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A highly selective, colorimetric and ratiometric fluorescent probe for NH2NH2 and its bioimaging
Author’s Accepted Manuscript A highly selective, colorimetric and ratiometric fluorescent probe for NH2NH2 and its bioimaging Hai Xu, Biao Gu, Yaqian Li, Zheng Huang, Wei Su, Xiaoli Duan, Peng Yin, Haitao Li, Shouzhuo Yao www.elsevier.com/locate/talanta
PII: DOI: Reference:
S0039-9140(17)31242-0 https://doi.org/10.1016/j.talanta.2017.12.039 TAL18175
To appear in: Talanta Received date: 5 August 2017 Revised date: 7 December 2017 Accepted date: 12 December 2017 Cite this article as: Hai Xu, Biao Gu, Yaqian Li, Zheng Huang, Wei Su, Xiaoli Duan, Peng Yin, Haitao Li and Shouzhuo Yao, A highly selective, colorimetric and ratiometric fluorescent probe for NH2NH2 and its bioimaging, Talanta, https://doi.org/10.1016/j.talanta.2017.12.039 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 galley proof before it is published in its final citable 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.
A highly selective, colorimetric and ratiometric fluorescent probe for NH2NH2 and its bioimaging
Hai Xua, Biao Gua,b, Yaqian Lia, Zheng Huanga, Wei Sua, Xiaoli Duana, Peng Yina*, Haitao Lia* and Shouzhuo Yaoa
a
Key Laboratory of Chemical Biology and Traditional Chinese Medicine Research (Ministry of Education), College of Chemistry and Chemical Engineering, Hunan Normal University, Changsha 410081, PR China. b
Key Laboratory of Functional Organometallic Materials of College of Hunan
Province, College of Chemistry and Materials Science, Hengyang Normal University, Hengyang 421008, PR China
1
* Corresponding
author:
Tel: +86-751-88865515;
Fax:
+86-731-88872531;
E-mail address: [email protected]
Abstract: In this work, we report a novel approach which employed substrate-triggered intramolecular addition-cyclization cascade to develop a highly selective fluorescent probe
(E)-3-(4-(1H-phenanthro[9,10-d]imidazol-2-yl)phenyl)-1-(2-hydroxylphenyl)
prop-2-en-1-one (P-OH) for NH2NH2. The new sensing mechanism of P-OH for NH2NH2 was investigated in detail by fluorescence spectroscopy, 1H NMR titration, mass spectrometry and control experiments. The synthesized probe showed ratiometric fluorescent response to NH2NH2 with naked-eye color changes from yellow to colorless. It’s noteworthy that this probe displayed high sensitivity and selectivity to NH2NH2 over other species, including primary amines, Cys, Hcy, GSH, HS- and HSO3-. Furthermore, the probe can not only detect NH2NH2 in real water samples but also image NH2NH2 in living cells, indicating its potential utility for NH2NH2 sensing in environmental and biological samples.
2
Key words: Addition-cyclization; Ratiometric; Selectivity; NH2NH2; Living cells
1. Introduction Hydrazine (NH2NH2), as a highly reactive base and reducing agent, plays an important role in the production of paints, emulsifiers, pharmaceuticals and pesticides [1]. In addition, NH2NH2 serves as an efficient propellant in rocket and missile propulsion systems due to its high enthalpy of combustion [2, 3]. Despite its usefulness, NH2NH2 displays deleterious effects on human health, which can be absorbed by skin and lungs, inducing serious damage to the kidneys, liver, lungs, and central nervous system [4, 5]. Moreover, research shows that NH2NH2 is carcinogenic and mutagenic [6]. The U.S. Environmental Protection Agency (EPA) standard for the threshold limit value of NH2NH2 in drinking water is 10 ppb [7]. Therefore, the development of reliable and sensitive analytical approaches for the selective determination of NH2NH2 is of great interest and importance. Several conventional methods have been exploited for NH2NH2 detection, including ultraviolet spectrometry [8], electrochemical methods [9], high performance liquid chromatography [10] and titration [11]. However, these methods often require 3
complicated instruments, time-consuming procedures and destruction of cell lysates or tissues, which are not suitable for analyzing NH2NH2 in vivo. Compared to these methods, fluorescence-based methods were more advantageous and attractive due to their simplicity, convenience, non-invasiveness, and great potential for biological applications [12, 13]. To date, a number of fluorescent probes for NH2NH2 have been developed based on three main mechanisms, including condensation with arylaldehydes [14, 15], selective reaction with arylidenemalononitrile [16-18], and selective deprotection of levulinoyl ester or acetyl groups [19-24]. Although great advances have been achieved in this respect, there still exist some issues of interest and concern. For example, the probes based on the reaction of NH2NH2 with arylaldehydes or arylidenemalononitrile often suffered the possible interference from other competitive amines (ammonia, diamines and alkylamines) and biologically related species (Cys, Hcy, GSH, HS-, HSO3-), especially GSH that is present at a millimolar level in living systems. The probes based on NH2NH2-induced deprotection of levulinoyl ester are highly selective, but they only work in low pH (pH< 5) condition, which greatly restricts their biological applications. Furthermore, most of them were fluorescence “turn-off” or “turn-on” type [25-30], and the measurement are using the simple change of fluorescence intensity, which are easily interfered by many factors such as excitation intensity, environmental factors, probe concentrations and instrumental efficiency. By contrast, ratiometric fluorescent probes, which employ the ratio of two emissions at different wavelengths for analysis, can effectively overcome the aforementioned problems and 4
thus make the results more accurate [31, 32]. Therefore, the design of new ratiometric fluorescent probes, which could highly selectively detect NH2NH2 in vitro and in vivo, is still in demand. Based on above consideration, we set out to develop a highly selective, colorimetric and ratiometric fluorescent probe for the detection of NH2NH2 (Scheme 1). The probe (E)-3-(4-(1H-phenanthro[9,10-d]imidazol-2-yl)phenyl)-1-(2-hydroxyl-phenyl)prop-2en-1-one (P-OH) was composed of the phenanthroimidazole moiety and 2'-hydroxychalcone moiety. The α, β-unsaturated carbonyl moiety as the recognition unit can be activated by the intramolecular hydrogen bond [33] and thus easily reacts with NH2NH2, resulting in optical responses. In absence of any analytes, probe P-OH itself exhibited strong yellow fluorescence due to the excited state intramolecular proton transfer process between the o-hydroxyl group and C=O group of the 2'-hydroxychalcone moiety [34, 35]. However, upon treatment with NH2NH2, P-OH is converted into the product 2-(5-(4-(1H-phenanthro[9,10-d]imidazol-2-yl)phenyl)4,5-dihydro-1H-pyrazol-3-yl)phenol
(P-OH-N2H4)
by
the
addition-cyclization
reaction of NH2NH2 to the activated α, β-unsaturated carbonyl moiety. Due to the difference in π-conjugation, P-OH and P-OH-N2H4 will show distinct emission spectra. The alterations of the ratio at two different emission wavelengths induced by NH2NH2 make the ratiometric measurement possible. As expected, the synthesized probe showed many sensing performance and preponderance including dual colorimetric and ratiometric fluorescence responses, high selectivity for NH2NH2 over other amines and reductive species (Cys, Hcy, GSH, HS-, HSO3-), applicability in 5
physiological pH range, low detection limit (2.36 ppb), the capability of sensing NH2NH2 in vitro and in vivo. To gain insight into the sensing mechanism of P-OH and NH2NH2, control probe without an o-hydroxyl group, (E)-3-(4-(1H-phenanthro[9,10-d] imidazol-2-yl)phenyl)-1-phenylprop-2-en-1-one (P-H), was also designed and synthesized.
2. Experimental 2.1. Materials and methods Phenanthrenequinone, 2'-hydroxyacetophenone, terephthalaldehyde, ammonium acetate, pyrrolidine and NH2NH2 were purchased from Guoyao Reagent. Acetic acid, tetrahydrofuran (THF), dimethyl sulfoxide (DMSO), ethanol (EtOH), ammonia (NH4OH), n-butylamine, hydroxylamine, triethylamine, diethylamine, aniline, cysteine (Cys), homocysteine (Hcy), glutathione (GSH), NaHSO3 and NaHS derived from Sigma-Aldrich Reagent Company. Unless otherwise stated, other reagents were used without further decontamination. The thin-layer chromatography (TLC) was carried out on silica gel plates (60F-254) using UV-light to monitor the reaction. The 1
H NMR and 13C NMR spectra of the compounds were measured by use of a Bruker
AVB-500 spectrometer (in DMSO-d6, TMS as an internal standard). UV–vis spectra were recorded with a Shimadzu UV-2550 spectrophotometer. High resolution mass spectra (HRMS) were determined with a QTOF6530 spectrograph (Agilent). Fluorescence emission spectra were obtained on a Hitachi F-7000 luminescence spectrophotometer. Ultrapure water was used throughout all experiments. 2.2. Synthesis 6
The synthetic routes of probe P-OH and P-H are shown in Scheme 1. They were readily prepared in two steps. Structural identification of the products was confirmed by NMR and HRMS spectrometry (Fig. S1-S8).
Inset Scheme 1 here
2.2.1. Synthesis of compound 2 A solution of phenanthrenequinone (832 mg, 4.00 mmol), terephthalaldehyde (536 mg, 4.00 mmol) and ammonium acetate (4.9 g, 64.00 mmol) in acetic acid (50 mL) was refluxed for 30 min (monitored by TLC). After cooling, the mixture was poured into a copious amount of water. The resulting precipitate was filtered, washed with water, and dried under vacuum conditions, which afforded a yellow solid (981 mg, 76%). 1H NMR (500 MHz, DMSO-d6) δ = 10.06 (s, 1H), 8.84 (d, J = 8.3 Hz, 2H), 8.62 (d, J = 7.8 Hz, 2H), 8.55 (d, J = 8.1 Hz, 2H), 8.08 (d, J = 8.1 Hz, 2H), 7.74 (t, J = 7.4 Hz, 2H), 7.64 (t, J = 7.5 Hz, 2H).
13
C NMR (126 MHz, DMSO-d6) δ = 192.95,
136.27, 130.58, 128.29, 127.60, 126.89, 125.87, 124.38, 122.60. 2.2.2. Synthesis of probe P-OH 2'-hydroxyacetophenone (240 µL, 2.00 mmol), compound 2 (646 mg, 2.00 mmol) and pyrrolidine (166 µL) were dissolved in EtOH (10 mL), and the reaction mixture was stirred at room temperature for 24 h (monitored by TLC). After the reaction was completed, red solid was formed. The resultant solid was collected by vacuum filtration in a Buchner funnel and dried under vacuum condition. The crude product 7
was purified by crystallization from THF/EtOH to afford the probe P-OH as red solid (577 mg, yield 65%). 1H NMR (500 MHz, DMSO-d6) δ = 13.58 (s, 1H), 12.56 (s, 1H), 8.87 (dd, J = 17.5, 8.2 Hz, 2H), 8.63 (d, J = 7.7 Hz, 1H), 8.58 (d, J = 7.8 Hz, 1H), 8.42 (d, J = 8.2 Hz, 2H), 8.31 (d, J = 7.9 Hz, 1H), 8.21 – 8.12 (m, 3H), 7.93 (d, J = 15.5 Hz, 1H), 7.82 – 7.73 (m, 2H), 7.66 (dd, J = 14.6, 7.2 Hz, 2H), 7.60 (t, J = 7.7 Hz, 1H), 7.05 (dd, J = 11.9, 6.3 Hz, 2H).
13
C NMR (126 MHz, DMSO-d6) δ = 193.99,
162.38, 148.77, 144.50, 136.86, 135.49, 132.76, 131.38, 130.31, 128.56, 127.72, 126.88, 126.11, 124.62, 124.25, 122.66, 121.32, 119.68, 118.25. HRMS calculated for C30H19N2O2- : 439.1452. Found: 439.1461. 2.2.3. Synthesis of control probe P-H Acetophenone (120 mg, 1.00 mmol), compound 2 (323 mg, 1.00 mmol) and pyrrolidine (83 µL) was dissolved in 5 mL EtOH. The resulting mixture was stirred at room temperature for 12 h (monitored by TLC). Then the precipitate was collected and dried under vacuum condition, which was purified by crystallization from EtOH to afford a yellow solid (168 mg, yield 55%). 1H NMR (500 MHz, DMSO-d6) δ = 8.88 (d, J = 7.9 Hz, 2H), 8.71 (d, J = 7.0 Hz, 2H), 8.49 (d, J = 7.4 Hz, 2H), 8.21 (d, J = 7.1 Hz, 2H), 8.16 (d, J = 7.2 Hz, 2H), 8.09 (d, J = 15.8 Hz, 1H), 7.84 (d, J = 15.7 Hz, 1H), 7.78 (d, J = 6.9 Hz, 2H), 7.70 (d, J = 6.7 Hz, 3H), 7.62 (d, J = 6.6 Hz, 2H). 13
C NMR (126 MHz, DMSO-d6) δ = 189.56, 143.43, 138.00, 133.71, 129.98, 129.30,
129.06, 128.50, 127.86, 127.53, 126.55, 124.47, 123.46, 122.86. HRMS calculated for C30H19N2O- : 423.1503. Found: 423.1515. 2.3. Spectral measurements 8
Stock solution of probe P-OH and P-H (1.0×10−3 mol/L) was prepared in DMSO. Stock solution of NH2NH2 (1.0×10−2 mol/L) was prepared in in double distilled water. In titration experiments, the test samples were prepared by adding 20 μL stock solution of probe P-OH (or control probe P-H) and varied amount of NH2NH2 in a test tube, and being diluted to 2 mL with PBS buffer (10 mM, containing 90% DMSO, pH = 8.0). The final concentration of probe P-OH (or control probe P-H) is 1.0×10−5 mol/L. After test solutions were incubated for 30 min, UV–vis absorption and fluorescence spectra were recorded (λex = 380 nm; slit width: dex = 10 nm, dem = 5 nm). The crude water samples were collected from the tap water in our laboratory, Xiangjiang River and Taozi Lake in Changsha city, which were filtered through a 0.22 μm membrane filter before use. In the sample analysis, the water samples were adjusted to pH 8.0 by using PBS buffer, and then different concentrations of NH2NH2 were introduced. The resulting samples were further treated with probe P-OH and DMSO to give the final mixtures (90 % DMSO, pH 8.0). Then, the fluorescence spectra of the mixtures containing probe P-OH (final concentration = 10 μM) and NH2NH2 (final concentration = 2.5, or 5.0 μM) were measured after 30 min of incubation. 2.4. Selectivity study To explore the selectivity of the new probe, a series of environment and biological-related species were examined in parallel under the same conditions, including amines, such as ammonia, n-butylamine, hydroxylamine, triethylamine, diaethylamine, aniline; reductive species, such as Cys, Hcy, GSH, HS-, HSO3-; and 9
cations, such as Mg2+, Pd2+, Zn2+, Al3+, Ag+, Fe3+, Hg2+, Sn2+, Cd2+, Ba2+ and Ca2+. Stock solutions of metal ions (1.0×10-2 mol/L) were prepared from the respective nitrate salts of the metal ions. Stock solutions of other analytes (1.0×10-2 mol/L) were prepared by dissolving the following compounds in double distilled water: NH4OH, n-butylamine, hydroxylamine, triethylamine, diaethylamin, aniline, Cys, Hcy, GSH, NaHSO3, and NaHS. Several solutions were prepared by adding the stock solution of probe P-OH (20 μL) and the stock solution of other possible interfering species (20 μL) in a test tube, and being diluted to 2 mL with PBS buffer (10 mM, containing 90 % DMSO, pH = 8.0). The fluorescence intensity ratio (I458 nm/I562 nm) of each sample was then recorded after 30 min. 2.5. Cell imaging experiments HeLa cells were grown up in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) in a 5% CO2 atmosphere at 37 °C. Before cell imaging experiments, HeLa cells were seeded in 96-well plates for 24 h. For the fluorescence microscopy imaging experiment, some of cells were incubated with 10 μM P-OH (with 5% DMSO, v/v) for 30 min at 37 °C, and slightly rinsed 3 times with PBS. Other cells preloaded with 10 μM P-OH were further incubated with 100 μM NH2NH2 for another 30 min, and washed 3 times with PBS. Fluorescence (excitation filter, 330-380 nm) and bright field images were observed under an inverted fluorescent microscope (Nikon, Eclipse Ti-S).
3. Results and discussion 3.1. Spectroscopic characterization 10
Initially, the spectroscopic properties of probe P-OH itself were studied by UV-vis absorbance and fluorescence spectroscopy. As illustrated in Fig. S9, the free probe solution showed one major absorption peak at 414 nm, which is related to the π–π* transition
of
the
conjugated
system
between
phenanthroimidazole
and
2'-hydroxychalcone dyes [34-36]. And it is found that probe P-OH exhibited two emission peaks at 458 and 562 nm under excitation at 415 nm. The emission peak at 458 nm is attributed to the enol emission while the other at 562 nm belongs to the keto emission [37, 38]. 3.2. Sensing properties The spectral responses of probe P-OH toward NH2NH2 were tested. As shown in Fig. 1A, probe P-OH itself displayed a major absorption peak at 414 nm. However, upon addition of NH2NH2, the maximum absorption peak of P-OH blue-shifted 94 nm from 414 nm to 320 nm. When excited at 380 nm (the optimal excitation wavelength for P-OH and the product of P-OH + NH2NH2 (Fig. S10)), the free probe gave keto emission at 562 nm. Addition of NH2NH2 to the solution of P-OH resulted in the decrease of the initial emission along with the appearance of a new emission at 458 nm (Fig. 1B). The large spectra shift should be assigned to the destruction of the π-conjugation of the probe P-OH molecule. Additionally, marked color changes could be observed. The colors of the reaction mixture changed from yellow to colorless under room light and from yellow to blue under UV light of 365 nm, respectively, which indicated that P-OH can be used as a dual colorimetric and ratiometric fluorescent probe for NH2NH2 (see Fig. 1 inset). 11
Inset Fig. 1 here
3.3. Response mechanism of probe P-OH for NH2NH2 The optical changes of probe P-OH in the presence of NH2NH2 suggested that the chemical reaction between the NH2NH2 and α, β-unsaturated carbonyl moiety interrupted the conjugation. To evidence the reaction mechanism of P-OH with NH2NH2, 1H NMR titration experiments were performed (Fig. 2). With the addition of NH2NH2 to probe P-OH in DMSO-d6, the original signals at 8.42 (Ha) and 8.16 ppm (Hb) corresponding to the alkene protons underwent up-field shift to 4.98 (Ha’) and 3.71 ppm (Hb’) respectively, while a new peak assigned to the methylene proton appeared at 3.09 ppm (Hc). As the addition of NH2NH2 was up to 2 equiv, the peak of the o-hydroxyl group at 12.56 ppm got broadened and shifted to upfielded (11.20 ppm), which indicated the intramolecular hydrogen bond (C=O…OH) was occluded. Additional evidence for the feasible reaction mechanism comes from the HRMS spectral analysis of the product of P-OH + NH2NH2. As shown in Fig. S11, a major peak at m/z 451.1583 ([M]-, cal’d 451.1564) was detected, which corresponds to the eventual product P-D resulting from the oxidative dehydrogenation of P-OH-N2H4 ([M]-, cal’d 453.1721) in the process of the mass spectrometry detection.
12
In order to explore whether the o-hydroxyl group plays an important role in the process of NH2NH2 sensing, control probe P-H without the corresponding hydroxyl group was also synthesized and examined under the same conditions upon addition of NH2NH2. Compared to P-OH, control probe P-H did not exhibit any remarkable fluorescence response to NH2NH2 (Fig. S12). From these results, it is supposed that the o-hydroxyl group plays a key role in activation of the carbonyl group for NH2NH2 addition through intramolecular hydrogen bond. Therefore, the recognition is implemented by the addition-cyclization reaction of the activated α, β-unsaturated carbonyl moiety triggered by NH2NH2 and the optical changes are attributed to the formation of compound P-OH-N2H4 (Scheme 2).
Inset Fig. 2 here
Inset Scheme 2 here
3.4. Optimization of experimental conditions In order to obtain better detection sensitivity, the optimization of experimental conditions such as solution pH value and reaction time is necessary. Firstly, the influence of pH on the fluorescence response of probe P-OH to NH2NH2 was examined. As shown in Fig. 3, P-OH itself was stable in the whole pH range and could respond to NH2NH2 effectively over a wide pH range including physiological conditions. Unlike most reported probes that could only be utilized in low pH (pH < 5)
13
conditions [1, 17, 19, 22, 25, 39, 40], probe P-OH could be applied in a wide pH range (7–10), which may extend its applications. Time dependent-fluorescence spectra of probe P-OH in the presence of NH2NH2 were investigated by monitoring the fluorescence intensity at 440 and 565 nm, respectively (Fig. 4). The result indicated that the interaction of P-OH with NH2NH2 was completed within 30 min. Based on those observations, a pH of 8.0 and an assay time of 30 min were used in further experiments.
Inset Fig. 3 here
Inset Fig. 4 here
3.5. Sensitive detection of NH2NH2 Under the optimum conditions discussed above, the fluorescence titrations of probe P-OH with NH2NH2 were performed. As shown in Fig. 5A, the free probe P-OH exhibited a maximal emission peak at 562 nm. Upon continuous addition of NH2NH2, a prominent new emission peak at 458 nm gradually enhanced in intensity, and a good ratiometric spectrum was observed with an isoemission point at 517nm. After the amount of NH2NH2 increased to 125 μM, there were no significant fluorescence changes (Fig. 5B). Therefore, about 125 μM of NH2NH2 was required to complete the reaction in the presence of 10 μM of P-OH within 30 min. Moreover, the fluorescence intensity ratio (I458 nm/I562 nm) was linearly proportional to concentrations of NH2NH2 in the range of 0-100 µM. And, the detection limit (defined as three times standard 14
deviation of probe intensity of the background) [41] determined to be 7.4×10-8 M (2.36 ppb), which is lower than the permitted level of NH2NH2 content in drinking water (10 ppb). The results demonstrated that probe P-OH could detect NH2NH2 levels both qualitatively and quantitatively by the fluorescence spectrometry method.
Inset Fig. 5 here
3.6. Selectivity studies of probe P-OH Selectivity is also an important index for probes. To evaluate the selectivity of probe P-OH toward NH2NH2, a series of species such as amines (ammonia, n-butylamine, hydroxylamine, triethylamine, diaethylamine, aniline) and reductive species (Cys, Hcy, GSH, HS-, HSO3-) was investigated. As shown in Fig. 6A, only addition of NH2NH2 resulted in a distinguished blue-shifted emission and remarkable intensity enhancement at 458 nm in the fluorescence spectrum, other species almost could not induced any obvious changes to the fluorescence spectra of P-OH. It’s noteworthy that P-OH was completely inert to ammonia and reductive species (Cys, Hcy, GSH, HS-, HSO3-) whereas a previously reported levulinate-type, aldehyde-type and arylidenemalononitrile-type fluorescent probes for NH2NH2 showed also high reactivity for these species [42-49]. The high selectivity of P-OH for NH2NH2 over other species may come from the efficient addition-cyclization reaction between NH2NH2 and α, β-unsaturated carbonyl moiety. In addition, other environmental and biological important metals ions such as Mg2+, Pd2+, Zn2+, Al3+, Ag+, Fe3+, Hg2+, Sn2+,
15
Cd2+, Ba2+ and Ca2+ were also tested, and almost no changes in the fluorescence intensity ratio (I458 nm/I562 nm) of P-OH were observed. To test practical applicability of our fluorescent probe for NH2NH2, we further investigated the fluorescence response of P-OH toward NH2NH2 in the presence of other species (amine-containing species, reductive species and metal ions). As shown in Fig. 6B and Fig. S13, other coexisting species had no apparent interference with the detection of NH2NH2. All the results indicated that P-OH has high selectivity for NH2NH2 and is promising for precise NH2NH2 detection in a complex environment.
Inset Fig. 6 here
3.7. Determination of NH2NH2 in Water Samples NH2NH2 is highly toxic and can cause serious environmental contamination and health problems during its manufacture, transport, use, and disposal procedures. Therefore, determination of NH2NH2 in real water samples is of great significance. The proposed method was applied to monitoring NH2NH2 in water samples from the tap water in our laboratory, Xiangjiang River and Taozi Lake in Changsha city. As shown in Table 1, recoveries of NH2NH2 were in the range of 98.2-103.4% for all the spiked samples. These demonstrated that our proposed probe could detect NH2NH2 in real water samples quantitatively.
Inset Table 1 here
16
3.8. Cell imaging Finally, we evaluated the ability of probe P-OH for fluorescent imaging of NH2NH2 in live cells. HeLa cells pre-incubated with P-OH (10 μM) for 30 min showed nearly no fluorescence in the blue channel (Fig. 7B). By contrast, after P-OH-loaded cells were incubated with NH2NH2 (100 μM) for another 30 min, strong fluorescence signals were observed in the blue channel (Fig. 7D). These cell experiments indicated that P-OH was both cell-permeable and capable of imaging NH2NH2 in living cells in a turn-on manner.
Inset Fig. 7 here
Moreover, the comparison of present probe with some previous reported NH2NH2 fluorescent probes has been listed in Table S1. Our probe shows many salient features including ratiometric detection, low detection limit, high specificity, applicability in physiological pH range, feasibility in aqueous samples and living cells. Therefore, probe P-OH could be used as a promising tool for the determination of NH2NH2 in environmental and biological samples.
4. Conclusion In summary, a novel fluorescent probe P-OH for NH2NH2 was synthesized via the NH2NH2-mediated addition-cyclization of α, β-unsaturated carbonyl moiety. P-OH can serve not only as a colorimetric probe but also as a ratiometic fluorescent probe for NH2NH2 with a low detection limit of 2.36 ppb at physiological pH. Notably, this
17
probe showed high selectivity towards NH2NH2 over other amines as well as other reductive species, which is more advantageous than levulinate-type, aldehyde-type and arylidenemalononitrile-type fluorescent probes for NH2NH2. Moreover, the probe possessed desirable cell permeability, and could successfully be applied for detection of NH2NH2 both in water samples and living cells. In addition, the proposed sensing mechanism in this work should be meaningful to the development of other new fluorescence probes for NH2NH2.
Acknowledgments This work was supported by the National Natural Science Foundation of China, China (21375037, 21675051), Hunan Provincial Innovation Foundation for Postgraduate (CX2017B226), Hunan Provincial Science and Technology Department of Hunan Province (NO: 2014FJ6034) and Scientific Research Project of Hengyang Normal University (17D05).
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[20] B. Liu, Q. Liu, M. Shah, J. Wang, G. Zhang, Y. Pang, Fluorescence monitor of hydrazine in vivo by selective deprotection of flavonoid, Sens. Actuators, B 202 (2014) 194-200. [21] Y. Sun, D. Zhao, S. Fan, L. Duan, A 4-hydroxynaphthalimide-derived ratiometric fluorescent probe for hydrazine and its in vivo applications, Sens. Actuators, B 208 (2015) 512-517. [22] K. Li, H. R. Xu, K. K. Yu, J. T. Hou, X. Q. Yu, A coumarin-based chromogenic and ratiometric probe for hydrazine, Anal. Methods 5 (2013) 2653-2656. [23] S. Zhu, W. Lin, L. Yuan, Development of a near-infrared fluorescent probe for monitoring hydrazine in serum and living cells, Anal. Methods 5 (2013) 3450-3453. [24] J. Zhou, R. Shi, J. Liu, R. Wang, Y. Xu, X. Qian, An ESIPT-based fluorescent probe for sensitive detection of hydrazine in aqueous solution, Org. Biomol. Chem. 13 (2015) 5344-5348. [25] H. L. Min, B. Yoon, J. S. Kim, J. L. Sessler, Naphthalimide trifluoroacetyl acetonate: a hydrazine-selective chemodosimetric sensor, Chem. Sci. 4 (2013) 4121-4126. [26] B. Chen, X. Sun, X. Li, H. Ågren, Y. Xie, TICT based fluorescence “turn-on” hydrazine probes, Sens. Actuators, B 199 (2014) 93-100. [27] Y. Qian, J. Lin, L. Han, L. Lin, H. Zhu, A resorufin-based colorimetric and fluorescent probe for live-cell monitoring of hydrazine, Biosens. Bioelectron. 58 (2014) 282-286. [28] M. G. Choi, J. O. Moon, J. Bae, J. W. Lee, S. K. Chang, Dual signaling of 21
hydrazine by selective deprotection of dichlorofluorescein and resorufin acetates, Org. Biomol. Chem. 11 (2013) 2961-2965. [29] D. Y. Qu, J. L. Chen, B. Di, A fluorescence “switch-on” approach to detect hydrazine in aqueous solution at neutral pH, Anal. Methods 6 (2014) 4705-4709. [30] J. Li, J. Liu, J. W. Y. Lam, B. Z. Tang, Poly(aryleneynonylene) with an aggregation-enhanced emission characteristic: a fluorescent sensor for both hydrazine and explosive detection, RSC. Adv. 3 (2013) 8193-8196. [31] B. Gu, L. Huang, N. Mi, P. Yin, Y. Zhang, X. Tu, X. Luo, S. Luo, S. Yao, An ESIPT-based fluorescent probe for highly selective and ratiometric detection of mercury (II) in solution and in cells, Analyst. 140 (2015) 2778-2784. [32] Y. Liu, D. Yu, S. Ding, Q. Xiao, J. Guo, G. Feng, Rapid and ratiometric fluorescent detection of cysteine with high selectivity and sensitivity by a simple and readily available probe, ACS Appl. Mater. Interfaces. 6 (2014) 17543-17550. [33] S. Park, H. J. Kim, Highly activated michael acceptor by an intramolecular hydrogen bond as a fluorescence turn-on probe for cyanide, Chem. Commun. 46 (2010) 9197-9199. [34] J. Wang, W. Lin, W. Li, Three-channel fluorescent sensing via organic white light-emitting dyes for detection of hydrogen sulfide in living cells, Biomaterials 34 (2013) 7429-7436. [35] Z. Song, R. T. Kwok, E. Zhao, Z. He, Y. Hong, J. W. Lam, B. Liu, B. Z. Tang, A ratiometric fluorescent probe based on ESIPT and AIE processes for alkaline phosphatase activity assay and visualization in living cells, ACS Appl. Mater. 22
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Figures:
Scheme 1. Synthesis routes of control probe P-H (R = H) and probe P-OH (R = OH).
25
Scheme 2. Proposed sensing mechanism of probe P-OH for NH2NH2.
26
Fig. 1. Absorption (A) and fluorescence (B) emission spectra of probe P-OH (10 µM) in DMSO/PBS buffer (9:1, v/v, 10 mM, pH = 8.0) in the absence (black) or presence (red) of NH2NH2 (100 µM). Each spectrum was acquired at 30 min after hydrazine addition. Excitation wavelength = 380 nm. Inset: color changes of the probe P-OH (10 µM) solution after addition of NH2NH2 (100 µM) under room light (A) and under a 365 nm light (B).
27
Fig. 2. Partial 1H NMR spectra of P-OH, P-OH + 0.5 equiv NH2NH2, P-OH + 1.0 equiv NH2NH2, P-OH + 2.0 equiv NH2NH2 in DMSO-d6 (500 MHz).
28
Fig. 3. The effect of pH on the fluorescence intensity ratio (I458 nm/I562 nm) of probe P-OH (10 μM) in the absence or presence of NH2NH2 (100 μM). Excitation wavelength = 380 nm.
29
Fig. 4. (A) Time-dependent changes in the fluorescence spectra of probe P-OH (10 μM) upon reaction with NH2NH2 (100 µM). Excitation wavelength = 380 nm. (B) Time-dependent fluorescence intensity at 562 nm (red) and 458 nm (black) of probe P-OH in the presence of NH2NH2 (100 µM).
30
Fig. 5. (A) Fluorescence spectra of P-OH (10 µM) in the presence of various concentrations of NH2NH2 (0-250 µM) in DMSO/PBS buffer (9:1, v/v, 10 mM, pH = 8). Excitation wavelength = 380 nm. (B) The fluorescence intensity ratio (I458 nm/I562 nm) of P-OH (10 µM) changes as concentrations of NH2NH2 (0-250 µM). Inset: a linear correlation between the fluorescence intensity ratio (I458 nm/I562 nm)
and concentrations of NH2NH2 (0-100 µM).
31
Fig. 6. (A) Fluorescence spectra of probe P-OH (10 μM) in the presence of various species (100 μM for ammonia, n-butylamine, hydroxylamine, triethylamine, diaethylamine, aniline, Cys, Hcy, GSH, HS-, HSO3- and NH2NH2). (B) The fluorescence intensity ratio (I458 nm/I562 nm) of probe P-OH (10 µM) upon addition of various substances (black bars) and then addition of NH2NH2 (red bars). The concentration of NH2NH2 and other interfering species are 100 μM. (1) None, (2) ammonia, (3) n-butylamine, (4) hydroxylamine, (5) triethylamine, (6) diaethylamine, (7) aniline, (8) Cys, (9) Hcy, (10) GSH, (11) HS-, (12) HSO3-. Excitation wavelength = 380 nm.
32
Fig. 7. Fluorescence images of HeLa cells. (A) Bright-field image, and (B) fluorescence image in blue channel after HeLa cells were incubated with probe P-OH (10 μM) for 30 min. (C) Bright-field image, and (D) fluorescence image in blue channel after HeLa cells being pre-treated with P-OH (10 μM) for 30 min, and then incubated with NH2NH2 (100 μM) for another 30 min.
33
Table 1. The measurement results of NH2NH2 in water samples
Water samples
NH2NH2 added (M)
Found (M)
Recovery (%)
2.5
2.53 ± 0.05
101.2
5.0
4.91 ± 0.10
98.2
2.5
2.56 ± 0.07
102.4
5.0
5.15 ± 0.12
103.0
2.5
2.48 ± 0.11
99.2
5.0
5.17 ± 0.14
103.4
Tap water
Xiangjiang River water
Taozi Lake
34
Highlights:
A
novel
fluorescent
probe
for
NH2NH2
was
developed
based
on
NH2NH2-triggered intramolecular addition-cyclization cascade.
It showed high sensitivity and selectivity toward NH2NH2 with obvious colorimetric and ratiometic fluorescence responses.
It could detect NH2NH2 in vitro and in vivo without interference from other amines and reductive species.
35
Graphical Abstract
36
PII: DOI: Reference:
S0039-9140(17)31242-0 https://doi.org/10.1016/j.talanta.2017.12.039 TAL18175
To appear in: Talanta Received date: 5 August 2017 Revised date: 7 December 2017 Accepted date: 12 December 2017 Cite this article as: Hai Xu, Biao Gu, Yaqian Li, Zheng Huang, Wei Su, Xiaoli Duan, Peng Yin, Haitao Li and Shouzhuo Yao, A highly selective, colorimetric and ratiometric fluorescent probe for NH2NH2 and its bioimaging, Talanta, https://doi.org/10.1016/j.talanta.2017.12.039 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 galley proof before it is published in its final citable 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.
A highly selective, colorimetric and ratiometric fluorescent probe for NH2NH2 and its bioimaging
Hai Xua, Biao Gua,b, Yaqian Lia, Zheng Huanga, Wei Sua, Xiaoli Duana, Peng Yina*, Haitao Lia* and Shouzhuo Yaoa
a
Key Laboratory of Chemical Biology and Traditional Chinese Medicine Research (Ministry of Education), College of Chemistry and Chemical Engineering, Hunan Normal University, Changsha 410081, PR China. b
Key Laboratory of Functional Organometallic Materials of College of Hunan
Province, College of Chemistry and Materials Science, Hengyang Normal University, Hengyang 421008, PR China
1
* Corresponding
author:
Tel: +86-751-88865515;
Fax:
+86-731-88872531;
E-mail address: [email protected]
Abstract: In this work, we report a novel approach which employed substrate-triggered intramolecular addition-cyclization cascade to develop a highly selective fluorescent probe
(E)-3-(4-(1H-phenanthro[9,10-d]imidazol-2-yl)phenyl)-1-(2-hydroxylphenyl)
prop-2-en-1-one (P-OH) for NH2NH2. The new sensing mechanism of P-OH for NH2NH2 was investigated in detail by fluorescence spectroscopy, 1H NMR titration, mass spectrometry and control experiments. The synthesized probe showed ratiometric fluorescent response to NH2NH2 with naked-eye color changes from yellow to colorless. It’s noteworthy that this probe displayed high sensitivity and selectivity to NH2NH2 over other species, including primary amines, Cys, Hcy, GSH, HS- and HSO3-. Furthermore, the probe can not only detect NH2NH2 in real water samples but also image NH2NH2 in living cells, indicating its potential utility for NH2NH2 sensing in environmental and biological samples.
2
Key words: Addition-cyclization; Ratiometric; Selectivity; NH2NH2; Living cells
1. Introduction Hydrazine (NH2NH2), as a highly reactive base and reducing agent, plays an important role in the production of paints, emulsifiers, pharmaceuticals and pesticides [1]. In addition, NH2NH2 serves as an efficient propellant in rocket and missile propulsion systems due to its high enthalpy of combustion [2, 3]. Despite its usefulness, NH2NH2 displays deleterious effects on human health, which can be absorbed by skin and lungs, inducing serious damage to the kidneys, liver, lungs, and central nervous system [4, 5]. Moreover, research shows that NH2NH2 is carcinogenic and mutagenic [6]. The U.S. Environmental Protection Agency (EPA) standard for the threshold limit value of NH2NH2 in drinking water is 10 ppb [7]. Therefore, the development of reliable and sensitive analytical approaches for the selective determination of NH2NH2 is of great interest and importance. Several conventional methods have been exploited for NH2NH2 detection, including ultraviolet spectrometry [8], electrochemical methods [9], high performance liquid chromatography [10] and titration [11]. However, these methods often require 3
complicated instruments, time-consuming procedures and destruction of cell lysates or tissues, which are not suitable for analyzing NH2NH2 in vivo. Compared to these methods, fluorescence-based methods were more advantageous and attractive due to their simplicity, convenience, non-invasiveness, and great potential for biological applications [12, 13]. To date, a number of fluorescent probes for NH2NH2 have been developed based on three main mechanisms, including condensation with arylaldehydes [14, 15], selective reaction with arylidenemalononitrile [16-18], and selective deprotection of levulinoyl ester or acetyl groups [19-24]. Although great advances have been achieved in this respect, there still exist some issues of interest and concern. For example, the probes based on the reaction of NH2NH2 with arylaldehydes or arylidenemalononitrile often suffered the possible interference from other competitive amines (ammonia, diamines and alkylamines) and biologically related species (Cys, Hcy, GSH, HS-, HSO3-), especially GSH that is present at a millimolar level in living systems. The probes based on NH2NH2-induced deprotection of levulinoyl ester are highly selective, but they only work in low pH (pH< 5) condition, which greatly restricts their biological applications. Furthermore, most of them were fluorescence “turn-off” or “turn-on” type [25-30], and the measurement are using the simple change of fluorescence intensity, which are easily interfered by many factors such as excitation intensity, environmental factors, probe concentrations and instrumental efficiency. By contrast, ratiometric fluorescent probes, which employ the ratio of two emissions at different wavelengths for analysis, can effectively overcome the aforementioned problems and 4
thus make the results more accurate [31, 32]. Therefore, the design of new ratiometric fluorescent probes, which could highly selectively detect NH2NH2 in vitro and in vivo, is still in demand. Based on above consideration, we set out to develop a highly selective, colorimetric and ratiometric fluorescent probe for the detection of NH2NH2 (Scheme 1). The probe (E)-3-(4-(1H-phenanthro[9,10-d]imidazol-2-yl)phenyl)-1-(2-hydroxyl-phenyl)prop-2en-1-one (P-OH) was composed of the phenanthroimidazole moiety and 2'-hydroxychalcone moiety. The α, β-unsaturated carbonyl moiety as the recognition unit can be activated by the intramolecular hydrogen bond [33] and thus easily reacts with NH2NH2, resulting in optical responses. In absence of any analytes, probe P-OH itself exhibited strong yellow fluorescence due to the excited state intramolecular proton transfer process between the o-hydroxyl group and C=O group of the 2'-hydroxychalcone moiety [34, 35]. However, upon treatment with NH2NH2, P-OH is converted into the product 2-(5-(4-(1H-phenanthro[9,10-d]imidazol-2-yl)phenyl)4,5-dihydro-1H-pyrazol-3-yl)phenol
(P-OH-N2H4)
by
the
addition-cyclization
reaction of NH2NH2 to the activated α, β-unsaturated carbonyl moiety. Due to the difference in π-conjugation, P-OH and P-OH-N2H4 will show distinct emission spectra. The alterations of the ratio at two different emission wavelengths induced by NH2NH2 make the ratiometric measurement possible. As expected, the synthesized probe showed many sensing performance and preponderance including dual colorimetric and ratiometric fluorescence responses, high selectivity for NH2NH2 over other amines and reductive species (Cys, Hcy, GSH, HS-, HSO3-), applicability in 5
physiological pH range, low detection limit (2.36 ppb), the capability of sensing NH2NH2 in vitro and in vivo. To gain insight into the sensing mechanism of P-OH and NH2NH2, control probe without an o-hydroxyl group, (E)-3-(4-(1H-phenanthro[9,10-d] imidazol-2-yl)phenyl)-1-phenylprop-2-en-1-one (P-H), was also designed and synthesized.
2. Experimental 2.1. Materials and methods Phenanthrenequinone, 2'-hydroxyacetophenone, terephthalaldehyde, ammonium acetate, pyrrolidine and NH2NH2 were purchased from Guoyao Reagent. Acetic acid, tetrahydrofuran (THF), dimethyl sulfoxide (DMSO), ethanol (EtOH), ammonia (NH4OH), n-butylamine, hydroxylamine, triethylamine, diethylamine, aniline, cysteine (Cys), homocysteine (Hcy), glutathione (GSH), NaHSO3 and NaHS derived from Sigma-Aldrich Reagent Company. Unless otherwise stated, other reagents were used without further decontamination. The thin-layer chromatography (TLC) was carried out on silica gel plates (60F-254) using UV-light to monitor the reaction. The 1
H NMR and 13C NMR spectra of the compounds were measured by use of a Bruker
AVB-500 spectrometer (in DMSO-d6, TMS as an internal standard). UV–vis spectra were recorded with a Shimadzu UV-2550 spectrophotometer. High resolution mass spectra (HRMS) were determined with a QTOF6530 spectrograph (Agilent). Fluorescence emission spectra were obtained on a Hitachi F-7000 luminescence spectrophotometer. Ultrapure water was used throughout all experiments. 2.2. Synthesis 6
The synthetic routes of probe P-OH and P-H are shown in Scheme 1. They were readily prepared in two steps. Structural identification of the products was confirmed by NMR and HRMS spectrometry (Fig. S1-S8).
Inset Scheme 1 here
2.2.1. Synthesis of compound 2 A solution of phenanthrenequinone (832 mg, 4.00 mmol), terephthalaldehyde (536 mg, 4.00 mmol) and ammonium acetate (4.9 g, 64.00 mmol) in acetic acid (50 mL) was refluxed for 30 min (monitored by TLC). After cooling, the mixture was poured into a copious amount of water. The resulting precipitate was filtered, washed with water, and dried under vacuum conditions, which afforded a yellow solid (981 mg, 76%). 1H NMR (500 MHz, DMSO-d6) δ = 10.06 (s, 1H), 8.84 (d, J = 8.3 Hz, 2H), 8.62 (d, J = 7.8 Hz, 2H), 8.55 (d, J = 8.1 Hz, 2H), 8.08 (d, J = 8.1 Hz, 2H), 7.74 (t, J = 7.4 Hz, 2H), 7.64 (t, J = 7.5 Hz, 2H).
13
C NMR (126 MHz, DMSO-d6) δ = 192.95,
136.27, 130.58, 128.29, 127.60, 126.89, 125.87, 124.38, 122.60. 2.2.2. Synthesis of probe P-OH 2'-hydroxyacetophenone (240 µL, 2.00 mmol), compound 2 (646 mg, 2.00 mmol) and pyrrolidine (166 µL) were dissolved in EtOH (10 mL), and the reaction mixture was stirred at room temperature for 24 h (monitored by TLC). After the reaction was completed, red solid was formed. The resultant solid was collected by vacuum filtration in a Buchner funnel and dried under vacuum condition. The crude product 7
was purified by crystallization from THF/EtOH to afford the probe P-OH as red solid (577 mg, yield 65%). 1H NMR (500 MHz, DMSO-d6) δ = 13.58 (s, 1H), 12.56 (s, 1H), 8.87 (dd, J = 17.5, 8.2 Hz, 2H), 8.63 (d, J = 7.7 Hz, 1H), 8.58 (d, J = 7.8 Hz, 1H), 8.42 (d, J = 8.2 Hz, 2H), 8.31 (d, J = 7.9 Hz, 1H), 8.21 – 8.12 (m, 3H), 7.93 (d, J = 15.5 Hz, 1H), 7.82 – 7.73 (m, 2H), 7.66 (dd, J = 14.6, 7.2 Hz, 2H), 7.60 (t, J = 7.7 Hz, 1H), 7.05 (dd, J = 11.9, 6.3 Hz, 2H).
13
C NMR (126 MHz, DMSO-d6) δ = 193.99,
162.38, 148.77, 144.50, 136.86, 135.49, 132.76, 131.38, 130.31, 128.56, 127.72, 126.88, 126.11, 124.62, 124.25, 122.66, 121.32, 119.68, 118.25. HRMS calculated for C30H19N2O2- : 439.1452. Found: 439.1461. 2.2.3. Synthesis of control probe P-H Acetophenone (120 mg, 1.00 mmol), compound 2 (323 mg, 1.00 mmol) and pyrrolidine (83 µL) was dissolved in 5 mL EtOH. The resulting mixture was stirred at room temperature for 12 h (monitored by TLC). Then the precipitate was collected and dried under vacuum condition, which was purified by crystallization from EtOH to afford a yellow solid (168 mg, yield 55%). 1H NMR (500 MHz, DMSO-d6) δ = 8.88 (d, J = 7.9 Hz, 2H), 8.71 (d, J = 7.0 Hz, 2H), 8.49 (d, J = 7.4 Hz, 2H), 8.21 (d, J = 7.1 Hz, 2H), 8.16 (d, J = 7.2 Hz, 2H), 8.09 (d, J = 15.8 Hz, 1H), 7.84 (d, J = 15.7 Hz, 1H), 7.78 (d, J = 6.9 Hz, 2H), 7.70 (d, J = 6.7 Hz, 3H), 7.62 (d, J = 6.6 Hz, 2H). 13
C NMR (126 MHz, DMSO-d6) δ = 189.56, 143.43, 138.00, 133.71, 129.98, 129.30,
129.06, 128.50, 127.86, 127.53, 126.55, 124.47, 123.46, 122.86. HRMS calculated for C30H19N2O- : 423.1503. Found: 423.1515. 2.3. Spectral measurements 8
Stock solution of probe P-OH and P-H (1.0×10−3 mol/L) was prepared in DMSO. Stock solution of NH2NH2 (1.0×10−2 mol/L) was prepared in in double distilled water. In titration experiments, the test samples were prepared by adding 20 μL stock solution of probe P-OH (or control probe P-H) and varied amount of NH2NH2 in a test tube, and being diluted to 2 mL with PBS buffer (10 mM, containing 90% DMSO, pH = 8.0). The final concentration of probe P-OH (or control probe P-H) is 1.0×10−5 mol/L. After test solutions were incubated for 30 min, UV–vis absorption and fluorescence spectra were recorded (λex = 380 nm; slit width: dex = 10 nm, dem = 5 nm). The crude water samples were collected from the tap water in our laboratory, Xiangjiang River and Taozi Lake in Changsha city, which were filtered through a 0.22 μm membrane filter before use. In the sample analysis, the water samples were adjusted to pH 8.0 by using PBS buffer, and then different concentrations of NH2NH2 were introduced. The resulting samples were further treated with probe P-OH and DMSO to give the final mixtures (90 % DMSO, pH 8.0). Then, the fluorescence spectra of the mixtures containing probe P-OH (final concentration = 10 μM) and NH2NH2 (final concentration = 2.5, or 5.0 μM) were measured after 30 min of incubation. 2.4. Selectivity study To explore the selectivity of the new probe, a series of environment and biological-related species were examined in parallel under the same conditions, including amines, such as ammonia, n-butylamine, hydroxylamine, triethylamine, diaethylamine, aniline; reductive species, such as Cys, Hcy, GSH, HS-, HSO3-; and 9
cations, such as Mg2+, Pd2+, Zn2+, Al3+, Ag+, Fe3+, Hg2+, Sn2+, Cd2+, Ba2+ and Ca2+. Stock solutions of metal ions (1.0×10-2 mol/L) were prepared from the respective nitrate salts of the metal ions. Stock solutions of other analytes (1.0×10-2 mol/L) were prepared by dissolving the following compounds in double distilled water: NH4OH, n-butylamine, hydroxylamine, triethylamine, diaethylamin, aniline, Cys, Hcy, GSH, NaHSO3, and NaHS. Several solutions were prepared by adding the stock solution of probe P-OH (20 μL) and the stock solution of other possible interfering species (20 μL) in a test tube, and being diluted to 2 mL with PBS buffer (10 mM, containing 90 % DMSO, pH = 8.0). The fluorescence intensity ratio (I458 nm/I562 nm) of each sample was then recorded after 30 min. 2.5. Cell imaging experiments HeLa cells were grown up in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) in a 5% CO2 atmosphere at 37 °C. Before cell imaging experiments, HeLa cells were seeded in 96-well plates for 24 h. For the fluorescence microscopy imaging experiment, some of cells were incubated with 10 μM P-OH (with 5% DMSO, v/v) for 30 min at 37 °C, and slightly rinsed 3 times with PBS. Other cells preloaded with 10 μM P-OH were further incubated with 100 μM NH2NH2 for another 30 min, and washed 3 times with PBS. Fluorescence (excitation filter, 330-380 nm) and bright field images were observed under an inverted fluorescent microscope (Nikon, Eclipse Ti-S).
3. Results and discussion 3.1. Spectroscopic characterization 10
Initially, the spectroscopic properties of probe P-OH itself were studied by UV-vis absorbance and fluorescence spectroscopy. As illustrated in Fig. S9, the free probe solution showed one major absorption peak at 414 nm, which is related to the π–π* transition
of
the
conjugated
system
between
phenanthroimidazole
and
2'-hydroxychalcone dyes [34-36]. And it is found that probe P-OH exhibited two emission peaks at 458 and 562 nm under excitation at 415 nm. The emission peak at 458 nm is attributed to the enol emission while the other at 562 nm belongs to the keto emission [37, 38]. 3.2. Sensing properties The spectral responses of probe P-OH toward NH2NH2 were tested. As shown in Fig. 1A, probe P-OH itself displayed a major absorption peak at 414 nm. However, upon addition of NH2NH2, the maximum absorption peak of P-OH blue-shifted 94 nm from 414 nm to 320 nm. When excited at 380 nm (the optimal excitation wavelength for P-OH and the product of P-OH + NH2NH2 (Fig. S10)), the free probe gave keto emission at 562 nm. Addition of NH2NH2 to the solution of P-OH resulted in the decrease of the initial emission along with the appearance of a new emission at 458 nm (Fig. 1B). The large spectra shift should be assigned to the destruction of the π-conjugation of the probe P-OH molecule. Additionally, marked color changes could be observed. The colors of the reaction mixture changed from yellow to colorless under room light and from yellow to blue under UV light of 365 nm, respectively, which indicated that P-OH can be used as a dual colorimetric and ratiometric fluorescent probe for NH2NH2 (see Fig. 1 inset). 11
Inset Fig. 1 here
3.3. Response mechanism of probe P-OH for NH2NH2 The optical changes of probe P-OH in the presence of NH2NH2 suggested that the chemical reaction between the NH2NH2 and α, β-unsaturated carbonyl moiety interrupted the conjugation. To evidence the reaction mechanism of P-OH with NH2NH2, 1H NMR titration experiments were performed (Fig. 2). With the addition of NH2NH2 to probe P-OH in DMSO-d6, the original signals at 8.42 (Ha) and 8.16 ppm (Hb) corresponding to the alkene protons underwent up-field shift to 4.98 (Ha’) and 3.71 ppm (Hb’) respectively, while a new peak assigned to the methylene proton appeared at 3.09 ppm (Hc). As the addition of NH2NH2 was up to 2 equiv, the peak of the o-hydroxyl group at 12.56 ppm got broadened and shifted to upfielded (11.20 ppm), which indicated the intramolecular hydrogen bond (C=O…OH) was occluded. Additional evidence for the feasible reaction mechanism comes from the HRMS spectral analysis of the product of P-OH + NH2NH2. As shown in Fig. S11, a major peak at m/z 451.1583 ([M]-, cal’d 451.1564) was detected, which corresponds to the eventual product P-D resulting from the oxidative dehydrogenation of P-OH-N2H4 ([M]-, cal’d 453.1721) in the process of the mass spectrometry detection.
12
In order to explore whether the o-hydroxyl group plays an important role in the process of NH2NH2 sensing, control probe P-H without the corresponding hydroxyl group was also synthesized and examined under the same conditions upon addition of NH2NH2. Compared to P-OH, control probe P-H did not exhibit any remarkable fluorescence response to NH2NH2 (Fig. S12). From these results, it is supposed that the o-hydroxyl group plays a key role in activation of the carbonyl group for NH2NH2 addition through intramolecular hydrogen bond. Therefore, the recognition is implemented by the addition-cyclization reaction of the activated α, β-unsaturated carbonyl moiety triggered by NH2NH2 and the optical changes are attributed to the formation of compound P-OH-N2H4 (Scheme 2).
Inset Fig. 2 here
Inset Scheme 2 here
3.4. Optimization of experimental conditions In order to obtain better detection sensitivity, the optimization of experimental conditions such as solution pH value and reaction time is necessary. Firstly, the influence of pH on the fluorescence response of probe P-OH to NH2NH2 was examined. As shown in Fig. 3, P-OH itself was stable in the whole pH range and could respond to NH2NH2 effectively over a wide pH range including physiological conditions. Unlike most reported probes that could only be utilized in low pH (pH < 5)
13
conditions [1, 17, 19, 22, 25, 39, 40], probe P-OH could be applied in a wide pH range (7–10), which may extend its applications. Time dependent-fluorescence spectra of probe P-OH in the presence of NH2NH2 were investigated by monitoring the fluorescence intensity at 440 and 565 nm, respectively (Fig. 4). The result indicated that the interaction of P-OH with NH2NH2 was completed within 30 min. Based on those observations, a pH of 8.0 and an assay time of 30 min were used in further experiments.
Inset Fig. 3 here
Inset Fig. 4 here
3.5. Sensitive detection of NH2NH2 Under the optimum conditions discussed above, the fluorescence titrations of probe P-OH with NH2NH2 were performed. As shown in Fig. 5A, the free probe P-OH exhibited a maximal emission peak at 562 nm. Upon continuous addition of NH2NH2, a prominent new emission peak at 458 nm gradually enhanced in intensity, and a good ratiometric spectrum was observed with an isoemission point at 517nm. After the amount of NH2NH2 increased to 125 μM, there were no significant fluorescence changes (Fig. 5B). Therefore, about 125 μM of NH2NH2 was required to complete the reaction in the presence of 10 μM of P-OH within 30 min. Moreover, the fluorescence intensity ratio (I458 nm/I562 nm) was linearly proportional to concentrations of NH2NH2 in the range of 0-100 µM. And, the detection limit (defined as three times standard 14
deviation of probe intensity of the background) [41] determined to be 7.4×10-8 M (2.36 ppb), which is lower than the permitted level of NH2NH2 content in drinking water (10 ppb). The results demonstrated that probe P-OH could detect NH2NH2 levels both qualitatively and quantitatively by the fluorescence spectrometry method.
Inset Fig. 5 here
3.6. Selectivity studies of probe P-OH Selectivity is also an important index for probes. To evaluate the selectivity of probe P-OH toward NH2NH2, a series of species such as amines (ammonia, n-butylamine, hydroxylamine, triethylamine, diaethylamine, aniline) and reductive species (Cys, Hcy, GSH, HS-, HSO3-) was investigated. As shown in Fig. 6A, only addition of NH2NH2 resulted in a distinguished blue-shifted emission and remarkable intensity enhancement at 458 nm in the fluorescence spectrum, other species almost could not induced any obvious changes to the fluorescence spectra of P-OH. It’s noteworthy that P-OH was completely inert to ammonia and reductive species (Cys, Hcy, GSH, HS-, HSO3-) whereas a previously reported levulinate-type, aldehyde-type and arylidenemalononitrile-type fluorescent probes for NH2NH2 showed also high reactivity for these species [42-49]. The high selectivity of P-OH for NH2NH2 over other species may come from the efficient addition-cyclization reaction between NH2NH2 and α, β-unsaturated carbonyl moiety. In addition, other environmental and biological important metals ions such as Mg2+, Pd2+, Zn2+, Al3+, Ag+, Fe3+, Hg2+, Sn2+,
15
Cd2+, Ba2+ and Ca2+ were also tested, and almost no changes in the fluorescence intensity ratio (I458 nm/I562 nm) of P-OH were observed. To test practical applicability of our fluorescent probe for NH2NH2, we further investigated the fluorescence response of P-OH toward NH2NH2 in the presence of other species (amine-containing species, reductive species and metal ions). As shown in Fig. 6B and Fig. S13, other coexisting species had no apparent interference with the detection of NH2NH2. All the results indicated that P-OH has high selectivity for NH2NH2 and is promising for precise NH2NH2 detection in a complex environment.
Inset Fig. 6 here
3.7. Determination of NH2NH2 in Water Samples NH2NH2 is highly toxic and can cause serious environmental contamination and health problems during its manufacture, transport, use, and disposal procedures. Therefore, determination of NH2NH2 in real water samples is of great significance. The proposed method was applied to monitoring NH2NH2 in water samples from the tap water in our laboratory, Xiangjiang River and Taozi Lake in Changsha city. As shown in Table 1, recoveries of NH2NH2 were in the range of 98.2-103.4% for all the spiked samples. These demonstrated that our proposed probe could detect NH2NH2 in real water samples quantitatively.
Inset Table 1 here
16
3.8. Cell imaging Finally, we evaluated the ability of probe P-OH for fluorescent imaging of NH2NH2 in live cells. HeLa cells pre-incubated with P-OH (10 μM) for 30 min showed nearly no fluorescence in the blue channel (Fig. 7B). By contrast, after P-OH-loaded cells were incubated with NH2NH2 (100 μM) for another 30 min, strong fluorescence signals were observed in the blue channel (Fig. 7D). These cell experiments indicated that P-OH was both cell-permeable and capable of imaging NH2NH2 in living cells in a turn-on manner.
Inset Fig. 7 here
Moreover, the comparison of present probe with some previous reported NH2NH2 fluorescent probes has been listed in Table S1. Our probe shows many salient features including ratiometric detection, low detection limit, high specificity, applicability in physiological pH range, feasibility in aqueous samples and living cells. Therefore, probe P-OH could be used as a promising tool for the determination of NH2NH2 in environmental and biological samples.
4. Conclusion In summary, a novel fluorescent probe P-OH for NH2NH2 was synthesized via the NH2NH2-mediated addition-cyclization of α, β-unsaturated carbonyl moiety. P-OH can serve not only as a colorimetric probe but also as a ratiometic fluorescent probe for NH2NH2 with a low detection limit of 2.36 ppb at physiological pH. Notably, this
17
probe showed high selectivity towards NH2NH2 over other amines as well as other reductive species, which is more advantageous than levulinate-type, aldehyde-type and arylidenemalononitrile-type fluorescent probes for NH2NH2. Moreover, the probe possessed desirable cell permeability, and could successfully be applied for detection of NH2NH2 both in water samples and living cells. In addition, the proposed sensing mechanism in this work should be meaningful to the development of other new fluorescence probes for NH2NH2.
Acknowledgments This work was supported by the National Natural Science Foundation of China, China (21375037, 21675051), Hunan Provincial Innovation Foundation for Postgraduate (CX2017B226), Hunan Provincial Science and Technology Department of Hunan Province (NO: 2014FJ6034) and Scientific Research Project of Hengyang Normal University (17D05).
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Figures:
Scheme 1. Synthesis routes of control probe P-H (R = H) and probe P-OH (R = OH).
25
Scheme 2. Proposed sensing mechanism of probe P-OH for NH2NH2.
26
Fig. 1. Absorption (A) and fluorescence (B) emission spectra of probe P-OH (10 µM) in DMSO/PBS buffer (9:1, v/v, 10 mM, pH = 8.0) in the absence (black) or presence (red) of NH2NH2 (100 µM). Each spectrum was acquired at 30 min after hydrazine addition. Excitation wavelength = 380 nm. Inset: color changes of the probe P-OH (10 µM) solution after addition of NH2NH2 (100 µM) under room light (A) and under a 365 nm light (B).
27
Fig. 2. Partial 1H NMR spectra of P-OH, P-OH + 0.5 equiv NH2NH2, P-OH + 1.0 equiv NH2NH2, P-OH + 2.0 equiv NH2NH2 in DMSO-d6 (500 MHz).
28
Fig. 3. The effect of pH on the fluorescence intensity ratio (I458 nm/I562 nm) of probe P-OH (10 μM) in the absence or presence of NH2NH2 (100 μM). Excitation wavelength = 380 nm.
29
Fig. 4. (A) Time-dependent changes in the fluorescence spectra of probe P-OH (10 μM) upon reaction with NH2NH2 (100 µM). Excitation wavelength = 380 nm. (B) Time-dependent fluorescence intensity at 562 nm (red) and 458 nm (black) of probe P-OH in the presence of NH2NH2 (100 µM).
30
Fig. 5. (A) Fluorescence spectra of P-OH (10 µM) in the presence of various concentrations of NH2NH2 (0-250 µM) in DMSO/PBS buffer (9:1, v/v, 10 mM, pH = 8). Excitation wavelength = 380 nm. (B) The fluorescence intensity ratio (I458 nm/I562 nm) of P-OH (10 µM) changes as concentrations of NH2NH2 (0-250 µM). Inset: a linear correlation between the fluorescence intensity ratio (I458 nm/I562 nm)
and concentrations of NH2NH2 (0-100 µM).
31
Fig. 6. (A) Fluorescence spectra of probe P-OH (10 μM) in the presence of various species (100 μM for ammonia, n-butylamine, hydroxylamine, triethylamine, diaethylamine, aniline, Cys, Hcy, GSH, HS-, HSO3- and NH2NH2). (B) The fluorescence intensity ratio (I458 nm/I562 nm) of probe P-OH (10 µM) upon addition of various substances (black bars) and then addition of NH2NH2 (red bars). The concentration of NH2NH2 and other interfering species are 100 μM. (1) None, (2) ammonia, (3) n-butylamine, (4) hydroxylamine, (5) triethylamine, (6) diaethylamine, (7) aniline, (8) Cys, (9) Hcy, (10) GSH, (11) HS-, (12) HSO3-. Excitation wavelength = 380 nm.
32
Fig. 7. Fluorescence images of HeLa cells. (A) Bright-field image, and (B) fluorescence image in blue channel after HeLa cells were incubated with probe P-OH (10 μM) for 30 min. (C) Bright-field image, and (D) fluorescence image in blue channel after HeLa cells being pre-treated with P-OH (10 μM) for 30 min, and then incubated with NH2NH2 (100 μM) for another 30 min.
33
Table 1. The measurement results of NH2NH2 in water samples
Water samples
NH2NH2 added (M)
Found (M)
Recovery (%)
2.5
2.53 ± 0.05
101.2
5.0
4.91 ± 0.10
98.2
2.5
2.56 ± 0.07
102.4
5.0
5.15 ± 0.12
103.0
2.5
2.48 ± 0.11
99.2
5.0
5.17 ± 0.14
103.4
Tap water
Xiangjiang River water
Taozi Lake
34
Highlights:
A
novel
fluorescent
probe
for
NH2NH2
was
developed
based
on
NH2NH2-triggered intramolecular addition-cyclization cascade.
It showed high sensitivity and selectivity toward NH2NH2 with obvious colorimetric and ratiometic fluorescence responses.
It could detect NH2NH2 in vitro and in vivo without interference from other amines and reductive species.
35
Graphical Abstract
36