for 5 h or 10 h, CCK-8 (10 % in serum free culture medium) was added to each well, and the plate was incubated for another 1 h. The absorbance of each well was measured at 450 nm on a microplate
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reader.
2.7. Imaging of HepG-2 cells The specific content of HepG-2 cells was cultured in DMEM medium (12 % Fetal Bovine Serum and 1 % antibiotics) at 37 °C and 5 % CO2 in the incubator. The cells
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were plated on 6-well plates and incubated overnight. Before the CLSM imaging, the cells were washed with PBS 3 times. Probe dissolved in DMSO was added to the cell medium (2 mL) with 10 μM final concentrations. After incubated for 30 min, excess probe was lightly washed with PBS (10 mM, pH = 7.4) three times. Meanwhile,
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another portion of HepG-2 cells pre-treated with probe (150 μM) was treated with N2H4 and incubated for further 15 min at 37 °C. Before imaging, the stained HepG-2
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cells were washed one time with PBS. By setting the same parameters, cell imaging was identified by blue channel and yellow channel. Fluorescence images were
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performed on a Zeiss LSM880 Airyscan confocal laser scanning microscope.
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3.1 Solvent effect on the spectrum test
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3. Results and Discussion
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As is known to all, solvent effect has a great influence on reaction rate and
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spectral intensity. UV and fluorescence spectra recorded the reaction of the probe with hydrazine in different solvent systems. Fig. S1a and Fig. S1b showed that the
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optical properties of this reaction were better in DMSO than that in other organic
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media. More, as shown in Fig. S2a and Fig. S2b, the probe and hydrazine reaction was optimal in PBS buffer (pH 7.4) when containing 50 % DMSO as a co-solvent, and we finally chose this system as the detection medium.
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3.2. UV and Fluorescence spectra of probe titrated with hydrazine As shown in Fig. 1(a), the free probe (20 μM) exhibited peaks at 325 nm. With the addition of hydrazine, the UV spectra had a wide absorption band (from 400 nm to 570 nm) with a peak at 470 nm. At this point, the colorless solution gradually turned
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orange which can be observed by naked eyes. At the same time, the absorption value at 470nm and the hydrazine concentration show a satisfactory linear relationship (Fig.1-b). As shown in Fig. 2a, the probe (10 μM) displayed a higher peak at 471 nm (λex = 400 nm), with the increasing concentration of hydrazine, the fluorescent
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intensity at 471 nm was quenched and at 560 nm was dramatically enhanced. The ratio of I560/I471 was applied to quantitatively monitor the hydrazine. As shown in Fig.
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2b, the graph of I560/I471 with the hydrazine concentration exhibited a great liner
relationship. The limit of detection of the probe for hydrazine was estimated to be
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(0.2032 µM) according to the formula (S/N = 3). In order to display the peak
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enhancement at 560 nm more clearly, λex = 470 nm was selected as the excitation to
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obtain the turn-on fluorescence spectrogram (Fig. 3a), a satisfying linear relation was
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also obtained for the quantitative determination of hydrazine (Fig. 3b).
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3.3. The Selective response of probe to hydrazine Good selectivity is one of the criteria for evaluating the specificity of a probe. The
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interferences of 15 common ions (1. Na+; 2. Mg2+; 3. K+; 4. Ca2+; 5. F-; 6. Cl-; 7. Br-;
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8. I-; 9. H2PO4-; 10. HPO42-; 11. NO3-; 12. CO32-; 13. SO32-; 14. SO42-; 15. NH4+) in the determination of hydrazine were investigated in this work. From the results, only hydrazine caused a significant red shift of probe from 471 nm to 560 nm (Fig. 4a). In
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addition, Fig. 4b shown that in the presence of these common ions, hydrazine can still react with the probe and reach the similar fluorescence intensity. Conversely, S2- and HSO3- exhibited fluorescence quenching at 471 nm, but no change at 560 nm (Fig. S3a). Some amines with similar structure to hydrazine were also investigated, most of
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these substance hardly react with the probe (Fig. S3b). Of course, as shown in the Fig. S3c, common metal ions did not react with the probe. The experimental results shown that probe was more specific to hydrazine than most ions.
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3.4. pH-dependent and time-dependent for hydrazine
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Selecting an optimal pH is one of the important steps in the detection process,
which is related to the stability of the sensor and the feasibility of the application to biological imaging. A normal pH range from 4.0 to 9.0 was studied in this experiment.
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As shown in Fig. 5, the probe was stable in a range of 4 to 7, but it began to
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decompose in alkaline media. Upon treatment with hydrazine, it shown that the probe
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reacted weakly with hydrazine in acidic environment. The fluorescence enhanced from pH 5.0 to 8.0, which shown that probe for hydrazine worked well during this pH
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range. At pH = 9.0, the probe was extremely unstable under strong alkaline
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environment. The same result between pH and I560 nm was shown in Fig. S4 when the excitation wavelength was 470 nm. The pH of normal physiological environment is
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7.4, so pH 7.4 PBS was selected as the appropriate solvent. Time-dependent modulation in the fluorescence spectra of probe (10 μM) was
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monitored in the presence of hydrazine (300 μΜ) in PBS buffer solution (pH = 7.4) (ex = 470 nm, em = 560 nm, slit: 5 nm /5 nm). Fig. S5 displayed that the reaction time of probe was within 10 min. 3.5. Proposed detection mechanism 10
The probe had cyan fluorescence emission in PBS buffer, pH 7.4/DMSO (v/v, 1: 1). After the hydrazine was added, nucleophilic addition took place, the lactone group of the probe was opened to form a large conjugated structure, which eventually led to a red shift and showed yellow fluorescence emission. To further understand the possible
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detection mechanism of probe for N2H4, mass spectrometry analysis and NMR spectroscopic analysis were studied. Fig. S8A and C clearly displayed the peaks
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position of partial hydrogen (Ha, Hb, Hc, Hd, He) moving toward the high field after hydrazine addition, at the same time, the most obvious change was that the peak of
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Hd (~8.77 ppm) broken into two separate peaks (Hd1~8.26 ppm, Hd2~8.04 ppm).
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Besides, another characteristic peaks (f1~6.00 ppm and f2~5.24 ppm) of the amino
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protons were observed (Fig. S8-C). The phenomenon can be inferred that the addition
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of hydrazine triggered a ring-opening reaction. Beyond that, the adduct structure was +
calculated for
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confirmed by mass spectrometry analysis, [Probe + hydrazine + H]
410.18283; found 410.18331 (Fig. S7). This result further confirmed that the possible
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mechanism in Scheme 2.
3.6. Detection of gaseous hydrazine To explore the practical applications of the probe, it was also utilized for the
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detection of hydrazine gas. For this test, filter paper strip were soaked in a trichloromethane solution of probe (2 mM) and dried. Then the filter paper strip hung in small closed glass bottles for 1h, which containing hydrazine hydrate in different concentrations. Distinguishing FL intensity (Fig. 6-a) and UV color (Fig.6-b) changes
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can be observed. The results indicated that the hydrazine gas in the environment can be expediently monitored by naked eyes. 3.6. Cytotoxicity Assay
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The cytotoxicity experiment laid a foundation for the next step of the cell imaging experiment. We investigated the potential toxicity of probe to HepG-2 cells.
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The living cells were incubated with various concentrations of probe (0-50 μM) for 5 h and 10 h, respectively. As shown in Figure 7, when the concentration of probe was
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50 μM, the cell viability decreased by less than 10%, the results demonstrated that the
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probe has low toxicity,
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3.6. Imaging of Living Cell
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HepG-2 cells were used to evaluate the practical application in cell imaging of this probe based on the results of cytotoxicity tests. HepG-2 cells were incubated with
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10 μM of probe for 30 min at 37℃, it appeared bright aquamarine cyan fluorescence
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which was taken between 450 nm to 490 nm (Fig. 8b), but yellow fluorescence was barely observed in the range of 540 nm to 580 nm (Fig. 8c). In a further experiment it was found that HepG-2 cells displayed bright yellow fluorescence (take between 540
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nm to 580 nm) when the cells were incubated with the probe (10 μM) for 30 min and then incubated with hydrazine (150 μM) for 15 min at 37℃ (Fig. 8g), at the same time, fluorescence was reduced in the range of 450 nm to 490 nm (Fig. 8f). These imaging experiments indicated that probe can be used to bioimaging in living Cells.
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4. Conclusions The above results suggest that this new ratiometric probe based on 1, 8-naphthalimide derivative was synthesized and successfully applied to environmental
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and biological detection. This work showed that hydrazine as a nucleophile can break off the lactone group, led to a ring-opening reaction that released bright orange
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fluorescence, which displayed good selective and sensitive for hydrazine over other
anions and amines. Moreover, compared with some reported sensors, the probe
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showed a rapid detection process, a good linearity range and a low detection limit of
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hydrazine (Table S1). Based on this specific identification, it's worth mentioning that
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demonstrated cell imaging experiments.
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the probe can not only be used for hydrazine vapor monitoring, but also successfully
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Acknowledgments
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We thank the National Natural Science Foundation of China (No. 21775096, 21672131, 21705102), Talents Support Program of Shanxi Province (2014401),
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Shanxi Province Foundation for Returness (2017-026), the Shanxi Province Science Foundation for Youths (No. 201701D221061), Shanxi Province "1331 project" key
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innovation team construction plan cultivation team (2018-CT-1)and Scientific Instrument Center of Shanxi University.
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applications, Org. Lett. 15 (2013) 4022–4025.
[37] Z.L. Lu, W.L. Fan, X.M. Shi, Y.N. Lu, C.H. Fan, Two distinctly separated
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highly selective ratiometric fluorescent probe for hydrogen peroxide displaying a large emission shift, Sens. Actuators, B 186 (2013) 681–686.
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[42] Y. Sun, D. Zhao, S.W. Fan, L. Duan, A 4-hydroxynaphthalimide-derived ratiometric fluorescent probe for hydrazine and its in vivo applications, Sens.
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[43] P.S. Zhang, H. Wang, D. Zhang, X.Y. Zeng, R.J. Zeng, L.H. Xiao, H.W. Tao, Long,
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[45] H.F. Zhang，C.X. Yin，T. Liu，J.B. Chao，Y.B. Zhang，F.J. Huo, Selective
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“off-on”detection of magnesium (II) ions using a naphthalimide-derived fluorescent probe, Dyes Pigments 146 (2017) 344–351.
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Figure captions
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Scheme 1 The synthesis of the compound 3 and probe.
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Fig. 1 (a) UV-Vis spectra of the probe (20 µM) in the presence of various
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concentrations of hydrazine (0-16 equive) in PBS buffer (pH 7.4, including 50 % DMSO); (b) The linearity of the UV absorption at 470 nm depending on the hydrazine
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concentrations .
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Fig. 2 (a) Fluorescence spectral change of the probe (10 µM) upon addition of hydrazine (0-16 equive) in PBS solution (pH 7.4, including 50% DMSO) (λex = 325
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nm, slit: 5 nm /5 nm). (b) The linearity of the Fluorescence intensity ratios I560 nm /I471 nm
versus partial hydrazine concentrations (6-16 equiv.).
Fig. 3 (a) Fluorescence spectral change of the probe (10 µM) upon addition of hydrazine (0-16 equive) in PBS solution (pH 7.4, including 50 % DMSO) (λex = 470 nm, λem = 560 nm, slit: 5 nm /5 nm). (b) The linearity of the fluorescence intensity at
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560nm depending on the hydrazine concentrations (0-10 equiv.). Fig. 4 (a) Fluorescence intensity ratios I560 nm /I471 nm of probe (10 µM) treated with various analytes in PBS-DMSO solution (pH 7.4). (b) Fluorescence intensity ratios I560 nm /I471 nm of probe (10 µM) in the presence of different ions (30 equive), followed
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by addition of hydrazine 16 equiv. Each spectrum was recorded within 10 min (λex = 325 nm, slit: 5 nm /5 nm).
absence (black line) and presence (red line) of hydrazine.
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Fig. 5 pH dependent fluorescence intensity ratios I560 nm /I471 nm of probe (10 µM) in
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Scheme 2 Proposed detection mechanism of the probe to hydrazine.
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Fig. 6 Photographs for probe coated filter paper upon exposure to gaseous hydrazine
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vapored from different concentrations of hydrazine aqueous solution. Upper: under a
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hand-hold UV lamp with an excitation at 365 nm in dark; Lower: under ambient light.
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Fig. 7 Viability of HepG-2 cells treated with probe. Fig. 8 Confocal fluorescence images in HepG-2 cells. Fluorescence image of HepG-2
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cells incubated with probe (10 µM) for 30 min at 37℃ (a–d); Cells pretreated with
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probe and then incubated with hydrazine for 15 min at 37℃ (e–h). Bright field images (a and e). Overlay (d and h) Emission is collected by blue channel at (b and f)
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and yellow channel at (c and g).
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Fig. 1
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Scheme 1
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Fig. 3
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Fig. 4
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(a)
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Scheme 2
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Biographies
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Xinrong Shi She obtained her BSC chemistry from Xin Zhou Teachers University in 2016. Now she is studying her master degree in Institute of Molecular Science at Shanxi University. Her current research interest is in molecular chemistry.
Caixia Yin She obtained her Doctor Degree is in chemistry for Shanxi University
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in 2005. Now she is a Professor in Key Laboratory of Chemical Biology and
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Molecular Engineering of Ministry of Education, Institute of Molecular Science at Shanxi University major in inorganic chemistry. Her current research interests are
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molecular recognition, sensors chemistry.
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Yongbin Zhang He obtained his Doctor degree in Research Institute of Applied
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Chemistry for Shanxi University in 2015. Now he is an Assoiate Professor in
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Research Institute of Applied Chemistry at Shanxi University major in organic chemistry. His current research interests are molecular recognition, sensors chemistry.
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Ying Wen She received her Ph.D. from the Chemistry Department of Fudan Unive rsity in 2016. Now she is a Lecturer in the Institute of Molecular Science at Shanxi U
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niversity with the major in inorganic chemistry. Her current research interests include
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optical materials and bioimaging. Fangjun Huo He obtained his Doctor Degree in chemistry for Shanxi University in
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2007. Now he is an Assoiate Professor in Research Institute of Applied Chemistry at Shanxi University major in organic chemistry. His current research interests are sensors, supramolecular chemistry.
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S0925-4005(19)30101-7 https://doi.org/10.1016/j.snb.2019.01.075 SNB 26001
To appear in:
Sensors and Actuators B
Received date: Revised date: Accepted date:
9 November 2018 14 January 2019 16 January 2019
Please cite this article as: Shi X, Yin C, Zhang Y, Wen Y, Huo F, A novel ratiometric and colorimetric fluorescent probe for hydrazine based on ring-opening reaction and its applications, Sensors and amp; Actuators: B. Chemical (2019), https://doi.org/10.1016/j.snb.2019.01.075 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.
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Graphical Abstract
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A novel ratiometric and colorimetric fluorescent probe for hydrazine
based
on
ring-opening
reaction
and
its
applications
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Highlights
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Xinrong Shi,a Caixia Yin,*,a Yongbin Zhang,b Ying Wen, a Fangjun Huo*,b a Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education, Key Laboratory of Materials for Energy Conversion and Storage of Shanxi Province, Institute of Molecular Science, Shanxi University, Taiyuan, 030006, China. b Research Institute of Applied Chemistry, Shanxi University, Taiyuan, 030006, China. *Corresponding author: F. J. Huo, E-mail: [email protected]； C. X. Yin, E-mail: [email protected].
The colorimetric and ratiometric probe can detect hydrazine with good sensitivity and selectivity.
The lactone ring opening of the probe is the sensing mechanism.
The probe can be used for hydrazine vapor monitoring and cell imaging experiments.
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Abstract: In recent years, with the increasing awareness of environmental protection, the detection of industrial emissions has become more and more strict. Hydrazine, as a common reactive group, the excessive emissions by the widespread use is inevitably
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harmful to the environment and human beings. To respond positively to environment protection policies at home and abroad, the development of a convenient and effective hydrazine probe has become an important subject for many research groups. In this work, a derivative containing lactone ring was synthesized based on naphthalimide.
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This probe displayed a fast colorimetric and ratiometric fluorescent response for hydrazine with high selectivity and sensitivity based on the ring-opening reaction. The detection limit of the probe for hydrazine is about 0.2032 μM (< 10 ppb). It is worth mentioning that the probe can successfully apply to detect gaseous hydrazine when
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the probe is loaded on the periopaper. Furthermore, the results of biological imaging experiment indicated that the probe can detect the hydrazine at the cellular level.
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Keywords: Lactone ring, Ratiometric and colorimetric, Ring-opening, Hydrazine, Cell
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imaging
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1. Introduction
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Hydrazine is a crucial reactive base which has been widely used in various
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industries，chemical，pharmaceutical, and agricultural [1]. It usually involves the
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preparation of catalysts, herbicides, preservatives, dyes and pharmaceuticals [2]. Due to its high enthalpy of combustion, flammable and detonable characteristics,
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hydrazine plays an important fuel in missile and rocket propulsion system [3–5]. And
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as such, the spread of hydrazine will inevitably bring harm to the environment and human beings. Medical research shows that inhalation or skin contact with hydrazine will injure organism and cytoarchitecture，even induce cancer, genetic mutations and
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neurological disorders [6]. In addition, the U.S. Environmental Protection Agency (EPA) identified hydrazine as a potential carcinogen with a threshold limit of 10 ppb [7]. Thus, it is imperative to explore a convenient and rapid sensor for detecting trace amounts of hydrazine in the environment and organisms.
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Some traditional methods have already been exploited, include chromatography [8, 9], chemiluminescence [10, 11], spectrophotometry [12, 13], electrochemistry [14–15], titrimetry [16] and surface-enhanced Raman spectroscopy [17]. But all of these techniques are time-consuming and insensitive [18]. By contrast, fluorescence
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detection has become one of the most popular detection methods because of its remarkable sensitivity and celerity [19]. The most ideal ones are turn-on probes and
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ratio probes, they are preferred due to their larger signal-to-noise ratio and less
interference from the nonspecific quenching by factors other than the analytes [20].
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Especially ratio fluorescent probes realize more effective quantitative detection
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through measuring the ratio of fluorescence intensities at two different wavelengths
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[21–23]. In consequence, a probe that can combine the visuality of visual colorimetry
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with the sensitivity and rapidity of ratio fluorescent to detect hydrazine is highly
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desirable.
Various dyes have been selected to design synthetic fluorescent probes, such as
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BODIPY [24, 25], coumarin [26–29], resorufin [30,31], benzothiazole [32–35],
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heptamethine cyanine and hemicyanine [36–38], fluorescein and rhodamine [39,40]. Beyond these fluorophore, 1, 8-naphthalimide is an ideal fluorophore for the construction of ratiometric fluorescent probes because of its desirable photophysical
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properties [41–44]. It is rarely reported that hydrazine induces the release of fluorophore based on the open-loop reaction. This work showed that hydrazine as a nucleophile can break the ester bonds to extend conjugate resulting in cyan fluorescence shifting orange
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fluorescence. Based on this specific identification, the probe successfully was applied to detect hydrazine vapor when the probe was loaded on the periopaper and hydrazine molecules at the cellular level. 2. Experimental
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2.1. Materials and instrumentations All chemicals were purchased from commercial suppliers and used without further
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purification. All solvents were of analytical grade also without further purification. Deionized water was used to prepare all aqueous solutions.
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A pH meter (Mettler Toledo, Switzerland) was used to determine the pH of buffer
spectrophotometer and
F-7000
FL fluorescence spectrophotometer
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UV-Vis
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solution. UV-Vis spectra and fluorescence spectra were measured on a Cary 50 Bio
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respectively. A PO-120 quartz cuvette (10 mm) was purchased from Shanghai 13
C NMR spectra were
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Huamei Experiment Instrument Plants, China. 1H NMR and
recorded on a Bruker AVANCE III-600 MHz and 150 MHz NMR spectrometer
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(Bruker, Billerica, MA) respectively, using tetramethylsilane as the internal standard
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for chemical shifts. ESI-MS was carried out on AB Triple TOF 5600 plus System (AB SCIEX, Framingham, USA). The ability of probe reacting to hydrazine in the living cells was evaluated by using a Zeiss LSM880 Airyscan confocal laser scanning
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microscope.
2.2. Preparation and characterization of compounds. 2.2.1. Synthesis of compounds The compound 1 and 2 was prepared according to our group's previous reports
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[45]. 2.2.2. Synthesis and Characterization of Probe Compound 2 (0.594g, 2.0 mmol) and ethyl acetoacetate (0.38 mL, 63.0 mmol) were dissoloved in dry EtOH (30 mL) with piperidine (40 µL). All of these liquid
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mixtures were heated at 85 degrees Celsius for four hours. Then cooled down to room temperature; the resulting precipitate was filtered; washed with cold ethanol and dried
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in vacuum, thus a desired light yellow solid was obtained (0.336g, 46 %). 1H NMR (600 MHz, CDCl3) δ = 8.89 (d, J = 8.4 Hz, 1H), 8.80 (d, J = 7.2 Hz, 1H), 8.77 (s, 2H),
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7.97 (t, J = 7.8 Hz, 1H), 4.21 (t, J = 7.5 Hz, 2H), 2.81 (s, 3H), 1.74 (dt, J = 14.5, 7.4
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Hz, 2H), 1.52 – 1.43 (m, 2H), 1.01 (t, J = 7.3 Hz, 3H). 13C NMR (151 MHz, CDCl3) δ
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= 147.6, 134.1, 131.3, 129.0, 128.5, 77.2, 77.0, 76.8, 40.6, 30.6, 30.2, 20.4, 13.9.
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ESI-MS m/z: [Probe + H]+ Calcd. for C21H18NO5 364.11850, Found 364.11823 (Fig.
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S6).
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2.3. Preparation of stock solutions for Probe and other analytes
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Stock solution of probe (2 mM) was dissolved in DMSO, which was further diluted with PBS solution (pH = 7.4, including 50 % DMSO) to the required concentration. Other analytes solutions (20 mM) were prepared in deionized water.
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2.4. General optical measurements For the optical measurement, probe (10 μL or 20 μL) was added in 2 mL solvent (PBS: DMSO = 1: 1, pH = 7.4) in a quartz cell for fluorescence and UV detection, respectively. The samples to be tested were placed for 15 minutes before testing. All
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optical spectra were recorded at room temperature. The fluorescence spectra were measured with the excitation at 325 nm and slit width are 5 nm/5 nm. 2.5. Detection of gaseous hydrazine The filter paper strips (5 cm × 5 mm) were tiled in a dish and soaked in a
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chloroform solution of 2 mM probe for 5 minutes, to ensure the probe can evenly distribute in every filter paper strip. After that, filter paper strips was taken out and
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dried for use. Different concentrations of hydrazine solution were prepared in glass
bottles, the above filter paper strips were remained into respective glass bottles at
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room temperature for 30 minutes.
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2.6. Cell viability assay
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HepG-2 cells were selected to verify the feasibility of the probe in biological
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system by Cell Counting Kit-8 (CCK-8). HepG-2 cells were cultured in DMEM
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medium (12% Fetal Bovine Serum and 1% antibiotics) at 37 °C and 5 % CO2 in the incubator. The cells were plated on 6-well plates and were incubated overnight..
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Different concentrations of probe (0 μM, 1 μM, 2.5 μM, 5 μM, 10 μM, 20 μM, 30 μM,
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50 μM) were then added to the wells. After incubation for 5 h or 10 h, CCK-8 (10 % in serum free culture medium) was added to each well, and the plate was incubated for another 1 h. The absorbance of each well was measured at 450 nm on a microplate
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reader.
2.7. Imaging of HepG-2 cells The specific content of HepG-2 cells was cultured in DMEM medium (12 % Fetal Bovine Serum and 1 % antibiotics) at 37 °C and 5 % CO2 in the incubator. The cells
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were plated on 6-well plates and incubated overnight. Before the CLSM imaging, the cells were washed with PBS 3 times. Probe dissolved in DMSO was added to the cell medium (2 mL) with 10 μM final concentrations. After incubated for 30 min, excess probe was lightly washed with PBS (10 mM, pH = 7.4) three times. Meanwhile,
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another portion of HepG-2 cells pre-treated with probe (150 μM) was treated with N2H4 and incubated for further 15 min at 37 °C. Before imaging, the stained HepG-2
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cells were washed one time with PBS. By setting the same parameters, cell imaging was identified by blue channel and yellow channel. Fluorescence images were
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performed on a Zeiss LSM880 Airyscan confocal laser scanning microscope.
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3.1 Solvent effect on the spectrum test
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3. Results and Discussion
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As is known to all, solvent effect has a great influence on reaction rate and
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spectral intensity. UV and fluorescence spectra recorded the reaction of the probe with hydrazine in different solvent systems. Fig. S1a and Fig. S1b showed that the
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optical properties of this reaction were better in DMSO than that in other organic
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media. More, as shown in Fig. S2a and Fig. S2b, the probe and hydrazine reaction was optimal in PBS buffer (pH 7.4) when containing 50 % DMSO as a co-solvent, and we finally chose this system as the detection medium.
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3.2. UV and Fluorescence spectra of probe titrated with hydrazine As shown in Fig. 1(a), the free probe (20 μM) exhibited peaks at 325 nm. With the addition of hydrazine, the UV spectra had a wide absorption band (from 400 nm to 570 nm) with a peak at 470 nm. At this point, the colorless solution gradually turned
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orange which can be observed by naked eyes. At the same time, the absorption value at 470nm and the hydrazine concentration show a satisfactory linear relationship (Fig.1-b). As shown in Fig. 2a, the probe (10 μM) displayed a higher peak at 471 nm (λex = 400 nm), with the increasing concentration of hydrazine, the fluorescent
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intensity at 471 nm was quenched and at 560 nm was dramatically enhanced. The ratio of I560/I471 was applied to quantitatively monitor the hydrazine. As shown in Fig.
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2b, the graph of I560/I471 with the hydrazine concentration exhibited a great liner
relationship. The limit of detection of the probe for hydrazine was estimated to be
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(0.2032 µM) according to the formula (S/N = 3). In order to display the peak
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enhancement at 560 nm more clearly, λex = 470 nm was selected as the excitation to
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obtain the turn-on fluorescence spectrogram (Fig. 3a), a satisfying linear relation was
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also obtained for the quantitative determination of hydrazine (Fig. 3b).
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3.3. The Selective response of probe to hydrazine Good selectivity is one of the criteria for evaluating the specificity of a probe. The
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interferences of 15 common ions (1. Na+; 2. Mg2+; 3. K+; 4. Ca2+; 5. F-; 6. Cl-; 7. Br-;
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8. I-; 9. H2PO4-; 10. HPO42-; 11. NO3-; 12. CO32-; 13. SO32-; 14. SO42-; 15. NH4+) in the determination of hydrazine were investigated in this work. From the results, only hydrazine caused a significant red shift of probe from 471 nm to 560 nm (Fig. 4a). In
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addition, Fig. 4b shown that in the presence of these common ions, hydrazine can still react with the probe and reach the similar fluorescence intensity. Conversely, S2- and HSO3- exhibited fluorescence quenching at 471 nm, but no change at 560 nm (Fig. S3a). Some amines with similar structure to hydrazine were also investigated, most of
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these substance hardly react with the probe (Fig. S3b). Of course, as shown in the Fig. S3c, common metal ions did not react with the probe. The experimental results shown that probe was more specific to hydrazine than most ions.
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3.4. pH-dependent and time-dependent for hydrazine
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Selecting an optimal pH is one of the important steps in the detection process,
which is related to the stability of the sensor and the feasibility of the application to biological imaging. A normal pH range from 4.0 to 9.0 was studied in this experiment.
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As shown in Fig. 5, the probe was stable in a range of 4 to 7, but it began to
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decompose in alkaline media. Upon treatment with hydrazine, it shown that the probe
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reacted weakly with hydrazine in acidic environment. The fluorescence enhanced from pH 5.0 to 8.0, which shown that probe for hydrazine worked well during this pH
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range. At pH = 9.0, the probe was extremely unstable under strong alkaline
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environment. The same result between pH and I560 nm was shown in Fig. S4 when the excitation wavelength was 470 nm. The pH of normal physiological environment is
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7.4, so pH 7.4 PBS was selected as the appropriate solvent. Time-dependent modulation in the fluorescence spectra of probe (10 μM) was
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monitored in the presence of hydrazine (300 μΜ) in PBS buffer solution (pH = 7.4) (ex = 470 nm, em = 560 nm, slit: 5 nm /5 nm). Fig. S5 displayed that the reaction time of probe was within 10 min.
The probe had cyan fluorescence emission in PBS buffer, pH 7.4/DMSO (v/v, 1: 1). After the hydrazine was added, nucleophilic addition took place, the lactone group of the probe was opened to form a large conjugated structure, which eventually led to a red shift and showed yellow fluorescence emission. To further understand the possible
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detection mechanism of probe for N2H4, mass spectrometry analysis and NMR spectroscopic analysis were studied. Fig. S8A and C clearly displayed the peaks
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position of partial hydrogen (Ha, Hb, Hc, Hd, He) moving toward the high field after hydrazine addition, at the same time, the most obvious change was that the peak of
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Hd (~8.77 ppm) broken into two separate peaks (Hd1~8.26 ppm, Hd2~8.04 ppm).
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Besides, another characteristic peaks (f1~6.00 ppm and f2~5.24 ppm) of the amino
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protons were observed (Fig. S8-C). The phenomenon can be inferred that the addition
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of hydrazine triggered a ring-opening reaction. Beyond that, the adduct structure was +
calculated for
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confirmed by mass spectrometry analysis, [Probe + hydrazine + H]
410.18283; found 410.18331 (Fig. S7). This result further confirmed that the possible
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mechanism in Scheme 2.
3.6. Detection of gaseous hydrazine To explore the practical applications of the probe, it was also utilized for the
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detection of hydrazine gas. For this test, filter paper strip were soaked in a trichloromethane solution of probe (2 mM) and dried. Then the filter paper strip hung in small closed glass bottles for 1h, which containing hydrazine hydrate in different concentrations. Distinguishing FL intensity (Fig. 6-a) and UV color (Fig.6-b) changes
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can be observed. The results indicated that the hydrazine gas in the environment can be expediently monitored by naked eyes.
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The cytotoxicity experiment laid a foundation for the next step of the cell imaging experiment. We investigated the potential toxicity of probe to HepG-2 cells.
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The living cells were incubated with various concentrations of probe (0-50 μM) for 5 h and 10 h, respectively. As shown in Figure 7, when the concentration of probe was
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50 μM, the cell viability decreased by less than 10%, the results demonstrated that the
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probe has low toxicity,
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3.6. Imaging of Living Cell
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HepG-2 cells were used to evaluate the practical application in cell imaging of this probe based on the results of cytotoxicity tests. HepG-2 cells were incubated with
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10 μM of probe for 30 min at 37℃, it appeared bright aquamarine cyan fluorescence
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which was taken between 450 nm to 490 nm (Fig. 8b), but yellow fluorescence was barely observed in the range of 540 nm to 580 nm (Fig. 8c). In a further experiment it was found that HepG-2 cells displayed bright yellow fluorescence (take between 540
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nm to 580 nm) when the cells were incubated with the probe (10 μM) for 30 min and then incubated with hydrazine (150 μM) for 15 min at 37℃ (Fig. 8g), at the same time, fluorescence was reduced in the range of 450 nm to 490 nm (Fig. 8f). These imaging experiments indicated that probe can be used to bioimaging in living Cells.
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and biological detection. This work showed that hydrazine as a nucleophile can break off the lactone group, led to a ring-opening reaction that released bright orange
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fluorescence, which displayed good selective and sensitive for hydrazine over other
anions and amines. Moreover, compared with some reported sensors, the probe
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showed a rapid detection process, a good linearity range and a low detection limit of
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hydrazine (Table S1). Based on this specific identification, it's worth mentioning that
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demonstrated cell imaging experiments.
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the probe can not only be used for hydrazine vapor monitoring, but also successfully
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Acknowledgments
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We thank the National Natural Science Foundation of China (No. 21775096, 21672131, 21705102), Talents Support Program of Shanxi Province (2014401),
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Shanxi Province Foundation for Returness (2017-026), the Shanxi Province Science Foundation for Youths (No. 201701D221061), Shanxi Province "1331 project" key
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innovation team construction plan cultivation team (2018-CT-1)and Scientific Instrument Center of Shanxi University.
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Figure captions
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Scheme 1 The synthesis of the compound 3 and probe.
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Fig. 1 (a) UV-Vis spectra of the probe (20 µM) in the presence of various
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concentrations of hydrazine (0-16 equive) in PBS buffer (pH 7.4, including 50 % DMSO); (b) The linearity of the UV absorption at 470 nm depending on the hydrazine
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concentrations .
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Fig. 2 (a) Fluorescence spectral change of the probe (10 µM) upon addition of hydrazine (0-16 equive) in PBS solution (pH 7.4, including 50% DMSO) (λex = 325
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nm, slit: 5 nm /5 nm). (b) The linearity of the Fluorescence intensity ratios I560 nm /I471 nm
versus partial hydrazine concentrations (6-16 equiv.).
Fig. 3 (a) Fluorescence spectral change of the probe (10 µM) upon addition of hydrazine (0-16 equive) in PBS solution (pH 7.4, including 50 % DMSO) (λex = 470 nm, λem = 560 nm, slit: 5 nm /5 nm). (b) The linearity of the fluorescence intensity at
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560nm depending on the hydrazine concentrations (0-10 equiv.). Fig. 4 (a) Fluorescence intensity ratios I560 nm /I471 nm of probe (10 µM) treated with various analytes in PBS-DMSO solution (pH 7.4). (b) Fluorescence intensity ratios I560 nm /I471 nm of probe (10 µM) in the presence of different ions (30 equive), followed
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by addition of hydrazine 16 equiv. Each spectrum was recorded within 10 min (λex = 325 nm, slit: 5 nm /5 nm).
absence (black line) and presence (red line) of hydrazine.
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Fig. 5 pH dependent fluorescence intensity ratios I560 nm /I471 nm of probe (10 µM) in
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Scheme 2 Proposed detection mechanism of the probe to hydrazine.
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Fig. 6 Photographs for probe coated filter paper upon exposure to gaseous hydrazine
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vapored from different concentrations of hydrazine aqueous solution. Upper: under a
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hand-hold UV lamp with an excitation at 365 nm in dark; Lower: under ambient light.
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Fig. 7 Viability of HepG-2 cells treated with probe. Fig. 8 Confocal fluorescence images in HepG-2 cells. Fluorescence image of HepG-2
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cells incubated with probe (10 µM) for 30 min at 37℃ (a–d); Cells pretreated with
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probe and then incubated with hydrazine for 15 min at 37℃ (e–h). Bright field images (a and e). Overlay (d and h) Emission is collected by blue channel at (b and f)
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and yellow channel at (c and g).
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Fig. 1
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Scheme 1
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Fig. 2
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Fig. 3
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Fig. 4
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(a)
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Fig. 5
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Scheme 2
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Fig. 6
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Biographies
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Xinrong Shi She obtained her BSC chemistry from Xin Zhou Teachers University in 2016. Now she is studying her master degree in Institute of Molecular Science at Shanxi University. Her current research interest is in molecular chemistry.
Caixia Yin She obtained her Doctor Degree is in chemistry for Shanxi University
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in 2005. Now she is a Professor in Key Laboratory of Chemical Biology and
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Molecular Engineering of Ministry of Education, Institute of Molecular Science at Shanxi University major in inorganic chemistry. Her current research interests are
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molecular recognition, sensors chemistry.
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Yongbin Zhang He obtained his Doctor degree in Research Institute of Applied
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Chemistry for Shanxi University in 2015. Now he is an Assoiate Professor in
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Research Institute of Applied Chemistry at Shanxi University major in organic chemistry. His current research interests are molecular recognition, sensors chemistry.
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Ying Wen She received her Ph.D. from the Chemistry Department of Fudan Unive rsity in 2016. Now she is a Lecturer in the Institute of Molecular Science at Shanxi U
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niversity with the major in inorganic chemistry. Her current research interests include
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optical materials and bioimaging. Fangjun Huo He obtained his Doctor Degree in chemistry for Shanxi University in
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2007. Now he is an Assoiate Professor in Research Institute of Applied Chemistry at Shanxi University major in organic chemistry. His current research interests are sensors, supramolecular chemistry.
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