Mitochondria-targeted ratiometric fluorescent detection of hydrazine with a fast response time

Article abstract of DOI:10.1039/c7nj04212d

A ratiometric fluorescent probe 1 was synthesized, which exhibited high sensitivity and excellent selectivity for hydrazine recognition. Probe 1 can quantitatively detect hydrazine in the concentration range from 30 to 180 μM with a LOD of 2.1 μM. Furthermore, it displayed excellent selectivity, anti-interference over many nucleophilic species, biological species, metal ions and anions, and a fast response time (4 min). Due to its ability to target mitochondria, the response of hydrazine to this probe in a living cell was successfully tracked in a ratiometric manner via fluorescence imaging.

Full text of DOI:10.1039/c7nj04212d

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Jason B. Benedict et al.
The role of atropisomers on the photo-reactivity and fatigue of
diarylethene-based metal–organic frameworks
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Mitochondria-targeted ratiometric fluorescent detection of
hydrazine with fast response time
Yanan Ban,a∥ Ruihui Wang,b∥ Yang Li,a Zhen An,a Mingming Yu,a* Chenjie Fang,b* Liuhe Weia and
Zhanxian Lia
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
A ratiometric fluorescent probe 1 was synthesized, which exhibited high sensitivity and excellent selectivity in hydrazine
recognition. Probe 1 can quantitatively detect hydrazine in concentration range from 30 to 180 μM with the LOD of 2.1
μM. Further, it displayed excellent selectivity, anti-interference over many nucleophilic species, biological species, metal
ions and anions, and fast response time (4 min). Due to its ability to target mitochondria, the response of hydrazine to this
probe in a living cell was successfully tracked in a ratiometric manner via fluorescence imaging.
www.rsc.org/
Compared with turn-on or turn-off mode fluorescent
probes, ratiometric fluorescent probes have many advantages,
for example, they can eliminate many interference factors
Introduction
Hydrazine is not only a highly reactive base and reducing
agent, but also a high-energy fuel for rocket-propulsion
systems and missile systems.1 As a result, it plays an important
role in many fields such as the pharmaceutical, chemical and
agricultural industries.2–4 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 human health.5−9 Therefore, it is
urgently demanded to develop an efficient and simple method
for determining hydrazine level in both environmental and
biological science.
such as optical path length and the illumination intensity.
Moreover, when ratiometric fluorescent probes are applied in
biosystem, they can realize more effective detection through
measuring the ratio of fluorescence intensities at two different
wavelengths.37−40 It is
fluorescent hydrazine-probe
a
pity that very few ratiometric
have been reported,41−47 and it is
s
essential to develop ratiometric fluorescent probes toward
hydrazine.
In this paper, using hydrazine-induced fracture of C=C, a
highly sensitive, photo-stable ratiometric fluorescent probe
Scheme 1) was developed, and the application of which for
selective detection and imaging of hydrazine in living cells was
successfully demonstrated. The probe exhibited high
1
(
a
Up to now, in order to measure hydrazine, some methods
including chromatography-mass spectrometry,10−12 titration13
and electrochemical methods14−18 have been reported,
however their shortcomings such as complicated equipment,
sample handling and professional operating will limit their
application. Therefore, it is still needed to expand new
methods which can detect trace hydrazine in situ, with short
time and low cost in room temperature.
sensitivity (the LOD is 2.1 μM), a wide linear response range
from hydrazine concentration of 30 to 180 μM, excellent
selectivity and anti-interference against other various species,
and fast response time. The intrinsic ability of probe
mitochondrion was confirmed with a co-localization study.
The synthesis of the smart probe was accomplished in 4
1 to target
1
steps as shown in Scheme 1. The starting materials 2-methyl-
8-hydroxyquinoline and acryloyl chloride were reacted for 12
hours under 20°C to give compound 4 in a yield of 47.3%. The
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 and most
of them are turn-on types.30−36
product 4 was oxidized by SeO2 at 101°C to give product
yield of 71.6%. The reaction of
trimethylbenzo[e]indolenine and CH3I gave compound
yield of 52.6%. Probe was obtained in a yield of 45.1% by the
Knoevenagel condensation reaction of compound with
compound The detailed synthetic procedures and
characterization data are presented in the Experimental
section Supporting Information).
3
in a
1,1,2-
in a
2
1
3
2.
(
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Compounds
were dissolved in 10 mL ethanol and the mixture was heated
to 78 . After refluxing for 12 h, the reaction mixture was
cooled to room temperature. The final product probe as
brown solid was obtained after filtration (0.2528 g, 45.1%).
Characterization of : HRMS (EI) m/z: calcd for C29H25N2O2
[M-I], 433.1911; found, 433.1839. 1H NMR δH (DMSO, 400
MHz) 8.75(d, 1H), 8.66 (d, 1H), 8.51 (d,1H), 8.35 (t, 3H), 8.28
2 (0.3513 g, 1.0 mmol) and 3 (0.2771 g, 1.0 mmol)
℃
1
1
：
(d, 1H), 8.22 (d, 1H), 8.14 (d, 1H), 8.05 (m, 1H), 7.86 (t,
1H),7.79 (m, 3H), 6.78 (d, 2H), 6.36 (t, 1H), 4.25 (s, 3H), and
2.05 (s, 6H). 13C NMR: δC (100 MHz, DMSO): 182.49, 164.86,
152.03, 148.53, 147.75, 139.95, 138.48,134.68, 133.98, 131.61,
130.53, 130.20, 129.09, 128.69, 130.20,128.12, 127.11, 126.63,
125.29, 123.98, 123.06, 117.15, 114.02, 54.71, 36.01, 25.08.
Scheme 1 Synthetic route of probe
1 and its recognition
mechanism toward hydrazine. (1) Acryloyl chloride, CH2Cl2,
Et3N, rt, 12 h; (2) SeO2, 1,4-dioxane, 101°C, rf, 4 h; (3) CH3I,
acetonitrile, reflux, 12 h; (4) ethanol, reflux, 12 h.
Cell incubation and imaging
Cell viability, as a testing endpoint of cytotoxicity, was
determined
with
3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) assay. HepG2 cells were
seeded into 96-well plates at a density of 5 × 103 cells per well
in DMEM (10% FBS) in six replicates and incubated for 24 h.
Experimental section
Methods and materials
After that the cells were exposed to 2, 4, 6, 8, 10 μM probe
1
8-hydroxy-2-methylquinoline, acryloyl chloride, CH2Cl2, Et3N,
SeO2, 1,4-dioxane, CH3I, acetonitrile, ethanol, NaCl, MgCl2, KCl,
CaCl2, MnCl2, Co(NO3)2, Ni(NO3)2, ZnCl2, Cd(NO3)2, NaF, NaBr,
NaI, NaNO3, NaClO4, Na2SO4, Na2HPO4, NaClO3, Na2S2O3,
Na2C2O4, Na2SO3, CH3COONa, NaHS, Na2N3 lle, Val, Ser, Gly, Trp,
Thr, Phe, Cys, Met, Pro, Leu, Ala, Gln, Tyr, His, Hcy, Glu, and
H2O2. All commercial grade chemicals and solvents were
purchased and were used without further purification.
for 1 h, then medium was discard and replaced with 100 μL
fresh medium for 24 h, then 20 μL MTT (5 mg/mL) was added,
and the cells were further incubated for 4 h at 37°C. The
medium was removed and the formazan crystals were
dissolved in DMSO. The optical density (OD) was measured at
490 nm.
The intrinsic ability of probe
investigated in living HepG2 cells. The cells were stained with
probe
(1.0 × 10−5 M, 30 min, 37°C) and washed twice with
1 to target mitochondrion was
Mass spectra were obtained on high resolution mass
spectrometer (IonSpec4.7 Tesla FTMS-MALDI/DHB). H and 13
1
1
C
PBS, then co-stained further with a commercially available
mito tracker (100 nM, 30 min) in the culture medium. After
removing the culture medium and washed twice with PBS, The
imaging results were obtained, which show that the green
image for the 1 channel was obtained upon excitation at 488
nm and the red image for the 2 channel was obtained upon
excitation at 495 nm.
NMR spectra were recorded on
a Bruker 400 NMR
spectrometer. Chemical shifts were reported in parts per
million using tetramethylsilane (TMS) as the internal standard.
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 spectroscopy
was determined on a Hitachi F-4600 spectrometer.
The HepG2 cells were incubated with probe
1 for ratio
imaging. The fluorescent images were recorded at green and
red channels with 525±50 nm and 595±50 nm filters separately,
and the ratio images were got with the intensity ratio of the
Synthesis of Compound 2
1
(1.0 × 10−5 M,
1,1,2-trimethylbenzo[e]indolenine (1.0452 g, 5 mmol) and CH3I
(1 mL) were dissolved in 15 mL acetonitrile, and the mixture
green/red after the cells incubated with probe
37°C) for 30 min. After adding 1 × 10−3 M hydrazine hydrate
and incubation for 30 min, the fluorescent and ratio images
were taken at the same situation. The excitation was
performed at 405 nm.
was heated to 45℃. After 13 hours of reaction, the mixture
was cooled to room temperature and a large amount of
diethyl ether was poured into the reaction solution. The final
product was obtained after filtration (0.9234 g, yield: 52.6%).
1
: H NMR δH (DMSO, 400 MHz): 8.38
Characterization of
2
Results and discussion
(d, 1H), 8.31 (d, 1H), 8.22 (d, 1H), 8.12 (d, 1H), 7.78 (t, 1H),
7.76(t, 1H), 4.10 (s, 3H), 2.88 (s, 3H), 1.76 (s, 6H). 13C NMR: δC
(100 MHz, DMSO): 196.37, 139.94, 136.97, 133.49, 130.98,
130.21, 128.84, 127.59, 127.58, 123.88, 113.62, 55.72, 35.59,
21.75, and 14.48.
Sensing of the probe 1 to hydrazine
To study the recognition properties of probe
1 toward
hydrazine, UV-vis and fluorescence titration experiments
(Figures S1 and S2 in Supporting Information and Fig. 1) were
conducted with 0.1 M hydrazine in aqueous solution of probe
Synthesis of compound probe 1
1
[3.0 × 10−5 M in 100:1 (v/v) water/DMSO]. Upon addition of
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indicating the decomposition of probe
compounds 2 and 5 (Scheme 1).
1
and the formation of
hydrazine, the peaks at 375 and 430 nm in the UV-vis
spectrum decreased gradually while a new band developed at
240 nm, and then, the band reached the highest intensity at
6.0 equiv of Hydrazine (Fig. S1 in Supporting Information).
Meanwhile, one clear isosbestic point was observed at 331 nm,
indicating that only one product was generated from the
reaction of 1 with hydrazine (Fig. S1 in Supporting Information).
The solution color changed by degrees from yellow to colorless
in the presence of different concentrations of hydrazine (Fig.
S2 in Supporting Information). As shown in Fig. 1a, upon
In order to further study its recognition mechanism, using
the reaction of compound 3 and hydrazine, compound 5 was
obtained, which was characterized with 1H NMR (Fig. S3 in
Supporting Information). In addition, the emission spectra of
compounds
Information), which are in accordance with the emission
spectral change of probe upon addition of hydrazine (Fig. 1a).
2 and 5 were also tested (Fig. S4 in Supporting
1
pH range in application of probe 1 toward hydrazine
excitation at 405 nm, one emission band centered at 590 nm The pH value of solution was found to be essential to the
reaction between probe and hydrazine. To investigate the pH
effect, the fluorescence of 3.0 × 10−5 M probe
in the absence
and presence of 1.8 × 10−4 M hydrazine were examined at pH
range from 0 to 14.0. As shown in Fig. S5, probe itself is
1
[3.0 × 10−5
1
appeared in the fluorescence spectrum of probe
1
M in 100:1 (v/v) water/DMSO]. Upon gradual addition of
hydrazine, the emission at 590 nm decreased while a new peak
at 467 nm increased with no change of the emission intensity
at 520 nm observed (Fig 1a). As demonstrated in Fig. 1b, in the
concentration range of 30 to 180 μM, the ratio of I590/I520 was
in good linear relationship with hydrazine concentration,
implying that hydrazine can be quantitatively detected in a
wide concentration range in a ratiometric fluorescence mode.
From the linear calibration graph with the fluorescence
1
stable and its emission spectra hardly changed in a wide pH
range from 1.0 to 10.0. However, upon addition of hydrazine,
there was a significant fluorescence change in pH range of 5.0–
10.0. Notably, the fluorescence changes of probe 1 to
hydrazine reached a maximum at about 5.0 and became
almost constant in the pH range of 5.0–10.0. Such result
implies that probe
wide pH range.
1 is able to detect hydrazine in a relatively
titration experiment (Fig. 1b), the detection limit of probe 1 for
hydrazine was figured out to be about 2.1 μM based on signal-
to-noise ratio (S/N) = 3,48,49 Further experiments indicated that
Selectivity and competition studies
To evaluate the selectivity of probe 1 for hydrazine, various
species including nucleophilic molecules (DIPEA, Phenol,
Thiourea, TEA, and Urea), metal ions (Na+, Mg2+, K+, Ca2+, Mn2+,
the fluorescence color of probe
from orange red to colorless under 365 nm light excitation (Fig.
1c). These results led us to conclude that probe could be an
effective ratiometric fluorescent probe for hydrazine.
1 aqueous solution changed
1
Co2+, Ni2+, Zn2+, and Cd2+), anions (F−, Cl−, Br−, I−, NO3 , ClO4 ,
−
−
−
−
SO42−, H2PO4 , ClO3 , S2O32−, C2O42−, SO32−, HS−, CH3COO−, and
N3 ) and biological species (Ile, Val, Ser, Gly, Trp, Thr, Phe, Cys,
−
Met, Pro, Leu, Ala, Gln, Tyr, His, Hcy, Glu, and H2O2) were
tested. As shown in figures S6, S7, S8, S9, red bars of figures
S10 S11 S12 in Supporting Information and Fig. 2, only
,
,
hydrazine induced a significant fluorescence quenching, whilst,
other tested species did not induce any obvious fluorescence
quenching. Some reported probes containing C=C can detect
some anions such as ONOO−, ClO−, SO32− 50-52 while ONOO− and
,
2−
SO3 have no such influence on our probe, indicating the high
selectivity toward hydrazine. Addition of ClO− can make the
emission change in both the peak and intensity of probe 1 (Fig.
S9), which needs us to study further in our future work.
To further assess its utility as
fluorescent probe, its fluorescent response to hydrazine in
complicated surroundings as mentioned above was also tested
(green bars of figures S10, S11, S12 in Supporting Information
a hydrazine-selective
Fig. 1 (a) Emission spectra of probe
(v/v) water/DMSO] upon titration of hydrazine (0–6.0 equiv to
) with excitation at 405 nm. (b) The linearity of emission
intensity ratio of I590/I520 with hydrazine concentration increase.
1
[3.0 × 10−5 M in 100:1
1
and Fig. 2). The results evidenced that all of the selected
species have no interference in the detection of hydrazine.
(c) Photographs of probe
1
[3.0 × 10−5 M in 100:1 (v/v)
water/DMSO] upon addition of hydrazine at various
concentrations (0 M, 3.0 × 10−5 M, 6.0 × 10−5 M, 1.2 × 10−4 M,
1.5 × 10−4 M, 1.8 × 10−4 M, from left to right) in water and
under a UV lamp (365 nm). The reaction time was 4 min.
This result strongly indicated that probe 1 could be an
excellent fluorescent probe towards hydrazine with strong
anti-interference ability.
Mechanism studies
The recognition mechanism was studied by Mass spectrometry.
For pure probe
obtained which corresponds to the species
1
, a characteristic peak at m/z = 433.1839 was
1
[M-I], whilst after
reaction with hydrazine, the peak at 433.1839 disappeared
and two new peaks appeared at m/z = 224.1441 and 188.0802,
which are corresponding to compounds 2 (M-I) and 5 (M+1),
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Fig. 2 Fluorescence responses of
1 1 M
[3.0 × 10−5 M in 100:1 (v/v) Fig. 3 Emission intensity ratio I590/I520 of probe [3.0 × 10−5
water/DMSO] upon addition of different species (6 equiv of in 100:1 (v/v) water/DMSO] with irradiation (λex = 405 nm)
species relative to ) (red bars) and fluorescence changes of time (a) and temperature (b) change. Kinetic curve of probe
the mixture of
and hydrazine (1.8 × 10−4 M in water) after [3.0 × 10−5 M in 100:1 (v/v) water/DMSO] at 590 nm and 520
addition of an excess of the indicated species (6 equiv relative nm with hydrazine at different concentrations (the
1
1
1
to 1
) (green bars). The excitation wavelength was 405 nm. I590 concentrations of hydrazine are 6.0 × 10−5 M (black line), 1.2 ×
and I520 represent the emission intensity at 590 and 520 nm. 10−4 M (red line), and 1.8 × 10−4 M (green line). The excitation
The reaction time was 4 min. The biomolecules used were Ile, wavelength was 405 nm (c). Viability assay for HepG2 cells
Val, Ser, Gly, Trp, Thr, Phe, Cys, Met, Pro, Leu, Ala, Gln, Tyr, His, treated with probe
Hcy, Glu, and H2O2.
1 in dark (d).
Application of the probe 1 in living cells
The intrinsic ability of probe to target mitochondrion was
investigated in living HepG2 cells. The cells stained with probe
An ideal probe ought to have good photostability and
thermal stability. As shown in Fig. 3a, the emission intensity
ratio of probe
590 and 520 nm (I590/I520) hardly changes with irradiation time
extending, indicating excellent photostability. When the
temperature increased from 30 to 42°C, the emission intensity
ratio I590/I520 doesn’t change (Fig. 3b). Such results suggest that
1
1
[3.0 × 10−5 M in 100:1 (v/v) water/DMSO] at
5
1
(1.0 × 10– M 30 min, 37°C) were co-stained further with the
commercially available mitochondrion-Tracker (100 nM, 30
min) in the culture medium. The imaging results show that the
red image for 1 channel obtained upon excitation at 595 nm is
almost identical to the green image for the mitochondrion-
Tracker channel obtained upon excitation at 488 nm (Fig. 4),
suggesting the ability of probe 1 to target mitochondrion. The
targeting ability of this probe toward mitochondrion may be
ascribed to the cationic cyanine moiety.
probe
1
as
a
photostability and thermal stability.
hydrazine probe possesses excellent
hydrazine in time-dependent fluorescence spectra of probe
probe 1hydrazine is completely accomplished within 4 min
1
and hydrazine can be detected within 2 min when the
concentration of hydrazine is higher or equal to 1.8 × 10–4 M.
Such result further proved the fast response and high
sensitivity of probe
1
for detection of hydrazine.
The cytotoxicity of probe
1
toward HepG2 cells was
measured using the
diphenyltetrazilium bromide (MTT) assay to evaluate the
potential application of probe in live cell imagining. The
cellular viability was estimated to be greater than 86% after 1
h at the concentration of 10−5 M for probe
, suggesting the
3-(4,5-dimethylthiazol-2-yl)-2,5-
1
1
low cytotoxicity of probe 1 (Fig. 3d).
Fig. 4 Confocal fluorescence images of HepG2 cells stained with
1
(1.0 × 10−5 M) for
mitochondrion-Tracker for 30 min (a) and
30 min (b). (c) Merged image of (a) and (b). (d) Bright-field
image.
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In summary, a ratiometric fluorescent hydrazine-probe was
synthesized and demonstrated. This probe can not only
quantitatively detect hydrazine in wide concentration range,
exhibits excellent selectivity, anti-interference, fast response
time, good location ability toward mitochondrion and the
ability of selective detection of hydrazine in living cells.
Acknowledgements
We are grateful for the financial supports from National
Natural Science Foundation of China (U1704161, U1504203,
21601158, 21571133, and 21171120), Zhengzhou University
(32210431), Beijing Natural Science Foundation Program and
Scientific Research Key Program of Beijing Municipal
Commission of Education (KZ201710025024), and Natural
Science Foundation of Beijing Municipality (7132020).
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Fig. 5 Ratiometric confocal fluorescence images in live HepG2
cells constained by 10−5 M probe
1
(a–c) and cells of
pretreated with 10−4 M probe
1 followed by incubation with
10−3 M hydrazine for 30 min (d–f). Emission was collected by
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a 405 nm diode laser. The confocal fluorescence microscopy
images are shown in Fig. 5. After incubation of HepG2 cells
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fluorescence was detected at green (525±25 nm) and strong
fluorescence at red (595±25 nm) channels in the cytoplasm,
demonstrating the good cell permeability of probe 1. Following
addition of hydrazine (1.0 × 10−3 M), although no obvious
change was observed at the green and red channels, a rapid
and obvious change at the ratiometric confocal fluorescence
was observed (figures 5c and 5f), suggesting the reaction of
probe
1 to hydrazine. The emission ratio at the green channel
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A fluorescent hydrazine-probe was synthesized, which exhibited high sensitivity,
excellent selectivity and anti-interference ability.