Turn on fluorescent detection of hydrazine with a 1,8-naphthalimde derivative

Article abstract of DOI:10.1016/j.dyepig.2017.08.010

It is crucial to develop highly sensitive and selective probes toward hydrazine because it is a class of highly toxic and pollutant compound. Herein, using fracture of carbon carbon double bond and dissociation of amide by hydrazine, a novel off-on fluorescent probe was developed for hydrazine. The probe can quantitatively detect hydrazine in concentration range from 0 to 20 μM with the LOD of 140 nM. Further, it displayed excellent selectivity and anti-interference ability over many neutral molecules, metal ions, anions, and biological species. The ability to target lysosome and the response of hydrazine to this probe in a living cell was successfully tracked via fluorescence imaging.

Full text of DOI:10.1016/j.dyepig.2017.08.010

Accepted Manuscript
Turn on fluorescent detection of hydrazine with a 1,8-naphthalimde derivative
Bingjie Shi, Yangyang He, Panpan Zhang, Yulong Wang, Mingming Yu, Hongyan
Zhang, Liuhe Wei, Zhanxian Li
PII:
S0143-7208(17)31185-3
10.1016/j.dyepig.2017.08.010
DYPI 6172
DOI:
Reference:
To appear in: Dyes and Pigments
Received Date: 22 May 2017
Revised Date: 24 July 2017
Accepted Date: 9 August 2017
Please cite this article as: Shi B, He Y, Zhang P, Wang Y, Yu M, Zhang H, Wei L, Li Z, Turn on
fluorescent detection of hydrazine with a 1,8-naphthalimde derivative, Dyes and Pigments (2017), doi:
10.1016/j.dyepig.2017.08.010.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Turn on fluorescent detection of hydrazine with a 1,8-naphthalimde
derivative
Bingjie Shia, Yangyang Hea, Panpan Zhangb, Yulong Wanga, Mingming Yu*a, Hongyan
Zhang*c, Liuhe Weia, Zhanxian Li*a
* Corresponding author.
a. College of Chemistry and Molecular Engineering, Zhengzhou University, Zhengzhou,
450001, China.
E-mail address: yumm@zzu.edu.cn (M. Yu), lizx@zzu.edu.cn (Z. Li).
Tel: +(86)371-67781205
Fax: +(86)371-67781205
b. Institute of Functional Nano & Soft Materials (FUNSOM), Collaborative Innovation
Center of Suzhou Nano Science and Technology, Jiangsu Key Laboratory for
Carbon-Based Functional Materials & Devices, Soochow University, Suzhou 215123,
China
c. Key Laboratory of Photochemical Conversion and Optoelectronic Materials, Technical
Institute of Physics and Chemistry, Chinese Academy of Sciences, Beijing, 100190,
China
E-mail address: zhanghongyan@mail.ipc.ac.cn (H. Zhang).

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Abstract
It is crucial to develop highly sensitive and selective probes toward hydrazine because it
is a class of highly toxic and pollutant compound. Herein, using fracture of carbon carbon
double bond and dissociation of amide by hydrazine, a novel off-on fluorescent probe was
developed for hydrazine. The probe can quantitatively detect hydrazine in concentration
range from 0 to 20 µM with the LOD of 140 nM. Further, it displayed excellent
selectivity and anti-interference ability over many neutral molecules, metal ions, anions,
and biological species. The ability to target lysosome and the response of hydrazine to
this probe in a living cell was successfully tracked via fluorescence imaging.
Keywords: Fluorescent probe; Off-on; 1,8-Naphthalimde; Hydrazine; Fluorescence
imaging.

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1. Introduction
Hydrazine is widely used not only in the pharmaceutical, chemical and agricultural
industries, but also in rocket-propulsion and missile systems [1–4]. On the other hand, as
a class of highly toxic and pollutant compound, hydrazine can potentially lead to serious
environmental contamination during its manufacture, use, transport and disposal. In
addition, as a neurotoxin, hydrazine has several mutagenic effects, which can cause
damage to the liver, lungs, kidneys and human central nervous system [5–9]. Therefore, it
is urgently necessary to develop an efficient and simple method for determining hydrazine
level in both environmental and biological science.
Some approaches such as chromatography-mass spectrometry [10−12], titration [13]
and electrochemical methods [14−18] have been reported to measure hydrazine, but their
shortcomings such as complicated equipment, sample handling and professional operating
will limit their application. Therefore, it is difficult to detect trace hydrazine in situ, with
short time and low cost in room temperature. On the other hand, fluorescence-based
method has been widely used to detect various analytes such as metal ions, anions and
biomolecules due to its simplicity, high sensitivity, rapid response, and capacity of
real-time and in situ monitoring of the dynamic biological processes in living cells
[19–29]. However, available fluorescent probes for hydrazine are still very limited [30–40].
Hence, it is needed to develop fluorescent probes toward hydrazine.
In this paper, using hydrazine-induced fracture of C=C and amide bonds, a highly
sensitive fluorescent off-on probe 1 was developed (Scheme 1), and the application of
which for selective detection and imaging of hydrazine in living cells was successfully
demonstrated. The probe exhibited a high sensitivity (the LOD is 140 nM), a wide linear
response range from hydrazine concentration of 0-20 µM, excellent selectivity and

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anti-interference ability against other various species.
2. Experimental
2.1. Methods and materials
All commercial grade chemicals and solvents were purchased and used without further
purification.
Mass spectra were obtained on high resolution mass spectrometer (IonSpec4.7 Tesla
1
13
FTMS-MALDI/DHB). H and C NMR spectra were recorded on a Bruker 400 NMR
spectrometer. Chemical shifts are reported in parts per million using tetramethylsilane
(TMS) as the internal standard.
2.2. Spectral characterizations
All spectral characterizations were carried out in HPLC-grade solvents at 20°C within
a 10 mm quartz cell. UV-vis absorption spectra were measured with a TU-1901
double-beam UV-vis spectrophotometer. Fluorescence spectra were determined on a
Hitachi F-4600 spectrometer.
2.3. Cell viability assay
The dark toxicity (The survival rate of cells which have been incubated with certain
probe for 1 hour without excitation light.) of the material: the stock solutions were diluted
with fresh medium to various concentrations (5, 10, 15 and 20 µM). The cell medium was
then exchanged for different concentrations of material medium solution. The cells were
then incubated with these solutions at 37 °C in 5% CO2 for 1 h before removing the
material solution and adding fresh medium. Subsequently, the plates were incubated at
37 °C in 5% CO2 for 24 h. The cell medium solutions were exchanged for 100 µL of fresh
medium, followed by the addition of 20 µL of MTT solution to each well. The culture

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plates were then incubated at 37 °C in 5% CO2 for 4 h. The culture medium was
discarded, and 100 µL of dimethylsulfoxide was added. The absorbance of an untreated
cell population under the same experimental conditions was used as the reference point to
establish 100% cell viability. The ratiometric confocal fluorescence images are recorded
by a Nikon A1 RSiMP Confocal Microscopy with a 60 ×WI objective (NA 1.27) and 457
nm diode laser.
2.4. Cell incubation and imaging
The HeLa cells were incubated with the synthesized probe at 37°C for fluorescence
imaging with Nikon A1Rmp Confocal microscopy. The fluorescent images were recorded
at green with 500-550 nm filters after the cells incubated with probe (10−5 M) for 30 min.
After adding 1.0 × 10−4 M hydrazine and incubation for 1 h, the fluorescent images were
taken at the same situation. The fluorescence excitation was performed at 457 nm using a
diode laser with pin hole and 60x oil objective lens.
2.5. Synthesis of compound 7
(Insert: Scheme 1)
4-Diethylaminosalicylaldehyde (0.4827 g, 2.5 mmol), diethylmalonate (750 µL, 5
mmol) and piperidine (250 µL) were combined in absolute ethanol (15 mL) and stirred
for 6 hours under refluxing conditions. Ethanol was evaporated under reduced pressure,
and then concentrated HCl (10 mL) and glacial acetic acid (10 mL) were added to
hydrolyze the reaction with stirring for another 6 hours. The solution was cooled to room
temperature and poured into 15 mL ice water. NaOH solution (40%) was added dropwise
to modulate pH of the solution to 5, and a pale precipitate was formed immediately. After
stirring for 30 min, the mixture was filtered, washed with water, and then the final
product was purified by silica gel column chromatography using DCM/ethanol (1/100 v/v)

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as eluent with a yield of 48% (Scheme 1). 1H NMR (400 MHz, CDCl3-d6, TMS):δH 7.54
(d, 1H), 7.27 (t, 1H), 6.50 (d, 1H), 6.50 (m, 1H), 6.03 (d, 1H), 3.42 (m, 4H), 1.24 (t,
6H).13C NMR (100 MHz, CDCl3-d6):δC 162.28, 156.74, 150.66, 143.70, 128.77, 109.19,
108.67, 108.29, 97.53, 44.82, 12.44.
2.6. Synthesis of compound 2
o
Fresh distilled DMF (1 mL) was added dropwise to POCl3 (1 mL) at 20-50 C within
N2 atmosphere and stirred for 30 minutes to yield a red solution. This solution was
combined with a portion of 7 (0.2543 g, 1.2 mmol, dissolved in 2 mL DMF) to yield a
scarlet suspension. The mixture was stirred at 60 oC for 12 hours and then poured into 15
mL of ice water. NaOH solution (20%) was added to adjust the pH of the mixture to yield
a large amount of precipitate. The crude product was filtered, thoroughly washed with
water, dried and then the final product was purified by silica gel column chromatography
1
using DCM as eluent with a yield of 58.5%. H NMR (400 MHz, CDCl3-d6, TMS): δH
10.13 (s, 1H), 8.26 (s, 1H), 7.43 (d, 1H), 6.67 (m, 1H), 6.51 (d, 1H), 3.50 (m, 4H), 1.27 (t,
6H). 13C NMR (100 MHz, CDCl3-d6): δC 187.92, 161.89, 158.93, 153.47, 145.37, 132.52,
110.20, 108.23, 97.15, 45.29, 29.70,.12.46.
2.7. Synthesis of compound 5
4-bromo-1,8-naphthalic
anhydride
(1.0002
g,
3.6
mmol)
and
4-(2-aminoethyl)-morpholine (945 µL, 7.2 mmol) were dissolved in 40 mL ethanol, and
the solution was refluxed for 2 hours. After cooling to room temperature, the yellowish
sediments were collected by filtration and then dried overnight at room temperature in a
vacuum oven to obtain 5 (1.3611 g, 3.5 mmol, yield: 97.2%). 1H NMR (400 MHz,
DMSO-d6, TMS): δH 8.85 (m, 1H), 8.57 (m, 1H), 8.41 (m, 1H), 8.05 (m, 1H), 7.86 (m,
1H), 4.37 (t, 2H), 3.70 (t, 4H), 2.72 (t, 2H), 2.62 (s, 4H). 13C NMR (100 MHz, DMSO-d6):

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δC 163.62, 163.60, 133.31, 132.04, 131.23, 131.12, 130.64, 130.32, 129.03, 128.10,
123.05, 122.18, 67.02, 56.09, 53.81, 37.30.
2.8. Synthesis of compound 4
A mixture of compound 5 (1.0113 g, 2.6 mmol), (3030 µL, 13 mmol) 80% hydrazine
hydrate and 15 mL 2-Methoxyethanol were refluxed for 8 h. After cooling to room
temperature, some of the 2-Methoxyethanol was evaporated under reduced pressure, and
then massive ether was added. The orange solid product was obtained by filtration and
being washed with ether three times with a yield of 92.3%. HRMS (EI) m/z: C18H20N4O3
[M+H], 341.1535, found, 341.1612.
2.9. Synthesis of compound 3
Compound 4 (0.3403 g, 1 mmol) and ethyl acetoacetate (255 µL, 2mmol) were
dissolved in 16 mL ethanol, and the solution was refluxed for 8 hours. The reaction
solution was then cooled to room temperature. The crude product 3 was purified by silica
gel column chromatography using aceticether as eluent. The yield was 75%. This product
was used without characterization.
2.10. Synthesis of compound 1
Compound 3 (0.1681 g, 0.69 mmol), and compound 2 (0.1681 g, 0.69 mmol) were
mixed in absolute alcohol (10 mL). Triethylamine (50 µL, 1.7 mmol) was added to it and
stirred at room temperature for 30 min. After refluxing for 20 h, the mixture solution was
then allowed to cool to room temperature and stirred for 5 h at room temperature. The
purple solid was filtered and washed with ethanol. The final product was obtained by
vacuum drying at 25 °C for 12 h (0.1823 g，0.28 mmol, 41%). HRMS (EI) m/z:
C36H35N5O6 [M+H], 634.2587; found, 634.2612. Elemental analysis data: C (68.23%), H

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1
(5.57%), N (11.05%); found C (67.96%), H (5.59%), N (11.01%). H NMR (400 MHz,
CDCl3-d6, TMS): δH 10.39 (d, 1H), 8.68 (m, 2H), 8.50 (m, 1H), 8.07 (m, 1H), 7.93 (m,
1H), 7.82 (m, 1H), 7.46 (m, 1H), 6.66 (m, 1H), 6.64 (m, 1H), 4.39 (d, 2H), 3.7 (s, 4H),
3.5 (m, 4H), 2.75(s, 2H), 2.63(s, 4H), 2.45(s, 3H), 1.29(t, 6H). 13C NMR (100 MHz,
CDCl3-d6): δC 164.21, 164.13, 163.73, 161.98, 158,71, 153.90, 152.85, 150.30, 142.30,
139.90, 133.14, 131,62, 131.51, 131.22, 127.10, 126.85, 123.83, 123.05, 121.45, 112.52,
110.49, 109.62, 97.20, 67.09, 56.09, 53.83, 45.45, 37.28.
3. Results and Discussion
3.1. Synthesis of probe 1
The synthesis of the smart probe 1 was accomplished in four steps as shown in Scheme
1 [41–44]. The starting materials 4-bromo-1,8-naphthalic anhydride and morpholine were
reacted in ethanol for 2 hours under 80°C to give compound 5 in a yield of 97.2%. The
product 5 and hydrazine hydrate reacted in ethylene glycol monomethyl ether to give
product 4 in a yield of 92.5%. Compound 4 reacted with ethyl acetoacetate in ethanol for
8 h to give compound 3 in a yield of 75.0%. Probe 1 was obtained by the reaction of
compounds 3 and 2 in a yield of 41%. The detailed synthetic procedures are presented in
the Experimental section. All new compounds were carefully characterized by1H NMR,
13C NMR and HRMS in supporting information.
(Insert: Fig. 1)
3.2. Hydrazine recognition properties of 1
To study the recognition properties of probe 1 toward hydrazine, fluorescence titration
experiments (Figures 1a and 1b) were conducted with 0.1 M hydrazine in aqueous
solution of 1 (the molar extinction coefficient is 7.46 × 104 M-1cm-1, 1.0 × 10-5 M,

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VHEPES:VDMSO = 4:6, pH = 7.40). Upon excitation at 440 nm, the characteristic emission
band of 1,8-naphthalic anhydride centered at 517 nm underwent an increase of about 24
times (the fluorescent quantum yield changed from 1.04% to 10.5%) as the concentration
of hydrazine increased from 0 to 10 equiv. As demonstrated in Fig. 1b, in the
concentration range of 0-20 µM, the fluorescence intensity at 517 nm were in good linear
relationship with hydrazine concentration, implying that hydrazine can be quantitatively
detected in a wide concentration range in fluorescence mode. From the linear calibration
graph with the fluorescence titration experiment (Fig. 1b), the detection limit of probe 1
for hydrazine was figured out to be about 140 nM based on signal-to-noise ratio (S/N) = 3
[45−47], which is much lower than the threshold limit value of hydrazine permitted for
human health (312 nM). These results led us to conclude that probe 1 could be an
effective off-on fluorescent probe for hydrazine.
hydrazine in time-dependent fluorescence spectra of probe 1 1hydrazine is completely
accomplished within 1 h and hydrazine can be detected within 8 min when the
concentration of hydrazineis higher or equal to 8.0 × 10−6 M. Such result further proved
the fast response and high sensitivity of 1 for detection of hydrazine.
3.3. pH range in application of probe 1 toward hydrazine
The pH value of solution was found to be essential to the reaction between probe 1 and
hydrazine. To investigate the pH effect, the fluorescence of 10.0 µM probe 1 in the
absence and presence of 100 µM hydrazine were examined at pH range from 4.0 to 10.0.
As shown in Fig.1d, probe 1 itself is stable and its emission spectra are not changed in a
wide pH range from 4.0 to 10.0. Although there was a significant fluorescence change of
probe 1 in pH ranges from 4.0 to 10.0 upon addition of hydrazine. Probe 1 is able to
detect hydrazine in a relatively wide pH range.

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3.4. Recognition mechanism of probe 1 toward hydrazine
The recognition mechanism was studied by Mass spectrometry. For pure probe 1, a
characteristic peak at m/z = 634.2665 was obtained which corresponds to the specie 1 [M
+ H], whilst after reaction with hydrazine, the peak at 634.2665 disappeared and two new
peaks appeared at m/z = 341.1514 and 242.3710, which are corresponding to compounds
4 (M + H) and 6 (M + H) indicating the decomposition of probe 1 and the formation of
new compounds 4 and 6 (Scheme 1). To further clarify the recognition mechanism, we
have separated one product from the reaction of probe 1 and hydrazine hydrate, which has
the same Rf value as that of compound 6. In addition, we have also measured the
fluorescence quantum yields of probe 1 (1.04%), the reaction solution of probe 1 and
hydrazine hydrate (10.5%), compound 4 (2.51%), and compound 6 (19.4%) (Scheme 1),
indicating that the fluorescence mainly comes from compound 6 upon addition of
hydrazine hydrate into the solution of probe 1. Besides, we have also characterized the
product from the reaction of probe 1 and hydrazine hydrate with HRMS, 1HNMR, and
13CNMR. Characterization data were as follows: HRMS (EI) m/z: C14H15N3O [M+H],
242.1293; found, 242.3710. 1H NMR (400 MHz, CDCl3-d6, TMS): δH 11.82 (s, 1H), 8.47
(s, 1H), 7.12 (s, 1H), 6.27 (t, 2H), 3.42 (q, 4H), 1.23 (t, 6H). 13C NMR (100 MHz,
CDCl3-d6): δC 161.45, 160.91, 151,21, 133.28, 106.95, 103.97, 97.87, 44.57, 29.71, 12.71.
(Insert: Fig. 2)
3.5. The selectivity of 1 forhydrazine
To evaluate the selectivity of probe 1 for hydrazine, various species including neutral
molecules including Et3N, NH3•H2O, C2H4(NH2)2, thiourea, urea, and aniline, metal ions
2−
−
(Na+, Mg2+, Al3+, K+, Ca2+, Cr3+, Mn2+, Zn2+, Pt2+, and Pb2+), anions (HPO4 , H2PO4 ,
3−
−
−
4−
2−
2−
2−
−
2−
2−
−
PO4 , N3 , HSO3 , P2O7 , CO3 , C2O4 , SiO3 , Cl−, F−, NO3 , SO3 , S2O3 , ClO3 , I−,

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2−
SO4 , CH3COO−, and HS−), and nucleophilic biological species (Gly, Gin, GSH, Asp,
Cys, Thr, Ala, Phe, Met, Lys, Leu, Ile, Val, Glu, Ser, Arg, Pro, Hcy and Tyr) were tested.
As shown in Figures S1, S2, S3 and S4 in supporting information and red bars of Figures
2 and 3, only hydrazine induced a significant fluorescence change, whilst, other tested
species did not induce any obvious fluorescence change, indicating the good selectivity of
probe 1 toward hydrazine.
3.6. The anti-disturbance effect of 1 for hydrazine detection
To further assess its utility as ahydrazine-selective fluorescent probe, its fluorescent
response to hydrazinein complicated surroundings as mentioned above was also tested
(figures 2 and 3). The results evidenced that all of the selected species have no
interference in the detection of hydrazine. This result strongly indicated that probe 1
could be an excellent fluorescent probe towards hydrazine with strong anti-interference
ability.
(Insert: Fig. 3)
3.7 Imaging of 1 to hydrazine in live cells
The intrinsic ability of probe 1 to target lysosome was investigated in living Hela cells
after addition of hydrazine (1.0 × 10−4). The cells stained with probe 1 (1.0 × 10−5 M, 30
min, 25°C) were co-stained further with a commercially available lysosome-specific dye
Neutral Red (Lysosome-Tracker) (1 mM, 30 min) in the culture medium. The imaging
results show that the green image for the probe 1 channel obtained upon excitation at 457
nm is almost identical to the red image for the lysosome-Tracker channel obtained upon
excitation at 640 nm (Fig. 4). The overlay between the fluorescence images of probe 1
and lysosome-Tracker discloses a Pearson's correction coefficient of 0.82, suggesting the
ability of probe 1 to target lysosome.

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The cytotoxicity of probe 1 toward HeLa cells was measured using the
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazilium bromide (MTT) assay to evaluate
the potential application of probe 1 in live cell imagining. The cellular viability was
estimated to be greater than 87% after 1 h at the concentration of 10−5 M for probe 1,
suggesting the low cytotoxicity of probe 1 (Fig. 5a). Practical applications of 1 in
imaging live HeLa cells were investigated using confocal fluorescence microscopy with a
457 nm diode laser. The confocal fluorescence microscopy images are shown in Figures
5b and 5c. After incubation of Hela cells with 1 (10−5 M) for 30 min at 37°C, no
fluorescence was detected at green (525±25 nm) channel in the cytoplasm. Following
addition of hydrazine (1.0 × 10−4), an obvious increase at the green channel was observed,
suggesting the reaction of 1 to hydrazine.
(Insert: Fig. 4)
(Insert: Fig. 5)
4. Conclusion
A turn-on fluorescent hydrazine-probe was synthesized and demonstrated. This probe
can not only quantitatively detect hydrazine in wide concentration range, but also exhibits
good selectivity and anti-interference ability over other various species. The good
location ability toward lysosome, the low cytotoxicity and the application for selective
detection of hydrazine in living cells has been successfully demonstrated, which offered a
designing approach for recognizing of hydrazine.
Acknowledgements
We are grateful for the financial supports from National Natural Science Foundation of
China (21601158 and U1504203), special foundation for outstanding young teachers of
Zhengzhou University.

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Figure Captions
Scheme 1 Synthesis of probe 1 and its recognition mechanism toward hydrazine.
Fig. 1. (a) Emission spectra of probe 1 (1.0 × 10-5 M, VHEPES:VDMSO = 4:6, pH = 7.40)
upon titration of hydrazine (0–10.0 equiv to 1) (b) The linearity of emission intensity at
517 nm with the concentration of hydrazine. (c) Kinetic curves of probe 1 (1.0 × 10-5 M,
VHEPES:VDMSO = 4:6, pH = 7.40) at 517 nm with hydrazine at different concentrations [the
concentrations of hydrazine are 0 M (black line), 4.0 × 10−6 M (red line), 8.0 × 10−6 M
(green line), 1.2 × 10−5 M (blue line), and 1.6 × 10−5 M (cyan line)]. (d) Fluorescence
change of 1 (1.0 × 10-5 M, VHEPES:VDMSO = 4:6, pH = 7.40) before (black line) and after
(red line) addition of hydrazine (100 µM) in different pH condition. The excitation
wavelength was 440 nm and the reaction time was 1 h.
Fig. 2. Fluorescence responses of 1 (1.0 × 10-5 M, VHEPES:VDMSO= 4:6, pH=7.40) upon
addition of different species (10 equiv of species relative to 1) (red bars) and fluorescence
changes of the mixture of 1 and hydrazine (1.0 × 10−4 M in water) after addition of an
excess of the indicated species (10 equiv relative to 1) (green bars) with excitation at 440
nm. The reaction time was 1 hour. I517 and I0 represent the emission intensity at 517 nm
after and before addition of hydrazine. The neutral moleculesused were blank, Et3N,
NH3.H2O, C2H4(NH2)2, thiamine, urea, and Aniline. The metal ions used were blank, Na+,
Mg2+, Al3+, K+, Ca2+,Cr3+, Mn2+, Zn2+, Pt2+, and Pb2+. The anions used were blank,
2−
−
3−
−
−
4−
2−
2−
2−
−
2−
HPO4 , H2PO4 , PO4 , N3 , HSO3 , P2O7 , CO3 , C2O4 , SiO3 , Cl−, F−, NO3 , SO3 ,
2−
−
2−
S2O3 , ClO3 , I−, SO4 , CH3COO−, and HS−.
Fig. 3. Fluorescence responses of 1 (1.0 × 10-5 M, VHEPES:VDMSO = 4:6, pH = 7.40)
upon addition of different species (10 equiv of species relative to 1) (red bars) and
fluorescence changes of the mixture of 1 and hydrazine (1.0 × 10−4 M in water) after

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addition of an excess of the indicated species (10 equiv relative to 1) (green bars) with
excitation at 440 nm. The reaction time was 1 hour. I517 and I0 represent the emission
intensity at 517 nm after and before addition of hydrazine.
Fig. 4. (a), (b) Confocal fluorescence images of Hela cells stained with probe
1
for 15 min (Channel 1: λex=457 nm, λem=525±25 nm nm, (a)) and
Lysosome-Tracker (2.0 mm) for 15 min (Channel 2: λex=640 nm, λem=663-738 nm,
(b)). (c) Merged image of (a) and (b). (d) Bright-field image. (e) Intensity profile of
regions of interest (ROIs) across Hela cells. (f) Intensity Correlation plot of probe
and Lysosome-Tracker (Rr = 0.82).
1
Fig. 5. (a) Viability assay for HeLa cells treated with probe 1 in dark. Confocal
fluorescence image in live HeLa cells contained by 10−5 M probe 1 (b) and cells of
pretreated with 10−5 M probe 1 followed by incubation with 1.0 × 10−4 M hydrazine for 1
h (c). The excitation wavelength was 457 nm.

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Scheme 1 Synthesis of probe 1 and its recognition mechanism toward hydrazine.

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Fig. 1. (a) Emission spectra of probe 1 (1.0 × 10-5 M, VHEPES:VDMSO = 4:6, pH = 7.40)
upon titration of hydrazine (0–10.0 equiv to 1) (b) The linearity of emission intensity at
517 nm with the concentration of hydrazine. (c) Kinetic curves of probe 1 (1.0 × 10-5 M,
VHEPES:VDMSO = 4:6, pH = 7.40) at 517 nm with hydrazine at different concentrations [the
concentrations of hydrazine are 0 M (black line), 4.0 × 10−6 M (red line), 8.0 × 10−6 M
(green line), 1.2 × 10−5 M (blue line), and 1.6 × 10−5 M (cyan line)]. (d) Fluorescence
change of 1 (1.0 × 10-5 M, VHEPES:VDMSO = 4:6, pH = 7.40) before (black line) and after
(red line) addition of hydrazine (100 µM) in different pH condition. The excitation
wavelength was 440 nm and the reaction time was 1 h.

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ACCEPTED MANUSCRIPT
Fig. 2. Fluorescence responses of 1 (1.0 × 10-5 M, VHEPES:VDMSO= 4:6, pH=7.40) upon
addition of different species (10 equiv of species relative to 1) (red bars) and fluorescence
changes of the mixture of 1 and hydrazine (1.0 × 10−4 M in water) after addition of an
excess of the indicated species (10 equiv relative to 1) (green bars) with excitation at 440
nm. The reaction time was 1 hour. I517 and I0 represent the emission intensity at 517 nm
after and before addition of hydrazine. The neutral moleculesused were blank, Et3N,
NH3.H2O, C2H4(NH2)2, thiamine, urea, and Aniline. The metal ions used were blank, Na+,
Mg2+, Al3+, K+, Ca2+,Cr3+, Mn2+, Zn2+, Pt2+, and Pb2+. The anions used were blank,
2−
−
3−
−
−
4−
2−
2−
2−
−
2−
HPO4 , H2PO4 , PO4 , N3 , HSO3 , P2O7 , CO3 , C2O4 , SiO3 , Cl−, F−, NO3 , SO3 ,
2−
−
2−
S2O3 , ClO3 , I−, SO4 , CH3COO−, and HS−.

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ACCEPTED MANUSCRIPT
Fig. 3. Fluorescence responses of 1 (1.0 × 10-5 M, VHEPES:VDMSO = 4:6, pH = 7.40)
upon addition of different species (10 equiv of species relative to 1) (red bars) and
fluorescence changes of the mixture of 1 and hydrazine (1.0 × 10−4 M in water) after
addition of an excess of the indicated species (10 equiv relative to 1) (green bars) with
excitation at 440 nm. The reaction time was 1 hour. I517 and I0 represent the emission
intensity at 517 nm after and before addition of hydrazine.

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ACCEPTED MANUSCRIPT
Fig. 4. (a), (b) Confocal fluorescence images of Hela cells stained with probe
1
for 15 min
(Channel 1: λex=457 nm, λem=525±25 nm nm, (a)) and Lysosome-Tracker (2.0 mm) for 15 min
(Channel 2: λex=640 nm, λem=663-738 nm, (b)). (c) Merged image of (a) and (b). (d)
Bright-field image. (e) Intensity profile of regions of interest (ROIs) across Hela cells. (f)
Intensity Correlation plot of probe
1 and Lysosome-Tracker (Rr = 0.82).

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ACCEPTED MANUSCRIPT
Fig. 5. (a) Viability assay for HeLa cells treated with probe 1 in dark. Confocal
fluorescence image in live HeLa cells contained by 10−5 M probe 1 (b) and cells of
pretreated with 10−5 M probe 1 followed by incubation with 1.0 × 10−4 M hydrazine for 1
h (c). The excitation wavelength was 457 nm.

ACCEPTED MANUSCRIPT
Highlights
►A highly selective fluorescent probe for hydrazine shows off-on property.
►The probe can quantitatively detect hydrazine in the concentration range from 0 to
20 µM.
►The detection limit on fluorescence response of the probe can be as low as 140 nM.