A coumarin-fused ‘off-on’ fluorescent probe for highly selective detection of hydrazine

Article abstract of DOI:10.1016/j.saa.2020.118075

Hydrazine is a kind of widely used industrial raw material and a toxic biochemical reagent. Due to its toxic to organisms, hydrazine has been classified to be a hazardous environmental pollutant. It is urgent to develop fluorescent probe tools for selective sensitivity detection of hydrazine in the environment and the body. We developed here a new coumarin-based fluorescent probe for hydrazine detection. The probe can selectively detect hydrazine over other environmental and endogenous interfering analytes with a large off-on fluorescence response. The detection limit is 8.55 ppb, which is well below the allowed threshold limit value. The sensing mechanism is hydrazine-induced pyrazole ring formation, which is confirmed by HRMS and DFT calculation methods. Additionally, the probe could also be applied for hydrazine imaging in living HeLa cells.

Full text of DOI:10.1016/j.saa.2020.118075

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 230 (2020) 118075
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Short Communication
A coumarin-fused ‘off-on’ fluorescent probe for highly selective detection
of hydrazine
Mei Wang a, Xiaoli Wang a, Xueyan Li a,b, Ziqi Yang a, Zhenbo Guo a,b, Jiangyan Zhang a,c
,
a,b,c,
Jingjun Ma d, , Chao Wei
⁎⁎
⁎
a
College of Chemistry and Environmental Science, Hebei University, Baoding 071002, China
b
c
Key Laboratory of Chemical Biology of Hebei Province, Hebei University, Baoding 071002, China
Key Laboratory of Medicinal Chemistry and Molecular Diagnosis of Ministry of Education, Hebei University, Baoding 071002, China
College of Science and Technology, Hebei Agricultural University, Huanghua 061100, China
d
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 13 August 2019
Received in revised form 10 January 2020
Accepted 15 January 2020
Available online 16 January 2020
Hydrazine is a kind of widely used industrial raw material and a toxic biochemical reagent. Due to its toxic to or-
ganisms, hydrazine has been classified to be a hazardous environmental pollutant. It is urgent to develop fluores-
cent probe tools for selective sensitivity detection of hydrazine in the environment and the body. We developed
here a new coumarin-based fluorescent probe for hydrazine detection. The probe can selectively detect hydra-
zine over other environmental and endogenous interfering analytes with a large off-on fluorescence response.
The detection limit is 8.55 ppb, which is well below the allowed threshold limit value. The sensing mechanism
is hydrazine-induced pyrazole ring formation, which is confirmed by HRMS and DFT calculation methods. Addi-
tionally, the probe could also be applied for hydrazine imaging in living HeLa cells.
Keywords:
Hydrazine
Highly selective
Large off-on response
Fluorescent probe
Cellular imaging
© 2020 Published by Elsevier B.V.
1. Introduction
and chemiluminescence [8]. However, comparing with traditional
methods, fluorescence analysis has been widely applied for the detec-
Hydrazine, a colorless liquid inorganic compound, is highly reduc-
tive and basic, which is used as corrosion inhibitors in heating system
[1]. As an important industrial raw material, hydrazine is widely applied
to manufacture pharmaceuticals, pesticides, and chemical dyes, etc.
Meanwhile, it is also used as a high-energy fuel for rocket-propulsion
and missile systems. However, hydrazine is famous for its high toxicity,
which can enter the body through breathing, osmosis, and other ways,
and further damage the lungs, liver, kidneys and nervous system. It
has been classified as a probable human carcinogen by the U.S. Environ-
mental Protection Agency (EPA), and the minimum threshold limit
value is set as 10 ppb [2]. Although hydrazine is not endogenously pro-
duced, some drugs can be metabolized to hydrazine in the human body
and further endangers health. Therefore, the selective recognition and
sensitive detection of trace hydrazine in environment and biological
systems have been attracting increasing attention.
tion of hydrazine because of its excellent selectivity, high sensitivity, es-
pecially non-invasive damage to biological samples [9–10]. To date,
Based on its basicity and nucleophilicity, many sensing strategies for hy-
drazine have been found to design fluorescent probes, mainly including
the hydrazinolysis of phenol acetate [11–19], the nucleophilic addition-
intramolecular cyclization-elimination cascade reaction of keto ester
[20–24] or halogen-ester [25–29], the Gabriel amine synthesis mecha-
nism [30–34], hydrazone formation [35–45], pyrazole forming
[46–50], and others [51–59]. High selectivity is the major challenge for
hydrazine probes. However, the ester-based fluorescent probes might
suffer from chemically unstable, and hydrazone formation-based
probes might be interfered by other amines and hydrazine analogs. In
contrast, the pyrazole forming and the Gabriel amine synthesis strategy
would be the better choice for construction hydrazine fluorescent probe
with high selectivity.
Conventional detection techniques for hydrazine mainly include
titrimetry [3], electrochemical method [4–5], chromatography [6–7],
Herein, we reported a coumarin-fused hydrazine fluorescent probe
1 using the pyrazole forming strategy. Coumarin was used as
fluorophore because of its high fluorescence quantum yield and excel-
lent light stability. The ynone group in the 3-position of coumarin can
not only quench fluorescence by photo-induced electron transfer
(PET) effect but also serve as a reactive unit of hydrazine. The selectivity
and sensitivity of the probe were examined in the PBS buffer solution,
the sensing mechanism was confirmed by HRMS and DFT calculation
⁎
Correspondence to: C. Wei, College of Chemistry and Environmental Science, Hebei
University, Baoding 071002, China.
⁎⁎ Correspondence to: J. Ma, College of Science and Technology, Hebei Agricultural
University, Huanghua 061100, China.
E-mail addresses: mjjwjpmartin@sina.com (J. Ma), weichao@hbu.edu.cn (C. Wei).
https://doi.org/10.1016/j.saa.2020.118075
1386-1425/© 2020 Published by Elsevier B.V.

2
M. Wang et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 230 (2020) 118075
Scheme 1. The sensing mechanism of probe 1 with hydrazine.
methods, and the bioimaging of hydrazine in HeLa cells using probe 1
was finally evaluated.
chromatography to give yellow solid (250 mg, yield: 80%). 1H NMR
(600 MHz, CDCl3) δ 7.74 (s, 1H), 7.20 (d, J = 12.0 Hz, 1H), 6.55 (dd,
J = 9.0, 2.4 Hz, 1H), 6.45 (d, J = 1.8 Hz, 1H), 3.41 (dd, J = 13.8,
6.6 Hz, 4H), 1.20 (t, J = 7.2 Hz, 6H), 0.25 (s, 9H).
2. Experimental section
2.1. Materials and instruments
2.2.2. Synthesis of 7-diethylamino-3-ethynylcoumarin (4)
Compound 3 (313 mg, 1.00 mmol) and K2CO3 (415 mg, 3.00 mmol)
were dissolved in CH3OH (15 mL). The mixture was stirred at room
temperature overnight. After reaction completion, the mixture was di-
luted with CH2Cl2, washed with water and saturated NaCl solutions.
The organic layer was dried, evaporated, and the residue was purified
with column chromatography to give yellow solid (205 mg, yield:
85%). 1H NMR (600 MHz, CDCl3) δ 7.73 (s, 1H), 7.19 (d, J = 9.0 Hz,
1H), 6.55 (d, J = 8.4 Hz, 1H), 6.41 (s, 1H), 3.40 (dd, J = 14.4, 7.4 Hz,
4H), 3.22 (s, 1H), 1.19 (t, J = 7.2 Hz, 6H).
All reagents including salicylaldehyde, trimethylsilylacetylene, PdCl2
(PPh3)2, CuI, and benzoyl bromide were purchased from J&K Scientific
Co. Ltd. with the purity N95%. All the solvents were produced by profes-
sional suppliers. All reagents and solvents were used directly without
further purification.
NMR spectra were recorded on Advance 600 MHz (Bruker). High-
Resolution Mass Spectrometry (HRMS) spectra were determined
using 7.0 T FTICR-MS (Varian). UV–vis spectra and fluorescence spectra
were obtained using Cary 5000 Bio (Agilent) and F-7000 spectropho-
tometer (Hitachi), respectively. Bioimaging of cells was collected on a
confocal microscope (Zeiss LSM 880).
2.2.3. Synthesis of 7-diethylamino-3-(3-oxo-3-phenyl prop-1-yn-1-yl)
coumarin (1)
The theoretical calculations were performed based on DFT (density
functional theory) at the M062X/6-311d level in Gaussian 09 software.
The solvent effect on molecular geometries was included by means of
the polarizable continuum model (PCM).
Compound 4 (241 mg, 1.00 mmol), PdCl2(PPh3)2 (35 mg,
0.05 mmol, 5.0 mol%), CuI (9.5 mg, 0.05 mmol, 5.0 mol%) and 1-
bromo-4-nitrobenzene (222 mg, 1.20 mmol) were dissolved in anhy-
drous DMF (10 mL), and then anhydrous TEA (5 mL) was added to
the system. The mixture was stirred at 80 °C for 5 h. After cooling to
room temperature, the solution was diluted with CH2Cl2, washed with
saturated NH4Cl solutions, water, and saturated NaCl solutions. The or-
ganic layer was dried, evaporated, and the residue was purified with
column chromatography to give a bright yellow solid (187 mg, yield:
54%). 1H NMR (600 MHz, CDCl3) δ 8.32 (d, J = 7.2 Hz, 2H), 7.97 (s,
1H), 7.60 (t, J = 7.2 Hz, 1H), 7.51 (t, J = 7.2 Hz, 2H), 7.27 (t, J =
9.0 Hz, 1H), 6.60 (d, J = 9.0 Hz, 1H), 6.47 (s, 1H), 3.44 (dd, J = 14.4,
7.2 Hz, 4H), 1.23 (t, J = 7.2 Hz, 6H). 13C NMR (151 MHz, CDCl3) δ
177.64, 160.38, 157.35, 152.49, 149.94, 136.94, 133.89, 130.07, 129.78,
128.60, 109.71, 108.36, 100.83, 97.26, 91.34, 89.85, 45.10, 12.43. HRMS
2.2. Synthesis
2.2.1. Synthesis of 7-diethylamino-3-(trimethylsilyl)ethynylcoumarin (3)
Compound 2 (296 mg, 1.00 mmol) was dissolved in DMF (10 mL).
PdCl2(PPh3)2 (35 mg, 0.05 mmol, 5.0 mol%), CuI (9.5 mg, 0.05 mmol,
5.0 mol%), and (trimethylsilyl)acetylene (424 μL, 3.00 mmol) were
added. The mixture was stirred at 60 °C for 5 h. After cooling to room
temperature, the solution was diluted with CH2Cl2, washed with satu-
rated NH4Cl solutions, water and saturated NaCl solutions. The organic
layer was dried, evaporated, and the residue was purified with column
Scheme 2. Synthetic route of probe 1.

M. Wang et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 230 (2020) 118075
3
0.6
0.5
0.4
0.3
0.2
0.1
0.0
probe
probe
1
a)
b)
1
+ Hydrazine
2000
1500
1000
500
0
0 equiv
N2H4
10 equiv
400
450
500
550
450
500
550
600
650
700
Wavelength (nm)
Wavelength (nm)
Fig. 1. (a) UV–vis spectra of probe 1 (20 μM) to hydrazine (0, 3, 5, 7, 10 Equiv) and fluorescence emission of probe 1 (20 μM) to hydrazine (10 Equiv) (b) in PBS (20 mM, pH 7.4, 30%
CH3CN).
(ESI): [M + Na]+, calcd C22H19NNaO3: m/z = 368.1263, found
368.1260.
modifiability, and excellent biocompatibility [60]. Meanwhile, the PET
effect is a general strategy for constructing fluorescent probes. In this
work, the ynone structure was introduced into the 3-position of the 7-
diethylamino-coumarin. The carbonyl group was expected to quench
the fluorescence of coumarin by PET effect, and at the same time was
served as the recognizing group. After reaction with hydrazine, the
pyrazole ring formed could not quench the fluorescence, and the intra-
molecular hydrogen bonding formed could be contributing to produc-
ing a large “turn-on” fluorescence response (Scheme 1).
2.3. MTT assay
HeLa cells were cultured in DMEM supplemented with 10% fetal calf
serum at 37 °C in 5% CO2. Cells were seeded in a 96-well plate. After
overnight culture, cells were incubated with each concentration of
probe for 12 h. To identify cell viability, 0.5 mg/mL of MTT (Sigma)
media was added to cells for 4 h and the produced formazan was dis-
solved in 150 μL of dimethyl sulfoxide (DMSO) and read at OD
490 nm with a Spectramax microwell plate reader.
As shown in Scheme 2, the synthetic route was referred to as the re-
ported literature [61]. Compound
2
was coupled with
trimethylsilylacetylene to provide compound 3, which was transformed
into compound 4 by removing trimethylsilyl. The alkynyl group was fur-
ther coupled with benzoyl bromide to provide probe 1. The structure of
all compounds was confirmed by 1H NMR, 13C NMR spectra and HRMS.
The detailed synthesis of compounds 2 was shown in the Supporting
Information.
2.4. Cell imaging
The cell imaging experiments were divided into three groups. First
group cells were only treated with probe 1 (10 μM) for 60 min. The
next two groups' cells were pro-incubated N2H4 (50 μM or 150 μM)
for 20 min, washed with PBS three times to remove excess hydrazine,
and then incubated with probe 1 (10 μM) for 60 min. After washing as
descript above, cells were used for imaging immediately. The cells
were imaged with the cyan channel (475–550 nm, excitation at
405 nm).
3.2. Spectra response of probe 1 toward hydrazine
With probe 1 in hand, we first studied the UV–vis spectra and emis-
sion spectra of probe 1. As shown in Fig. 1a, the probe solution showed
the maximum absorption peak at 464 nm. With the addition of hydra-
zine, the intensity of the absorption peak at 464 nm decreased, and a
new absorption band appeared at 415 nm appeared. The isosbestic
point at 430 nm suggested the formation of a new product. In the fol-
lowing experiment, we chose 430 nm as the excitation wavelength.
Probe 1 exhibited weak fluorescence emission (quantum yield Φ =
0.001) in PBS buffer solution (20 mM, pH 7.4, 30% CH3CN), while after
the addition of hydrazine, the obvious fluorescence enhancement at
508 nm could be observed (Φ = 0.093) (Fig. 1b).
3. Results and discussion
3.1. Design and synthesis
The coumarin scaffold has been widely applied in the design of
small-molecule fluorescent chemosensors because of its high fluores-
cence quantum yield, strong light stability, good structural
Fig. 2. Fluorescence responses of probe 1 (20 μM) to hydrazine (10 Equiv) and other species (20 Equiv). Each spectrum was recorded after 12 h of reaction in PBS (20 mM, pH 7.4, 30%
CH3CN) at room temperature. (1. Probe 1 only, 2. K+, 3. Ag+, 4. Ca2+, 5. Cu2+, 6. Mg2+, 7. Zn2+, 8. Pb2+, 9. Ni2+, 10. Cd2+, 11. Mn2+, 12. Hg2+, 13. Fe2+, 14. Fe3+, 15. Al3+, 16. Co3+
,
17. NH4+, 18. Cl−, 19. Br−, 20. I−, 21. NO−3 , 22. NO2−, 23. ClO−4 , 24. AcO−, 25. t-BuOO−, 26. N−3 , 27. HSO3−, 28. SO24−, 29. CO23−, 30. SO32−, 31. PO34−, 32. Vc, 33. H2O2, 34. HClO, 35. NO, 36.
H2S, 37. Cys, 38. Hcy, 39. GSH, 40. ammonia, 41. hydroxylamine, 42. aniline, 43. methylamine, 44. triethylamine, 45. ethylenediamine, 46. urea, 47. thiourea, 48. lysine, 49.
hydrazinobenzene, 50. hydrazine).

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M. Wang et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 230 (2020) 118075
Fig. 3. (a) The titration experiments of probe 1 (20 μM) to different concentrations of hydrazine (0–350 μM). (Each spectrum was recorded after 12 h of reaction in PBS (20 mM, pH 7.4, 30%
CH3CN) at room temperature.) (b) The linear relationship between the fluorescence intensity of probe 1 at 480 nm and the concentration of hydrazine (0–350 μM). (c) The changes in the
fluorescence intensity at 508 nm observed for probe 1 as a function of hydrazine concentration (0–30 μM). (d) The fluorescence intensity of probe 1 at 508 nm during different pH values in
presence and absence of hydrazine (200 μM).
The selectivity is the crucial property of the fluorescent probe. We
then examined the fluorescence response of probe 1 toward a range of
environmental and biological interfering compounds. These related
and found a broad working pH range from 6.0 to 10.0 for the application
of the probe (Fig. 3d).
compounds conclude cations (K+, Ag+, Ca2+, Cu2+, Mg2+, Zn2+, Pb2+
,
,
,
3.3. Mechanism of probe 1 toward hydrazine
Ni2+, Cd2+, Mn2+, Hg2+, Fe2+, Fe3+, Al3+, Co3+, NH4+), anions (Cl−
Br−, I−, NO−3 , NO2−, ClO4−, AcO−, t-BuOO−, N−3 , HSO3−, SO42−, CO32−
To further get insight into the relationship between the optical re-
sponse of probe 1 to hydrazine with the structure, we carried out DFT
calculations using a b3lyp/6-311d method [62]. As shown in Fig. 4, for
probe 1, the level of the lowest unoccupied molecular orbital (LUMO)
of coumarin (−0.66 eV) is lower than that of ynone structure
(−1.30 eV), thus coumarin served as the electron acceptor, and its fluo-
rescence could be quenched by PET effect. After reaction with hydra-
zine, the level of LUMO of coumarin (−0.66 eV) is higher than that of
pyrazole structure (−0.16 eV), PET effect disappeared. We also tested
the HRMS spectra of the product of the probe reaction with hydrazine.
A peak at m/z = 360.1710 (M + H+) also suggested that hydrazine in-
duced pyrazole structure formation (Scheme 1).
SO23−, PO34−), ascorbic acid (Vc), endogenous active species (H2O2,
HClO, NO, H2S, GSH, Cys, Hcy), amine compounds (ammonia, hydroxyl-
amine, aniline, methylamine, triethylamine, ethylenediamine, urea,
thiourea, lysine) and hydrazine analogues (hydrazinobenzene). As
shown in Figs. 2, 20 equivalents of interfering substances could not
cause obvious fluorescence changes except Cys and ethylenediamine.
On the contrary, 10 equivalents of hydrazine could induce drastic emis-
sion changes. These results suggested that probe 1 can detect hydrazine
with high selectivity.
We further performed the titration experiments to evaluate the sen-
sitivity of the probe. As shown in Fig. 3a, with the increase of hydrazine
concentration, the emission gradually enhanced in intensity. The fluo-
rescence intensity increased about 600 times in the presence of 17.5
equivalent of hydrazine, and even in the presence of 0.5 equivalent hy-
drazine, the fluorescence intensity could be enhanced 10 times. By fur-
ther data analysis, the fluorescence intensity at 508 nm is linearly
dependent (r = 0.9950) with the concentration of hydrazine between
0 and 350 μM (Fig. 3b). The detection limit is calculated to be 267 nM
(8.55 ppb) according to the 3σ/k method, which was lower than the
EPA accepted the minimum threshold limit value (Fig. 3c). These results
demonstrated that probe 1 is highly sensitivity to hydrazine. We also
evaluated the suitable working pH range for the application of probe 1
3.4. Fluorescence microscopy imaging
Encouraged by the above results, we then sought to examine
whether probe 1 can detect hydrazine in living cells. The MTT assay
was first carried out to evaluate the cytotoxicity. The results suggest
that the probe 1 has no obvious toxicity to the living cells (Fig. S1).
Next, HeLa cells were treated with the probe in the absence or presence
of hydrazine and then fluorescence imaging. As shown in Fig. 5, the cells
treated with only probe 1 showed no fluorescence, while cells pre-
treated with hydrazine and then incubated with probe 1 displayed

M. Wang et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 230 (2020) 118075
5
Fig. 4. HOMO-LUMO energy levels and the interfacial plots of the molecular orbitals for probe 1 and 1-N2H4. The orbitals of 1 and 1-N2H4 are calculated at the DFT level using a b3lyp/6-
311d method.
obvious green fluorescence. Moreover, the higher the concentration of
hydrazine pre-treated, the stronger the fluorescence emerged (Fig. 5b
and c). The above results indicated that probe 1 can be used for
bioimaging hydrazine in living cells.
shown in Table 1, the concentration of hydrazine detected by probe 1
showed a good match with the concentration of hydrazine added. The
recovery values are moreover 90%, which implied probe 1 is capable
of quantitative determination of hydrazine in real water samples.
3.5. Hydrazine detection in real water samples
4. Conclusions
To evaluate the efficacy of probe 1 in real samples, the probe was
In summary, we developed here a coumarin-fused fluorescence
used to monitoring hydrazine in spring water and river water. As
probe for the detection of hydrazine. The coumarin moiety with high
Fig. 5. Confocal fluorescence imaging of HeLa cells. Cells were only treated with probe 1 (5 μM) for 60 min (a). Cells were first treated with hydrazine (50 μM, b) or (150 μM, c) for 20 min,
then washed with DMEM, and treated with probe 1 (5 μM) for 60 min. Excitation 405 nm, emission 475–550 nm, and scale bar 20 μm.

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Spring water
River water
2.5
10
50
2.5
10
50
2.29
10.7
45.1
2.51
9.1
0.12
91.6
107
90.2
100.4
91
0.32
2.32
0.08
0.51
1.19
53.3
106.6
fluorescence quantum yield was served as the fluorophore, and the
ynone part was chosen as hydrazine reactive group. The probe showed
high selectivity (fluorescence off-on response was approx. 600 fold) and
sensitivity (the detection limit was 267 nM). The sensing mechanism
was confirmed by DFT calculation and HRMS spectra. The probe can
be successfully used to image hydrazine in cells and detect hydrazine
in real water samples. We further expect that the probe could be applied
in tissue imaging.
Declaration of competing interest
We declare that we do not have any commercial or associative inter-
est that represents a conflict of interest in connection with the work
submitted.
Acknowledgments
This work was supported by the National Natural Science Founda-
tion of China (21605034), the Natural Science Foundation of Hebei
Province (B2019201918, B2018201234), the Foundation of Hebei Edu-
cation Department (QN2017015), and Science Technology Research
and Development Guidance Program Project of Baoding City (18ZF315).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.saa.2020.118075.
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