A new aggregation-induced emission active red-emitting fluorescent sensor for ultrarapidly, selectively and sensitively detecting hydrazine and its multiple applications

Article abstract of DOI:10.1016/j.molliq.2020.113845

In this work, a novel carbazole-indandione based red-emitting fluorescent sensor CBI was developed, which exhibited typical and interesting aggregation-induced emission (AIE) characteristics with a large Stokes shift in mixed aqueous media. CBI as a colorimetric and fluorimetric sensor for sensing hydrazine in ~100percent aqueous media shows ultrarapid response, excellent selectivity, and great sensitivity with a low detection limit of 1.18 ppb. The mechanism of CBI for highly sensing hydrazine was proved by fluorescence, FTIR, ¹H NMR and HRMS spectra. Interestingly, the CBI was not only applied to sensitively detect aqueous N₂H₄ in water, human urine and live cells, but also was conveniently utilized to visualize gaseous N₂H₄ with sensitive naked-eye color diversification. Additionally, owing to its intense fluorescence irradiated by UV light, the CBI was significantly used as a new fluorescent ink for writing and drawing as well as a writable fluorescent display material.

Full text of DOI:10.1016/j.molliq.2020.113845

Journal Pre-proof
A new aggregation-induced emission active red-emitting
fluorescent sensor for ultrarapidly, selectively and sensitively
detecting hydrazine and its multiple applications
Pengcheng Yin, Qingfen Niu, Tianduo Li, Tao Wei, Jianbin Chen,
Xuyang Qin
PII:
S0167-7322(20)33528-5
DOI:
https://doi.org/10.1016/j.molliq.2020.113845
MOLLIQ 113845
Reference:
To appear in:
Journal of Molecular Liquids
Received date:
Revised date:
Accepted date:
2 June 2020
10 July 2020
14 July 2020
Please cite this article as: P. Yin, Q. Niu, T. Li, et al., A new aggregation-induced
emission active red-emitting fluorescent sensor for ultrarapidly, selectively and sensitively
detecting hydrazine and its multiple applications, Journal of Molecular Liquids (2020),
https://doi.org/10.1016/j.molliq.2020.113845
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© 2020 Published by Elsevier.

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A new aggregation-induced emission active red-emitting fluorescent
sensor for ultrarapidly, selectively and sensitively detecting hydrazine
and its multiple applications
Pengcheng Yin, Qingfen Niu*, Tianduo Li, Tao Wei, Jianbin Chen, Xuyang Qin
Shandong Provincial Key Laboratory of Molecular Engineering, School of Chemistry
and Pharmaceutical Engineering, Qilu University of Technology (Shandong Academy
of Sciences), Jinan 250353, People‟s Republic of China
ABSTRACT
In this work, a novel carbazole-indandione based red-emitting fluorescent sensor CBI
was developed, which exhibited typical and interesting aggregation-induced emission
(AIE) characteristics with a large Stokes shift in mixed aqueous media. CBI as a
colorimetric and fluorimetric sensor for sensing hydrazine in ～100% aqueous media
shows ultrarapid response, excellent selectivity, and great sensitivity with a low
detection limit of 1.18 ppb. The mechanism of CBI for highly sensing hydrazine was
1
proved by fluorescence, FTIR, H NMR and HRMS spectra. Interestingly, the CBI
was not only applied to sensitively detect aqueous N2H4 in water, human urine and
live cells, but also was conveniently utilized to visualize gaseous N2H4 with sensitive
naked-eye color diversification. Additionally, owing to its intense fluorescence
irradiated by UV light, the CBI was significantly used as a new fluorescent ink for
writing and drawing as well as a writable fluorescent display material.
Keywords: Aggregation-induced emission; Fluorescent sensor; Red-emitting;
Hydrazine detection; Bio-imaging; Fluorescent ink.
* To whom the correspondence should be addressed
E-mail: qf_niu1216@qlu.edu.cn
Telephone: +86(531)89631760
Fax number: +86(531)89631208
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1. Introduction
Hydrazine (N2H4), not only was widely used for the manufacture of
dyes, photosensitizers, pharmaceuticals, emulsifiers and pesticides
because of its strong reducing properties [1–3], but also applied as a
high-energy fuel in rocket propulsion system due to its explosive
characteristics [4–6]. Additinally, ascring to its excellent water solubility
and high toxicity, hydrazine is easy to cause environmental pollution and
serious damage to the human health [7,8]. The U.S. Environmental
Protection Agency (USEPA) has set the threshold limit of hydrazine is 10
ppb [9]. Hence, it is urgently demanded to develop an effective,
convenient and reliable method for sensitively determining hydrazine in
enviro/biological systems.
In recent years, several methods have been reported for detecting
hydrazine, such as high-performance liquid chromatography (HPLC) [10],
coulometry [11], and electrochemistry [12]. Fluorescent sensors are more
attractive and advantageous over other conventional methods for
biological and environmental cations and anions as well as active
molecules in terms of their rapidity, reliability, simplicity and significant
potential for bioimaging [13–33]. Up to now, numerous fluorescent
sensors for N2H4 detection have been developed [34–40]. However, some
fluorescent sensors still has some shortcomings such as sluggish response,
poor selectivity and inferior sensitivity, which suffer from their practical
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applications. Thus, the exploitation of novel fluorescent sensors for N2H4
detection with outstanding features and multiple practical applications,
which could effectively overcome the existent shortcomings, are still
highly demanded.
Luminogens of aggregation-induced emission (AIE) have attracted
tremendous attention as that they are poor mission in solution but show
highly emissive upon aggregation [41]. Because of the interesting
fluorescence characteristics and great application potentials, AIE-active
luminogens have been extensively developed and applied as
chemo/biosensors and smart materials [42–44]. Recently, great efforts
have been devoted to the extensively developing new AIE-active
fluorescent sensors for sensitive and quantitative recognization of various
environmental and biological analytes since they display a remarkable
advantage of high adaptability to working conditions [41,456–47].
Unfortunately, most of the reported AIE-active fluorescent sensors
dominantly emit green/blue fluorescence [48–50], while quite few are
red-emitting. Hence, developing new AIE-active red-emitting fluorescent
sensors are highly desirable.
Herein, we report a new carbazole-indandione based AIE active
red-emitting fluorescent sensor CBI, and was utilized for sensitive,
specific, colorimetric and fluorimetric detection of N2H4 in ～100%
aqueous solution. Interestingly, CBI exhibits good features of
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red-emitting fluorescence, large Stokes shift (170 nm), ultrarapid
fluorescence response (50 s), low cytotoxicity (cell viability > 90%) and
outstanding biocompatibility. In addition, CBI displays a highly selective
fluorescence quenching effect toward N2H4 with low detection limit of
1.18 ppb. Interestingly, sensor CBI can be utilized as a new fluorescent
ink and a writable molecular smart fluorescent display material. More
importantly, the CBI is applied to monitor and image aqueous N2H4 in
real water, human urine, live cells, as well as visualize gaseous hydrazine.
2. Experimental part
2.1. Reagents and instruments
All chemicals (including active small molecules: N2H4, Cys, Hcy, GSH,
urea, thiourea, NH2OH, EDA, Ph-NH2, TEA, NH3, n-BuNH2, various
anions including F–, Br–, Cl–, CN– and AcO– obtained from their
tetrabutylammonium or sodium salts, and various cations including Na+,
Al3+, Cr3+, Cd2+, Co2+, Mg2+, Hg2+ and Fe2+ obtained from their nitrate
salts) were purchased from Alfa Aesar Co. (Tianjin, China), which were
1
of analytical grade and were used without further purification. H and 13C
nuclear magnetic resonance (NMR) spectra were recorded on a Bruker
400 MHz spectrophotometer using DMSO-d6 and tetramethylsilane
(TMS) as a solvent and an internal standard, respectively. High-resolution
mass spectra (HRMS) were obtained using a Q-TOF6510 spectrograph
(Agilent). Infrared spectra (IR) were performed using a Shimadzu
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FTIR-8100 spectrophotometer. Optical absorption spectra were measured
using a Shimadzu UV-2600 spectropolarimeter at room temperature.
Fluorescence spectra were recorded using an Edinburgh Ltd-FLS920
spectrofluorophotometer. For all fluorescent measurements, both
excitation and emission slit widths were 5 nm. The pH was tested on a
Model PHS-25 pH meter. The bioimaging was tested using a Leica TCS
SP8 confocal-laser scanning microscope (CLSM) (λex = 552 nm).
2.2. General spectroscopic procedures
Sensor CBI was dissolved in EtOH to prepare the stock solution of 1
× 10–3 M. The other various testing species, including primary amines
such as N2H4, Cys, Hcy, GSH, urea, thiourea, NH2OH, EDA, Ph-NH2,
TEA, NH3, n-BuNH2, representative cations (Na+, Al3+, Cr3+, Cd2+, Co2+,
Mg2+, Hg2+, Fe2+) and representative anions (CN–, CH3COO–, F–, Br–, Cl–)
were subsequently dissolved in deionized water to provide the stock
solutions (1 × 10–3 M). The analytical solutions were prepared by diluting
reserve solution with the deionized water, and then shaken well and kept
for 0.5 h before taking the optical spectra. All spectral tests were
performed in ～100% aqueous solution at room temperature (λex = 445
nm).
2.3. Fluorescence quantum yield
The relative fluorescence quantum yield (Φ) was evaluated by
quinine sulfate as reference and calculated by the following formula:
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Φs = Φr (Ar/As)(Is/Ir)(ηs2/ηr2)
Where Φs and Φr represent the quantum yield of sample and the quinine
sulfate (Φ = 0.55, 0.1 N H2SO4), Ar and As represent the absorbance of
reference and sample, Is and Ir represent the integrated fluorescence
intensity of sample and reference, and η represents the refractive index of
the solvent, respectively.
2.4. Synthesis of 2-((9-propyl-9H-carbazol-3-yl)methylene)-1H-indene-
1,3(2H)-dione (CBI)
N-propylcarbazole (1) and N-propylcarbazole-3-carbaldehyde (2)
were prepared as the previously reported procedure [51,52]. The Scheme
1 clearly showed that the CBI was synthesized by the knoevenagel
condensation reaction between N-propylcarbazole-3-carbaldehyde and
indandione. Compound 2 (80 mg, 0.34 mmol), indandione (50 mg, 0.34
mmol) and dry ethanol (10 mL) were heated to reflux for 5 h. Then this
reaction system was cooled to room temperature, and the final product
was collected by the recrystallization to give compound CBI as an orange
1
solid (95 mg. 76.6% yield). H NMR (400 MHz, DMSO-d6, ppm): δ =
9.51 (s, 1H), 8.78 (d, J = 8.0 Hz, 1H), 8.16 (d, J = 8.0 Hz, 1H), 7.97 (s,
1H), 7.82-7.93 (m, 4H), 7.76 (d, J = 8.0 Hz, 1H), 7.66 (d, J = 8.0 Hz, 1H),
7.47 (t, J = 8.0 Hz, 1H), 7.27 (t, J = 8.0 Hz, 1H), 4.38 (t, J = 8.0 Hz, 2H),
1.72-1.81 (m, 2H), 0.81 (t, J = 8.0 Hz, 3H); 13C NMR (100 Hz, DMSO-d6,
ppm): δ = 197.4, 190.6, 189.6, 148.2, 143.9, 142.2, 141.3, 139.7, 136.0,
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135.9, 133.1, 129.2, 127.3, 125.7, 124.8, 123.3, 123.2, 123.1, 122.8,
121.0, 110.9, 110.4, 44.6, 22.4, 11.8; FTIR (KBr, cm–1): ν = 1560 (C=C),
1670 (C=O); HRMS (ESI): m/z [M+H]+ calcd. for: C25H19NO2: 366.1416,
found: 366.1494. The structure of CBI was confirmed by the spectra of
13
1H NMR, C NMR, FTIR and HRMS (Figs. S1–S4), demonstrating the
CBI was successfully prepared.
Scheme 1 The preparation of sensor CBI.
2.5. Bioimaging of living cells
The 20 μM CBI was firstly incubated in HeLa cells within culture
media for 0.5 h at 37°C, then washed 3 times using PBS. The cells were
further treated with 20 μM N2H4 in cell media for 0.5 h, then washed the
media using PBS. The CLSM cell imaging was collected under the red
channel (600-650 nm).
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3. Results and discussion
3.1. AIE property of CBI
To explore the AIE property of CBI, the emission spectra in
EtOH/H2O (0～99%, v/v) solution were investigated. CBI is highly
soluble in EtOH, but it is hardly to dissolve in water. As shown in Fig. 1,
when the H2O content was < 80%, the CBI (10 μM) was non-emissive
(λex = 445 nm). However, a distinct broad emission at 615 nm was
observed and red-shifted greatly (80 nm) when the H2O fraction exceeded
80%, and then reached the strongest emission intensity at fw = 99%, with
the relative emission intensity (I/I0) at 615 nm up to 44-fold (Fig. 1a,
inset), implying the typical AIE activity of CBI in aqueous media.
Moreover, the AIE feature of CBI can be directly observed by the
fluorescence color change from colorless to red when increasing the
proportion of water in the EtOH/H2O mixture (Fig. 1b). The fluorescence
quantum yields (Φ) of CBI in EtOH and EtOH/H2O (fw = 99%) were
determined to be 1.04% and 39.1%, respectively, using quinine sulfate (Φ
= 0.55) as standard [53]. In addition, the typical AIE characteristics were
also confirmed by its solid luminescence. As displayed in Fig. 1c, the CBI
exhibits an intense fluorescence emission at 609 nm with a large Stokes
shift (164 nm) in the solid state, and emits intense yellow fluorescence (Φ
= 0.37) under UV light. These findings revealed that CBI is a typical
AIE-active compound.
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2000
1500
1000
500
0
2000
1500
1000
500
0
(a)
0
20 40 60 80 100
f -Vol%
w
500
600
700
800
Wavelength/nm
(b)
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6000
5000
4000
3000
2000
1000
0
(c)
500
600
700
800
Wavelength/nm
Fig. 1 (a) Fluorescence emission spectra of 10 µM sensor CBI in
different EtOH/H2O (v/v) mixtures; (b) Photographs of CBI in
EtOH/H2O mixtures with various H2O fractions (fw 0～99%) taken under
365 nm UV illumination; (c) The fluorescence emission spectrum of the
CBI in solid state; Inset: Image of CBI in solid state under UV light.
3.2. Optical response to hydrazine
The optical behavior of chemosensor CBI toward hydrazine in ～
100% aqueous media were investigated by the absorption and emission
method. As shown in Fig. 2a, the absorption spectrum of CBI (10 µM)
shows two distinct bands at 339 and 445 nm corresponding to the π-π*
and intramolecular charge transfer (ICT) transitions, respectively, and its
solution color was pale yellow. The adding 2.0 equiv. of hydrazine to the
sensor CBI solution, the bands decreases drasticly in absorption along
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with its solution color distinctly changed to colorless, which enables it to
distinguish N2H4 by the naked eye. Whereas, the adding 2.0 equiv. of
with other various relevant analytes (Cys, Hcy, GSH, urea, thiourea,
NH2OH, EDA, Ph-NH2, TEA, NH3, n-BuNH2, Na+, Al3+, Cr3+, Cd2+, Co2+,
Mg2+, Hg2+, Fe2+, CN–, F–, Br–, Cl– and AcO–) to CBI solution only led to
a slight red shifted band at ca. 494 nm, no noticeable visual color was
found, which is due to the solid precipitation in the solution. As shown in
Fig. 2b, the fluorescence spectrum of CBI (10 µM) exhibits a strong red
emission at 615 nm when excited at 445 nm. The addition of various
analytes (2.0 equiv.) to CBI solution, only N2H4 induced a drastic
fluorescence quenching behavior, along with a distinct fluorescence color
change from red to colorless by the naked eyes, and no noticeable
fluorescence variation was observed by adding other analytes. These
investigations suggested the high selectivity of the CBI for N2H4. The
high specificity of CBI towards N2H4 was proved by the competitive
experiments. The CBI was treated with 20 µM N2H4 in the presence of 20
µM other potential interfering species such as Cys, Hcy, GSH, urea,
thiourea, NH2OH, EDA, Ph-NH2, TEA, NH3, n-BuNH2, Na+, Al3+, Cr3+,
Cd2+, Co2+, Mg2+, Hg2+, CN–, F–, Br–, Cl– and AcO–. The Fig. 2c shows a
great fluorescence quenching when CBI treated with N2H4, which was
hardly disturbed by other tested interfering species, demonstrating that
sensor CBI for N2H4 had excellent selectivity over a variety of interfering
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species.
1
2
3
4
5
6
7
8
9
10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
0.3
0.2
0.1
0.0
CBI
Urea, Thiourea, NH2OH,
(a)
NH3, EDA, Ph-NH2, TEA,
n-BuNH2, Cys, GSH, Hcy,
F-, Cl-, Br-, AcO-, CN-, Na+,
Mg2+, Al3+, Cd2+, Co2+, Cr3+,
Hg2+, Fe2+
N2H4
300
400
500
600
700
800
Wavelength/nm
1
2
3
4
5
6
7
8
9
10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
2500
2000
1500
1000
500
CBI, Urea, Thiourea, NH2OH,
NH3, EDA, Ph-NH2, TEA,
(b)
n-BuNH2, Cys, GSH, Hcy, F-,
Cl-, Br-, AcO-, CN-, Na+, Mg2+,
Al3+, Cd2+, Co2+, Cr3+, Hg2+, Fe2+
N2H4
0
500
600
700
800
Wavelength/nm
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CBI+test substance
CBI+test substance+N2H4
(c)
2500
2000
1500
1000
500
0
-
-
-
-
-
+
3+
2+
2+
2+
3+
2+
2+
4
3
2
2
A
N
11
N
8
H
2
+F
4
OH
2
+Cl
4
+Br
4
N
H
2
H
4
+Cys
4
+Na
4
+Hcy
4
+CN
4
+Al
4
+NH
4
+Fe
4
+Cr
4
+Co
4
H
2
+Cd
4
+Hg
4
+TE
4
+Urea
4
+GSH
4
iourea
N
+Mg
4
+AcO
4
H
2
H
2
H
2
H
2
H
2
H
2
H
2
H
2
H
2
N
H
2
H
2
H
2
H
2
+C
4
H
2
+C
4
H
2
H
2
H
2
+Ph-NH
H
2
+NH
4
N
N
N
N
4
N
N
N
N
+Th
N
N
N
N
N
N
H
2
H
N
N
N
H
2
4
H
2
2
H
2
N
N
N
N
Fig. 2 Absorption (a) and fluorescence emission (b) spectra of the CBI
(10 µM) when adding different test substances (2.0 equiv.); Insets:
Colorimetric and fluorimetric responses of CBI (10 µM) towards various
test substances (2.0 equiv.) (1-26: free CBI, N2H4, urea, thiourea, NH2OH,
NH3, EDA, Ph-NH2, TEA, n-BuNH2, Cys, GSH, Hcy, F–, Cl–, Br–, AcO–,
CN–, Na+, Mg2+, Al3+, Cd2+, Co2+, Cr3+, Hg2+, Fe2+); (c) Competitive
experiments of CBI (10 μM) at 615 nm toward N2H4 (2.0 equiv.) over
various test substances (2.0 equiv.).
To evaluate the sensitivity of CBI towards hydrazine, the
fluorescence titration of CBI treated with different amounts of hydrazine
(0-2.0 equiv.) was conducted. As illustrated in Fig. 3a, the emission at
615 nm gradually decreased with the increasing hydrazine and peaked
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when added 10 μM hydrazine. Fig. 3b demonstrated that fluorescence
intensity at 615 nm of CBI varying with the hydrazine concentration
showed good linearity (R2 = 0.99691) in the range of 0–10 µM,
accompanied with a prominent change in solution color. Moreover,
according to the DL = 3σ/k [54], the low detection limit of CBI was
calculated to be 37 nM (1.18 ppb). This DL value is not only quite lower
than the USEPA threshold concentration of 10 ppb [9], but only is
comparable with some reported sensors (Table S1), demonstrating that
CBI was indeed a sensitive sensor for quantitatively detecting hydrazine.
2500
0
(a)
2500
2000
1500
1000
500
2000
1500
1000
500
0
N2H4
2.0 equiv
0 2 4 6 8 101214161820
-6
[N2H4] (10 M)
0
500
600
700
800
Wavelength/nm
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Equation
y = a + b*x
0.99691
3000
2500
2000
1500
1000
500
Adj. R-Square
(b)
Value
Standard Error
24.67475
B
B
Intercept
Slope
2427.4472
7
-237.04182
4.17079
DL=3.7*10-8
0
0
2
4
6
8
10
[N2H4] (10-6 M)
Fig. 3 (a) Fluorescence emission spectra of CBI (10 µM) upon addition
of N2H4 (0–2.0 equiv.) in ~100% aqueous solution; Inset: Plot of
fluorescence emission intensity (615 nm) of CBI versus [N2H4]. (b)
Fluorescence emission intensity (615 nm) changes of the CBI with
gradual addition of N2H4 (0–10 µM); Inset: Photographs of CBI treated
with various N2H4 concentrations.
The time-dependent fluorescent response of CBI with 2.0 equivalent
of hydrazine was studied (Fig. S5). It was clearly found that the emission
intensity at 615 nm decreased immediately and achieved a stable state at
50 s, indicating that the reaction is complete within 50 s, and the CBI was
quite useful for ultrarapidly detecting hydrazine.
The recognition behavior of the CBI for hydrazine at varied pH was
also studied (Fig. S6). When the pH value was varied from 1.0 to 12.0,
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free CBI solution showed negligible fluorescence signal change,
indicating that CBI is pH-uninfluenced and high stable in a wide pH
range. However, when 2.0 equiv. hydrazine was added, the fluorescence
intensity of the CBI at 615 nm sharply decreased and kept stable in the
pH range of 5.0–10.0, which was ascribed to the nucleophilicity of
hydrazine under alkaline condition. Whereas, under the acidic conditions
(pH < 4), the fluorescence intensity had no apparent change due to the
protonation of hydrazine at low pH value, and weaken the nucleophilicity
of hydrazine [55]. It is indicated that CBI can selectively detect N2H4 in a
wide pH range of 5.0–10.0 including physiological conditions.
3.3. Response mechanism studies
To further confirm the interaction mechanism of CBI with N2H4, the
reaction of CBI with N2H4 was conducted. The product of CBI reacted
with N2H4 was subjected to 1H NMR, FTIR and HRMS spectral analysis.
1
The H NMR of CBI in DMSO-d6 with/without hydrazine was firstly
examined. As observed from Fig. 4, after the CBI being treated with
hydrazine (1.0 equiv.), the characteristic proton signal for Ha (at 9.51 ppm)
of CBI shifted to 8.09 ppm, which matched the characteristic chemical
shift for Ha' of the byproduct. The aromatic proton signals (Hb at 8.78
ppm and Hc at 8.16 ppm) of CBI completely disappeared, and a proton
signal Hd at 6.41 ppm (C=NNH2) obviously appeared, indicating the
decomposition of CBI by hydrazine and formation of a corresponding
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hydrazone. The FTIR spectra of CBI before and after N2H4 provided
another proof (Fig. S7). When treated with N2H4, it was clearly observed
that the characteristic stretching vibration absorption peak at 1670 cm–1
owing to carbonyl (C=O) group disappeared completely, together with a
new absorption band emerged at 1625 cm–1 due to aldimine (C=N) group.
Meanwhile, the appearance of new double absorption bands emerged at
3323 and 3394 cm–1 due to the hydrazone amine (C=N-NH2) group. The
reaction product of CBI and hydrazine was further identified by HRMS
spectrum (Fig. S8). The HRMS spectrum showed a strong peak at m/z =
252.1463, which was identical to the corresponding adduct product 3
([M+H]+ = 252.1422). Additionally, the Job‟s plot analysis further proved
the 1:1 stoichiometric reaction between CBI and N2H4 (Fig. S9). Based
on the above facts, we concluded that the new species 3 was the obtained
adduct product hydrazone by the CBI reacting with N2H4, which blocked
the ICT process and caused the fluorescence quenching because of the
weaken withdrawing ability of the hydrazone amine group (Scheme 2).
The proposed sensing mechanism was similar to that of the previously
reported literature [56–61].
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Ha'
d
Ha
HC
Hb
10
9
8
7
6
5
δ/ ppm
1
Fig. 4. H NMR spectra before and after adding N2H4 to CBI (1.0 equiv)
in DMSO-d6 solution.
Scheme 2 The proposed mechanism of CBI for sensing N2H4.
3.4. Application in water and urine samples
In terms of the excellent sensitivity of CBI for hydrazine sensing, the
ability of the CBI to quantitatively detect hydrazine in distilled water, tap
water, domestic water, Dacheng river water in Qilu University of
Technology, the Yellow River water, and human urine samples was
investigated applying a standard addition method. These crude samples
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were firstly passed via a microfiltration membrane (0.22 µm), and then
spiked with different hydrazine concentrations (2.5, 5 and 10 µM). As
shown in Table 1, the tested results were consistent with the actual added
amounts of hydrazine in each sample (recovery 98.2%～101.3% with low
RSD values). The spiked recoveries obtained were favorably compared
with those reported recoveries (97.5%～100.3%) using a standard method
[62], indicating the feasibility of the sensor CBI for sensitively
monitoring hydrazine in practical samples.
Table 1 Quantitative detection of N2H4 in real water and human urine
samples.
Sample
Added
(μM)
2.5
Recovery
(%)
RSD
(%)
Standard
error
Detected (x±SD)
(μM)
Distilled water
2.51±0.026
5.01±0.035
10.04±0.135
2.49±0.020
5.00±0.052
9.93±0.072
2.52±0.025
4.99±0.025
10.11±0.100
2.50±0.005
4.98±0.035
9.82±0.096
2.48±0.025
4.97±0.007
10.04±0.067
2.47±0.006
5.07±0.186
10.00±0.038
100.4
100.2
100.4
99.6
1.05
0.70
1.34
0.80
1.04
0.73
1.00
0.51
0.99
0.23
0.71
0.98
1.01
1.41
0.66
0.23
2.67
0.38
0.027
0.029
0.110
0.016
0.042
0.059
0.020
0.021
0.082
0.005
0.029
0.078
0.020
0.057
0.054
0.005
0.152
0.031
5
10
Dacheng river water 2.5
5
100
10
99.3
Tap water
2.5
5
100.8
99.8
10
101.1
100
The Yellow river 2.5
water
5
99.6
10
2.5
5
98.2
Domestic water
99.2
99.4
10
2.5
5
100.4
98.8
Human urine
101.3
99.6
10
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3.4. Application as fluorescent ink
Taking the advantage of the strong fluorescence, the CBI could be
used as a new type UV-actived fluorescent ink with a broad application
prospect. The homogeneous aqueous solution of the CBI (10 µM) was
injected into a vacant pen without any modification, which was directly
used as fluorescent ink to write Chinese characters and patterns on the
handwritten filter paper. As displayed in Fig. 5, all the Chinese characters
and patterns emitted yellow fluorescence under UV light, which are
highly distinct from the background. These results suggests that the CBI
ink could be an alternative for traditional fluorescent pens.
Fig. 5. The photographic images of the Chinese characters and
fluorescent patterns written on filter paper using aqueous CBI solution
under a UV light.
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3.5. Application as fluorescent display material
Considering its good fluorescent performance, the CBI could act as
a writable molecular smart fluorescent display material. The CBI thin
film was prepared by spreading out a CBI solution on the TLC plate.
After being air-dried, a writing brush dipped in aqueous hydrazine (10
µM) was used to write the chemical formula “N2H4” on the film.
Interestingly, under irradiation with 365 nm UV lamp, the bright yellow
fluorescence was immediately changed to blue at a specific position by
hydrazine exposure (Fig. 6). Thus, the simple CBI based thin-film could
not only be utilized as a convenient hydrazine-sensitive solid fluorescent
sensor but also as a writable fluorescent display material.
Fig. 6. The preparation and application of the sensor CBI based thin-film.
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3.6. Visualization of gaseous hydrazine
Inspired by its excellent performance, the potential utility of CBI for
sensitively visualize gaseous hydrazine was further evaluated. The
filter-paper was firstly soaked in a stock solution of CBI. After air-dried,
the CBI-loaded test strips were exposed to hydrazine vapor by being
placed in sealed bottles containing different hydrazine concentrations
(blank, 5%, 10%, 15%, 20%, 25%, w/w ) for 10 min [63]. As shown in
Fig. 7, as increasing the hydrazine concentration, distinctive color
changes of the CBI-coated filter-papers were displayed from yellow to
gray and the fluorescence color turn from bright yellow into white gray,
which were highly dependent on the hydrazine concentration and easily
distinguished by the naked eyes. These findings demonstrate that the
CBI-loaded test strips approach is more convenient and instant
visualization of gaseous hydrazine.
Fig. 7. The color variations of CBI-coated filter paper when exposed to
hydrazine vapor for 10 min under ambient light and UV lamp (365 nm).
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3.7. Bioimaging in live cells
The cytotoxicity of CBI was tested by a standard MTT assay toward
HeLa cells. As illustrated in Fig. 8a, the cell viability is above 90% at the
concentrations ranging from 0 to 30.0 µM for 24 hours. The results
proved that the CBI has no marked cytotoxicity and suitable for
bioimaging hydrazine in living cells. As displayed in Fig. 8b, after
incubating the 25 µM CBI to HeLa cells for 30 min, the cells showed a
remarkable red fluorescence, revealing CBI has good cell permeability.
However, after the cells being treated with N2H4 (1.0 equiv) for 30 min,
the red fluorescence was completely quenched, and almost no detectable
fluorescence signal was found (Fig. 8c). This implied that the CBI was
effectively attacked by N2H4 in live cells, and quenched drastically the
fluorescence. These results demonstrate that CBI can be used for imaging
N2H4 in cellular environment.
(a)
(b)
(c)
Fig. 8 (a) The cell viability of HeLa cells upon treatment of various
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concentrations of CBI; Confocal fluorescence images of HeLa cells
incubated with CBI before (b) and after (c) being treated with N2H4 for
0.5 h. Scale bar: 25 μm.
3.8. Comparison with other selective-N2H4 fluorescent sensors
Compared with some previously N2H4-selective fluorescent sensors
[59,64–67] (Table S1), our developed N2H4-selective sensor CBI showed
multitudinous attractive advantages of interesting AIE property,
red-emitting with large Stokes shift, wide pH-range tolerance, ultrafast,
colorimetric and fluorimetric responses, which was used for fluorescent
ink, writable fluorescent display material, aqueous and gaseous detection,
bioimaging, environmental and biological samples monitoring.
4. Conclusions
In summary, we reported a novel carbazole-indandione based
red-emitting fluorescent sensor CBI, which displayed an interesting AIE
active feature at a long wavelength of 615 nm and a large Stokes shift.
Compared with presently N2H4-selective fluorescent sensors, CBI shows
ultrafast response, specific selectivity, great sensitivity and
colorimetric/fluorimetric responses. The mechanism was confirmed by
1
fluorescence, H NMR and HRMS spectra. Interestingly, the CBI thin
film was prepared and displayed a remarkable luminescence response to
hydrazine with great sensitivity, which can serve as a writable fluorescent
display material. Besides, owing to its strong luminescence irradiated by
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UV light, the CBI was significantly utilized as a new fluorescent ink for
writing and drawing. Notably, the CBI-loaded test paper conveniently
and sensitively detect gaseous hydrazine by color and fluorescence
changes. Importantly, the CBI was used for sensitive detection of
hydrazine in water, urine samples and cellular imaging, demonstrating its
great potential in practical applications.
Acknowledgments
We greatly appreciate financial support from the National Natural
Science Foundation of China (21801144, 21776143, 51702178), the
Natural Science Foundation of Shandong Province (ZR2017LB009), the
Ji Nan Science & Technology Bureau (2019GXRC021), the Youth
Innovative Talents Recruitment and Cultivation Program of Shandong
Higher Education, and the Program for Scientific Research Innovation
Team in Colleges and Universities of Shandong Province.
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