A dual-analytes responsive fluorescent probe for discriminative detection of ClO? and N2H4 in living cells

Article abstract of DOI:10.1016/j.saa.2020.118953

Hydrazine (N₂H₄) and ClO^? are very harmful for public health, hence it is important and necessary to monitor them in living cells. Herein, we rationally designed and synthesized a dual-analytes responsive fluorescent sensor PTMQ for distinguishing detection of N₂H₄ and ClO^?. PTMQ underwent N₂H₄-induced double bond cleavage, affording colorimetric and green fluorescence enhancement with good selectivity and a low detection limit (89 nM). On the other hand, PTMQ underwent ClO^?-induced sulfur oxidation and displayed red fluorescence lighting-up response towards ClO^? with good selectivity, rapid response (<0.2 min) and a low detection limit (58 nM). Moreover, PTMQ was successfully employed for in-situ imaging of N₂H₄ and ClO^? in living cells.

Full text of DOI:10.1016/j.saa.2020.118953

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 246 (2021) 118953
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
journal homepage: www.elsevier.com/locate/saa
A dual-analytes responsive fluorescent probe for discriminative
detection of ClO− and N2H4 in living cells
c,
b,
Beitong Zhu a,b,1, Xiaoli Wu a,1, João Rodriguesc, Xichao Hua, , Ruilong Sheng , Guang-Ming Bao
⁎
⁎
⁎
a
School of Food and Drug, Luoyang Normal University, Luoyang 471934, PR China
Institute of Veterinary Drug, Jiangxi Agricultural University, Nanchang 330045, PR China
b
c
CQM-Centro de Quimica da Madeira, Universidade da Madeira, Campus da Penteada, 9000-390 Funchal, Madeira, Portugal
a r t i c l e i n f o
a b s t r a c t
Hydrazine (N2H4) and ClO− are very harmful for public health, hence it is important and necessary to monitor
them in living cells. Herein, we rationally designed and synthesized a dual-analytes responsive fluorescent sensor
PTMQ for distinguishing detection of N2H4 and ClO−. PTMQ underwent N2H4-induced double bond cleavage,
affording colorimetric and green fluorescence enhancement with good selectivity and a low detection limit (89
nM). On the other hand, PTMQ underwent ClO−-induced sulfur oxidation and displayed red fluorescence
lighting-up response towards ClO− with good selectivity, rapid response (<0.2 min) and a low detection limit
(58 nM). Moreover, PTMQ was successfully employed for in-situ imaging of N2H4 and ClO− in living cells.
© 2020 Elsevier B.V. All rights reserved.
Article history:
Received 14 August 2020
Received in revised form 6 September 2020
Accepted 9 September 2020
Available online 17 September 2020
Keywords:
Fluorescent probe
Dual-analytes
N2H4
ClO−
Bioimaging
Living cells
1. Introduction
in which the reactive sites including methylene malononitrile [12],
phthalimides [13,14] and carboxylates [15,16] served as hydrazine-
Hydrazine (N2H4) is an essential chemical substance, which has
been widely used in chemical synthesis and catalysis, as well as in the
pharmaceutical industry [1,2]. Hydrazine is generally produced by hy-
pochlorite (ClO−)-mediated oxidation of ammonia or urea. Hence, the
discharged industrial wastewater from hydrazine manufacture usually
contains hydrazine and hypochlorite, which would be taken up by
aquatic organisms/microorganisms and cause extremely toxic effect
on them. Besides, N2H4 can cause severe damage to the liver, kidneys,
lungs, central nervous system, and the respiratory system. For this rea-
son, N2H4 has been categorized into a highly toxic and carcinogenic sub-
stance (B2 grade) by US government [3], and the concentration of N2H4
in water should be lower than 10 ppb [4]. Therefore, it is of great impor-
tance to monitor hydrazine in living cells/organisms.
specific moieties. Recently, Zeng et al. developed a coumarin-based
chemosensor for discriminative detection of hydrazine and bisulfate
via hydrazine-induced ethyl cyanoacetate cleavage and bisulfate-
involved Michael addition [17]. Likewise, several fluorescent sensors
for visual detection of HOCl/ClO− have been explored on the basis of
the ClO−-mediated oxidation reaction of various functional groups
such as ether [18], p-methoxyphenol [19], thioether [20,21], oxime
[22–24], hydroxamic acid [25] and C_C double bond [26,27]. Although
the above-mentioned progresses have been made, until now, two
analytes ClO− and N2H4 hardly be distinguishingly determined by
using a simple, effective and visible chemosensor.
It is disclosed that integration of two different reaction/binding sites
into a single fluorescent probe could be an efficient approach to achieve
discriminative detection of two analytes [17,26–28]. In this study, we
designed a dual-analytes responsive fluorescent probe PTMQ for the
distinguishing detection of ClO− and N2H4. Probe PTMQ possesses
two reaction sites: (1) the sulfur atom (\\S\\) located at the 9-
position of phenothiazine serves as a reaction site for ClO−; (2) the
MQ moiety acts as the reactive site for N2H4. We anticipated that
PTMQ would separately react with HClO and N2H4, generating fluores-
cent products with different color/fluorescence signal output. We inves-
tigated the colorimetric and fluorescence responses, selectivity and
sensitivity, detection limit, as well as time-dependent fluorescence
Fluorescent probes have emerged as remarkable sensing tools for
the detection of cationic, anionic, and biomolecules [5–11] due to the
advantages of visual signal output, good selectivity, high sensitivity,
and real-time monitoring ability. Up to date, a great many of reaction-
based fluorescent probes were adopted for the detection of hydrazine,
⁎
Corresponding authors.
E-mail addresses: hxc30@163.com (X. Hu), ruilong.sheng@staff.uma.pt (R. Sheng),
bycb2005@gmail.com (G.-M. Bao).
1
These authors contributed equally to this work.
https://doi.org/10.1016/j.saa.2020.118953
1386-1425/© 2020 Elsevier B.V. All rights reserved.

B. Zhu, X. Wu, J. Rodrigues et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 246 (2021) 118953
response of PTMQ to ClO− and N2H4, respectively. Moreover, PTMQ was
used to in-situ image ClO− and N2H4 in living human cervical cancer
HeLa cells.
temperature. The precipitate was collected by filtration, followed by re-
crystallization in ethanol to give PTMQ as purple solid (332 mg, yield:
68%). 1H NMR (400 MHz, DMSO‑d6) δ 9.26 (d, J = 6.6 Hz, 1H), 9.03 (d,
J = 8.6 Hz, 1H), 8.52 (d, J = 8.9 Hz, 1H), 8.41 (d, J = 6.6 Hz, 1H), 8.25
(d, J = 7.2 Hz, 1H), 8.20 (d, J = 5.8 Hz, 2H), 8.03 (s, 2H), 7.30 (t, J =
7.7 Hz, 1H), 7.25 (d, J = 7.8 Hz, 2H), 7.10–7.04 (m, 1H), 6.94 (s, 1H),
4.99 (d, J = 7.2 Hz, 2H), 4.83 (s, 2H), 4.01 (s, 3H), 3.59 (s, 1H), 1.57 (t,
J = 7.1 Hz, 3H). 13C NMR (100 MHz, DMSO‑d6) δ 159.0, 153.4, 147.9,
147.3, 142.8, 138.1, 136.5, 135.5, 129.4, 128.2, 127.42, 127.2, 127.1,
126.6, 124.2, 122.4, 119.4, 119.2, 118.1, 116.3, 116.1, 113.8, 100.3, 79.5,
77.8, 56.6, 52.4, 38.0, 15.6. HR-MS (ESI, m/z): calcd. for
[C29H25N2OS]+: 449.1682; found: 449.1699.
2. Experimental
2.1. Chemicals and instrumentations
All chemicals were commercially purchased from Sigma-Aldrich and
used directly without further purification. Compound 1 (N-ethyl-4-
methylquinolinium iodide, MQ) was prepared according to the litera-
ture [29]. 1H NMR (400 MHz) and 13C NMR (100 MHz) spectra were
measured on a Bruker AV spectrometer. High resolution mass spec-
trometry (HRMS) was obtained on HP-1100 LC-MS spectrometer. UV–
Vis absorption spectra were performed on a Hitachi UV-3310 spectrom-
eter. Fluorescence spectra were obtained with a FL-4500 fluorescence
spectrometer. The cells imaging experiments were taken under a
Nikon A1 confocal laser-scanning microscope with a 100 × objective
lens.
2.5. General procedures for spectral measurements
10 μM of PTMQ solution in DMSO/PBS (v/v = 2/8, pH = 7.4) was
used for spectral measurements. Optical tests of PTMQ for ClO− and
N2H4 were performed as follows: a) PTMQ (10 μM) was pre-
incubated with ClO− for 5 min at room temperature. The UV–Vis ab-
sorption and the fluorescence spectra were recorded directly. Excitation
wavelength was 460 nm with slit widths 5/5 nm; b) PTMQ (10 μM) was
pre-incubated with N2H4 for 30 min at room temperature. Then the UV–
Vis absorption and the fluorescence spectra were measured directly. Ex-
citation was at 360 nm with slit widths 2.5/5 nm.
2.2. Synthesis of compound 2 (N-propagyl-8-methoxy-phenothiazine)
In a 250 mL flask, 2-methoxyphenothiazine (500 mg, 2.18 mmol), 3-
bromopropyne (1.30 g, 10.90 mmol) and KI (166 mg, 1.0 mmol) were
dissolved in 15 mL anhydrous DMF. The mixture was stirred at 100 °C
for 8 h under N2 atmosphere. After cooling down to room temperature,
the resulted mixture was poured into 100 mL H2O and extracted three
times with CH2Cl2. Then, solvent of the collected organic layer was re-
moved under reduced pressure. The obtained crude product was further
purified by silica gel flash chromatography (petroleum ether/ethyl
acetate = 15:1, v/v) to give compound 2 (N-propagyl-8-methoxy-
phenothiazine) as colorless solid (495 mg, yield: 85%). 1H NMR (400
MHz, DMSO‑d6) δ 7.24 (t, J = 7.7 Hz, 1H), 7.21–7.12 (m, 2H), 7.07 (d, J
= 8.4 Hz, 1H), 6.99 (t, J = 7.4 Hz, 1H), 6.79 (s, 1H), 6.62 (d, J = 8.4
Hz, 1H), 4.64 (s, 2H), 3.76 (s, 3H), 3.50 (s, 1H). 13C NMR (100 MHz,
DMSO‑d6) δ 159.9, 145.4, 143.8, 127.9, 127.7, 127.2, 123.4, 123.1,
115.6, 113.2, 107.8, 103.4, 79.9, 77.4, 55.8, 38.0.
2.6. Fluorescence imaging of ClO− and N2H4 by PTMQ in HeLa cells
HeLa cells were seeded in glass-bottomed dishes and cultured in
DMEM culture medium with 10% FBS at 37 °C for 24 h. For imaging
ClO− and N2H4, the HeLa cells were washed with PBS three times to re-
move culture medium and then pre-treated with PTMQ (10 μM) for 30
min. Afterwards, the cells-containing dishes were washed there times
with PBS, and mounted on the stage of confocal laser-scanning micro-
scope. Before fluorescence imaging, the HeLa cells were further co-
incubated with different concentrations of ClO− and N2H4 for another
30 min, respectively.
3. Results and discussion
2.3. Synthesis of compound 3 (N-propagyl-8-methoxy-phenothiazine-7-al-
dehyde, PT)
3.1. Design and synthesis of PTMQ
Phosphorous oxychloride (0.52 mL, 5.61 mmol) was dropwise
added to anhydrous dimethylformamide (0.43 mL, 5.61 mmol) at 0 °C
under a N2 atmosphere. After the mixture was stirred at 0 °C for 30
Herein, a simple synthetic route was established to prepare the de-
sired dual-analytes responsive fluorescent probe PTMQ, as depicted in
Scheme 1. 4-Methyl-quinoline was quaternized with ethyl iodide to
prepare N-ethyl-4-methyl-quinolinium iodide (compound 1, MQ) via
N-alkylation reaction. The fluorescent precursor 8-methoxy-phenothia-
zine was coupled with propagyl bromide to prepare compound 2, which
underwent a Vilsmeier reaction (POCl3/DMF) to prepare N-propagyl-8-
methoxy- phenothiazine-7-aldehyde (compound 3, PT). Furthermore,
MQ reacted with PT to prepare the dual-analytes responsive fluorescent
probe PTMQ via one-step condensation approach. The structure of
PTMQ was analyzed by 1H NMR, 13C NMR and HR-MS (shown in
Figs. S1–S7, Supporting information).
min, compound
2 (300 mg, 1.12 mmol) in 5 mL anhydrous
dimethylformamide was dropwise added to the above solution. The
reacting mixture was heated to 60 °C and stirred for 5 h. Then the
resulting mixture was poured into ice water, and the reaction solution
was neutralized to pH 7 by NaOH solution (20%) until a large amount
of solid precipitated. After filtration, the solid was washed with distilled
water (3 × 10 mL), and dried in vacuum and then recrystallized from
ethanol to offer compound 3 (N-propagyl-8-methoxy-phenothiazine-
7-aldehyde, PT) as yellow solid (252 mg, yield: 78%). 1H NMR (400
MHz, DMSO‑d6) δ 10.14 (s, 1H), 7.40 (s, 1H), 7.32–7.26 (m, 1H), 7.24
(d, J = 8.3 Hz, 1H), 7.21 (d, J = 6.2 Hz, 1H), 7.06 (t, J = 8.1 Hz, 1H),
6.91 (s, 1H), 4.83 (s, 2H), 3.97 (s, 3H), 3.58 (s, 1H)·13C NMR (100
MHz, DMSO‑d6) δ 186.8, 162.6, 151.0, 142.2, 128.3, 127.4, 125.9, 124.5,
122.3, 119.7, 116.3, 113.6, 100.3, 79.3, 77.9, 56.5, 38.6.
3.2. Sensing performance of PTMQ for ClO− and N2H4
The UV–Vis and fluorescent spectra titration of PTMQ with HClO and
N2H4 were measured, respectively (Figs. 1 and 2). As shown in Fig. 1,
PTMQ itself is non-fluorescence in DMSO/PBS solution due to the strong
intramolecular charge transfer (ICT) effect from sulfur (\\S\\) and ni-
trogen (\\N\\) atoms in phenothiazine to quinolinium [30]. Upon the
addition of an increasing amount of ClO−, the absorption band of
PTMQ at 518 nm gradually decreased with the increase of a new band
at 465 nm, and simultaneously the color of the PTMQ solution turned
from deep purple to light pink. The isosbestic point in the absorption ti-
tration spectra implied that PTMQ has transformed to a new compound
2.4. Synthesis of probe PTMQ
Compound 1 (MQ, 380 mg, 1.27 mmol) and compound 3 (PT, 250
mg, 0.85 mmol) were dissolved in 15 mL anhydrous EtOH. Afterward,
piperidine (0.1 mL) and AcOH (0.1 mL) were added to the solution as
catalysts. The mixture was refluxed at 80 °C for 6 h under nitrogen at-
mosphere. Precipitate was formed after cooling down to room
2

B. Zhu, X. Wu, J. Rodrigues et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 246 (2021) 118953
Scheme 1. Synthetic route of probe PTMQ
after reaction with ClO−. Meanwhile, a fluorescence band at 577 nm
was lighted-up, which progressively rose up with the increasing
amount of ClO− from 0 to 500 μM (Fig. 1B). Notably, a good linear cor-
relation between fluorescence intensity at 577 nm (F577, R2 = 0.9912)
and concentration of ClO− (0–500 μM) was observed (Fig. 1C), and
the detection limit was calculated as 58 nM (S/N = 3). Moreover, the
time-dependent fluorescence profile showed that F577 of PTMQ quickly
rose up and reached a stable plateau within 12 s (0.2 min) after addition
of ClO− (500 μM). This observation suggests that PTMQ could be used as
a rapid-response probe for ClO− detection in aqueous solution.
band at 285 nm, and the color of PTMQ solution changed from deep
purple to colorless in synchrony. Notably, a green fluorescence band
emerged at 500 nm, which were becoming much stonger with the in-
crease of N2H4 amount from 0 to 900 μM (Fig. 2B). Likewise, a perfect
linear correlation between fluorescence intensity at 500 nm (F500, R2
= 0.9930) and N2H4 concentration (0–900 μM) was observed
(Fig. 2C), and the detection limit was calculated as 89 nM (S/N = 3).
From the time-dependent fluorescence enhancement profile, it could
be observed that after the addition of N2H4 (900 μM), F500 of PTMQ
gradually enhanced and reached a stable plateau within 24 min, imply-
ing that PTMQ took a relatively slow chemical reaction with N2H4.
Therefore, it provides a temporal and spectral discriminative manner
to determine N2H4 and ClO−.
The spectral responses of PTMQ towards N2H4 were shown in Fig. 2.
With the addition of increasing amount of N2H4, the absorption band of
PTMQ at 518 nm gradually decreased along with the increase of a new
Fig. 1. (A) The UV–vis absorption spectra and (B) the fluorescence spectra of PTMQ (10 μM) upon addition of various concentrations of ClO− (0–50 eq) in DMSO/PBS solution (v/v = 2:8,
pH = 7.4). Inset: colorimetric or fluorescent responses of PTMQ towards ClO− under daylight or 365 nm light. (C) The linear relationship between fluorescence intensity of PTMQ (10 μM)
and concentrations of ClO−. (D) Time-dependent fluorescence response of PTMQ (10 μM) to ClO− (500 μM) in DMSO/PBS solution (v/v = 2/8, pH = 7.4). λex = 460 nm, slits: 5 nm/5 nm.
3

B. Zhu, X. Wu, J. Rodrigues et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 246 (2021) 118953
Fig. 2. (A) The UV–vis absorption spectra and (B) the fluorescence spectra of PTMQ (10 μM) upon addition of various concentrations of N2H4 (0–100 eq) in DMSO/PBS solution (v/v = 2:8,
pH = 7.4). Inset: colorimetric or fluorescent responses of PTMQ towards N2H4 under daylight or 365 nm light. (C) The linear relationship between fluorescence intensity of PTMQ (10 μM)
and concentrations of N2H4. (D) Time-dependent fluorescence response of PTMQ (10 μM) to N2H4 (900 μM) in DMSO/PBS solution (v/v = 2/8, pH = 7.4). λex = 360 nm, slits: 2.5 nm/5 nm.
3.3. Effect of pH on the sensing performance
within the pH range of 3.0–10.0, as shown in Fig. 3B. The addition of
N2H4 (900 μM) also led to a sharp fluorescence enhancement at pH
6.0–9.5. Therefore, PTMQ itself owns good pH stability and remarkable
fluorescence response to ClO− and N2H4 at pH 7.4, which is suitable for
intracellular bioimaging applications.
As an important environmental factor, pH value usually has a large
impact on the chemical stability and sensing capability of fluorescence
probes [31]. Herein, the pH-dependent fluorescence response of
PTMQ (10 μM) to ClO− (500 μM) and N2H4 (900 μM) were investigated,
respectively. As depicted in Fig. 3A, the fluorescence intensity at 577 nm
(F577) of PTMQ almost maintained constant within the pH range of
3.0–9.5. Upon the addition of ClO− (500 μM) to PTMQ solution, an obvi-
ous fluorescence enhancement was observed at pH 5.0–8.5. Whereas,
the fluorescence intensity at 500 nm (F500) of PTMQ also kept constant
3.4. The selectivity
For practical detection, a favorable fluorescent probe should have
high selectivity to the analyte. Herein, we tested the selectivity of
PTMQ (10 μM) to ClO−(500 μM), N2H4 (900 μM) and various species
(A)
(B)
1600
PTMQ
PTMQ + N2H4
PTMQ
PTMQ + HClO
3200
2400
1600
800
0
1200
800
400
0
3
4
5
6
7
8
9
10
3
4
5
6
7
8
9
10
PH
pH
Fig. 3. Fluorescence intensity of PTMQ (10 μM) to (A) ClO− (λex = 460 nm, slits: 5 nm/5 nm) and (B) N2H4 (λex = 360 nm, slits: 2.5 nm/5 nm) in DMSO/PBS solution (v/v = 2/8) at various
pH values.
4

B. Zhu, X. Wu, J. Rodrigues et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 246 (2021) 118953
Fig. 4. (A) The fluorescent responses of PTMQ (10 μM) to ClO− (500 μM) and other analytes (500 μM). λex = 460 nm, slits: 5 nm/5 nm; (B) The fluorescent responses of PTMQ (10 μM) to
N2H4 (900 μM) and other biological molecular species (900 μM). λex = 360 nm, slits: 2.5 nm/5 nm. Inset: Color and fluorescence photograms of PTMQ (10 μM) in the presence of various
analytes.
including anions (AcO−, Br−, Cl−, CO32−, HS−, HSO−3 , NO2−, S2O32−, SO23−
and SO24−), metal cations (Zn2+, Al3+, Ba2+, Ca2+, Cu2+, Fe2+, Fe3+, K+,
Mg2+, Na+, Ni2+) as well as biothiols (Cys and GSH). As shown in
Fig. 4A, negligible fluorescence and color changes of PTMQ were ob-
served in the presence of various species except ClO−. In contrast, the
addition of ClO− brought an evident bathochromic shift from purple
to light pink with an obvious fluorescence enhancement at 577 nm.
Likewise, PTMQ (10 μM) also demonstrated remarkable green fluores-
cence “turn-on” response to N2H4 (100 μM) along with color change
from purple to colorless (Fig. 3B). The results indicated that probe
PTMQ possesses excellent selectivity to ClO− and N2H4, which might
be employed as a potential tool for real sample detection.
(\\S\\) in phenothiazine was converted into electron-withdrawing
sulfoxide (-S=O) group after reaction with ClO− [30], which blocked
the intramolecular charge transfer effect (ICT), and thus leading to a sig-
nificant red fluorescence enhancement. For the sensing mechanism of
N2H4, the HRMS spectra (Figs. S9 and S10) showed two main peaks at
m/z values of 310.1026 and 172.1146 after reaction with N2H4, which
were identified as PT-NHNH2 (calcd: 310.1051) and MQ (calcd:
172.1027), respectively. These observations indicated that the polarized
C_C bridge of PTMQ could be disrupted by nucleophilic N2H4 to form
PT-NHNH2 [17], which gave rise to green fluorescence emission. Based
on these observations, the proposed working mechanisms of PTMQ
for HClO/ClO− and N2H4 were depicted in Scheme 2.
3.5. Working mechanism
3.6. Cell imaging
To further understand the working mechanism, we made great ef-
forts to isolate the major products generated from the reactions of
PTMQ with ClO− and N2H4, respectively. Unfortunately, we could not
obtain the pure products for NMR analysis due to their strong polarity
and some complex fragments. So, we can only measure the HRMS spec-
tra of main products. After PTMQ was treated with ClO−, the HRMS
spectra of PTMQ (Fig. S8) showed a dominant peak located at m/z
value of 465.1649 (calcd: 465.1612), which was corresponding to
[PTMQ-O+]. It was proposed that the electron-donating sulfur atom
In view of the aforementioned excellent sensing properties, PTMQ
was utilized to separately image HClO and N2H4 in human cervical can-
cer (HeLa) cells [32,33]. Firstly, the HeLa cells were pre-incubated with
PTMQ (10 μM) at 37 °C for 30 min, and the fluorescence images were
recorded by laser confocal scanning microscopy. As illustrated in
Fig. 5A and B, for the blank cells, non-fluorescence could be observed
in red and green channels. Then, these HeLa cells were co-incubated
with different amounts of ClO− (0, 80 and 200 μM) and N2H4 (0, 300,
and 900 μM) for 30 min, and their fluorescence images were recorded.
Scheme 2. The proposed mechanism of PTMQ for ClO− and N2H4
5

B. Zhu, X. Wu, J. Rodrigues et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 246 (2021) 118953
Fig. 5. Fluorescence images of living HeLa cells treated with ClO− or N2H4. HeLa cells were pre- treated with PTMQ (10 μM) at 37 °C for 30 min, then washed with PBS and further co-
incubated with different concentrations of (A) ClO− (0, 80 and 200 μM), fluorescence images from the red channel (λex = 543 nm, λem = 552–617 nm), and (B) N2H4 (0, 300 and
900 μM) for another 30 min, fluorescence images from the green channel (λex = 488 nm, λem = 500–530 nm). Scale bar: 20 μm.
6

B. Zhu, X. Wu, J. Rodrigues et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 246 (2021) 118953
The cells after incubation with 80 μM of ClO− displayed weak red fluo-
rescence emission, which became much brighter with the increase of
ClO− concentration from 80 μM to 200 μM. By contrast, the cells showed
clear green fluorescence at N2H4 concentration of 300 μM and remark-
able green fluorescence emission when the concentration rose up to
900 μM. These observations demonstrated that PTMQ could serve as
an efficient fluorescent probe for separately imaging of exogenous
ClO− and N2H4 in living cells. Moreover, it can be observed that the in-
tracellular green/red fluorescence of PTMQ induced by ClO− and N2H4
mainly localized in the cytoplasm within 30 min, suggesting that ClO−
and N2H4 can quickly penetrate and diffuse inside the HeLa cell, which
may eventually cause oxidative-injury and/or hepatic steatosis [34].
The dual-analytes responsive properties, high contrast ratio, and low
background interference, enable PTMQ to serve as a promising tool for
discriminatively bioimaging and monitoring ClO− and N2H4.
[2] Z. Zhao, G. Zhang, Y. Gao, X. Yang, Y. Li, A novel detection technique of hydrazine hy-
drate: modality change of hydrogen bonding-induced rapid and ultrasensitive col-
orimetric assay, Chem. Commun. 47 (2011) 12816–12818.
[3] J. Mondal, A.K. Manna, K. Rout, S.K. Singh, J.P. Naskar, G.K. Patra, A simple Schiff base
as selective and sensitive fluorescent-colorimetric hydrazine chemosensor, Int. J. En-
viron. Anal. Chem. 98 (2018) 1160–1174.
[4] J. Qian, C. Wen, J. Xia, Development of highly efficient chemosensors for Cu2+ and
N2H4 detection based on 2D polyaniline derivatives by template-free chemical po-
lymerization method, J. Hazard. Mater. 121902 (2019).
[5] N. Kwon, Y. Hu, J. Yoon, Fluorescent chemosensors for various analytes including re-
active oxygen species, biothiol, metal ions, and toxic gases, ACS Omega 3 (2018)
13731–13751.
[6] J. Yan, S. Lee, A. Zhang, J. Yoon, Self-immolative colorimetric, fluorescent and chemi-
luminescent chemosensors, Chem. Soc. Rev. 47 (2018) 6900–6916.
[7] D. Wu, A.C. Sedgwick, T. Gunnlaugsson, E.U. Akkaya, J. Yoon, T.D. James, Fluorescent
chemosensors: the past, present and future, Chem. Soc. Rev. 46 (2017) 7105–7123.
[8] M. Lan, S. Zhao, S. Wu, X. Wei, Y. Fu, J. Wu, P. Wang, W. Zhang, Optically tunable
fluorescent carbon nanoparticles and their application in fluorometric sensing of
copper ions, Nano Res. 12 (2019) 2576–2583.
[9] Y. Fu, S. Wu, H. Zhou, S. Zhao, M. Lan, J. Huang, X. Song, Carbon dots and a CdTe
quantum dot hybrid-based fluorometric probe for spermine detection, Ind. Eng.
Chem. Res. 59 (2020) 1723–1729.
[10] T. Xu, S. Zhao, X. Wu, L. Zeng, M. Lan, β-Cyclodextrin-promoted colorimetric and
fluorescence turn-on probe for discriminating highly toxic thiophenol from
biothiols, ACS Sustainable Chem. Eng. 8 (2020) 6413–6421.
[11] Y. Fu, S. Zhao, S. Wu, L. Huang, T. Xu, X. Xing, M. Lan, X. Song, A carbon dots-based
fluorescent probe for turn-on sensing of ampicillin, Dyes Pigments 172 (2020)
107846.
[12] X.-X. Zheng, S.-Q. Wang, H.-Y. Wang, R.-R. Zhang, J.-T. Liu, B.-X. Zhao, Novel
pyrazoline-based selective fluorescent probe for the detection of hydrazine,
Spectrochim, Acta A 138 (2015) 247–251.
[13] L. Wang, F.-y. Liu, H.-y. Liu, Y.-s. Dong, T.-q. Liu, J.-f. Liu, Y.-w. Yao, X.-j. Wan, A novel
pyrazoline-based fluorescent probe for detection of hydrazine in aqueous solution
and gas state and its imaging in living cells, Sens. Actuators B Chem. 229 (2016)
441–452.
[14] R. Maji, A.K. Mahapatra, K. Maiti, S. Mondal, S.S. Ali, P. Sahoo, S. Mandal, M.R. Uddin,
S. Goswami, C.K. Quah, H.-K. Fun, A highly sensitive fluorescent probe for detection
of hydrazine in gas and solution phases based on the Gabriel mechanism and its
bioimaging, RSC Adv. 6 (2016) 70855–70862.
[15] C. Hu, W. Sun, J. Cao, P. Gao, J. Wang, J. Fan, F. Song, S. Sun, X. Peng, A ratiometric
near-infrared fluorescent probe for hydrazine and its in vivo applications, Org.
Lett. 15 (2013) 4022–4025.
[16] G. Yu, Y. Cao, H. Liu, Q. Wu, Q. Hu, B. Jiang, Z. Yuan, A spirobenzopyran-based mul-
tifunctional chemosensor for the chromogenic sensing of Cu2+ and fluorescent
sensing of hydrazine with practical applications, Sens. Actuators B Chem. 245
(2017) 803–814.
[17] J. Wu, J. Pan, Z. Ye, L. Zeng, D. Su, A smart fluorescent probe for discriminative detec-
tion of hydrazine and bisulfite from different emission channels, Sens. Actuators B
Chem. 274 (2018) 274–284.
[18] J. Shepherd, S.A. Hilderbrand, P. Waterman, J.W. Heinecke, R. Weissleder, P. Libby, A
fluorescent probe for the detection of myeloperoxidase activity in atherosclerosis-
associated macrophages, Chem. Biol. 14 (2007) 1221–1231.
4. Conclusion
In conclusion, we have designed and synthesized a dual-analytes re-
sponsive fluorescent probe PTMQ for distinguishing detection of ClO−
and N2H4 in aqueous solution. PTMQ exhibited a colorimetric and
ratiometric fluorescence response to N2H4 with good selectivity and a
low detection limit (89 nM). Meanwhile, PTMQ displayed red fluores-
cence lighting-up response to ClO− with good selectivity, rapid re-
sponse (<0.2 min) and a low detection limit (58 nM). Moreover,
PTMQ showed good biocompatibility and could be applied for in-situ
imaging of ClO− and N2H4 in living HeLa cells. This work provides an ef-
ficient way to create dual-analytes responsive fluorescent probe with
the advantages of visual detection, good selectivity, as well as in situ dis-
criminative bioimaging of two analytes in cancer cells.
CRediT authorship contribution statement
Beitong Zhu: Investigation, Writing - original draft. Xiaoli Wu: Meth-
odology, Investigation, Data curation. João Rodrigues: Writing - review &
editing. Xichao Hu: Methodology, Writing - original draft. Ruilong
Sheng: Project administration, Writing - review & editing. Guang-Ming
Bao: Conceptualization, Project administration, Supervision.
[19] Z.-N. Sun, F.-Q. Liu, Y. Chen, P.K.H. Tam, D. Yang, A highly specific BODIPY-based
fluorescent probe for the detection of hypochlorous acid, Org. Lett. 10 (2008)
2171–2174.
Declaration of competing interest
[20] Y. Koide, Y. Urano, K. Hanaoka, T. Terai, T. Nagano, Development of an Si-
rhodamine-based far-red to near-infrared fluorescence probe selective for
hypochlorous acid and its applications for biological imaging, J. Am. Chem. Soc.
133 (2011) 5680–5682.
[21] S. Kenmoku, Y. Urano, H. Kojima, T. Nagano, Development of a highly specific
rhodamine-based fluorescence probe for hypochlorous acid and its application to
real-time imaging of phagocytosis, J. Am. Chem. Soc. 129 (2007) 7313–7318.
[22] W. Lin, L. Long, B. Chen, W. Tan, A ratiometric fluorescent probe for hypochlorite
based on a deoximation reaction, Chem. Eur. J. 15 (2009) 2305–2309.
[23] X. Cheng, H. Jia, T. Long, J. Feng, J. Qin, Z. Li, A “turn-on” fluorescent probe for
hypochlorous acid: convenient synthesis, good sensing performance, and a new de-
sign strategy by the removal of C_N isomerization, Chem. Commun. 47 (2011)
11978–11980.
[24] G. Cheng, J. Fan, W. Sun, K. Sui, X. Jin, J. Wang, X. Peng, A highly specific BODIPY-
based probe localized in mitochondria for HClO imaging, Analyst 138 (2013)
6091–6096.
[25] Y.-K. Yang, H.J. Cho, J. Lee, I. Shin, J. Tae, A rhodamine–hydroxamic acid-based fluo-
rescent probe for hypochlorous acid and its applications to biological imagings, Org.
Lett. 11 (2009) 859–861.
[26] Q. Hu, C. Qin, L. Huang, H. Wang, Q. Liu, L. Zeng, Selective visualization of hypochlo-
rite and its fluctuation in cancer cells by a mitochondria-targeting ratiometric fluo-
rescent probe, Dyes Pigments 149 (2018) 253–260.
[27] J. Zha, B. Fu, C. Qin, L. Zeng, X. Hu, A ratiometric fluorescent probe for rapid and sen-
sitive visualization of hypochlorite in living cells, RSC Adv. 4 (2014) 43110–43113.
[28] J.-T. Hou, H.S. Kim, C. Duan, M.S. Ji, S. Wang, L. Zeng, W.X. Ren, J.S. Kim, A ratiometric
fluorescent probe for detecting hypochlorite in the endoplasmic reticulum, Chem.
Commun. 55 (2019) 2533–2536.
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgements
This work was sponsored by the NSFC (no. 31960720), Fundação
para a Ciência e a Tecnologia (FCT Base Fund - UIDB/00674/2020),
ARDITI-Agência Regional para o Desenvolvimento da Investigação
Tecnologia e Inovação through the project M1420-01-0145-FEDER-
000005-Centro de Química da Madeira-CQM+ (Madeira 14-20 Pro-
gram) and ARDITI-2017-ISG-003.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.saa.2020.118953.
References
[29] A.J. Winstead, G. Nyambura, R. Matthews, D. Toney, S. Oyaghire, Synthesis of quater-
nary heterocyclic salts, Molecules 18 (2013) 14306–14319.
[30] H. Song, Y. Zhou, C. Xu, X. Wang, J. Zhang, Y. Wang, X. Liu, M. Guo, X. Peng, A dual-
function fluorescent probe: sensitive detection of water content in commercial
[1] M. Annalakshmi, P. Balasubramanian, S.-M. Chen, T.-W. Chen, One pot synthesis of
nanospheres-like trimetallic NiFeCo nanoalloy: a superior electrocatalyst for electro-
chemical sensing of hydrazine in water bodies, Sens. Actuators B Chem. 296 (2019)
126620.
7

B. Zhu, X. Wu, J. Rodrigues et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 246 (2021) 118953
products and rapid detection of hypochlorite with a large Stokes shift, Dyes Pig-
ments 162 (2019) 160–167.
[31] X. Wu, L. Zeng, B.-Q. Chen, M. Zhang, J. Rodrigues, R. Sheng, G.-M. Bao, A selective
cascade reaction-based probe for colorimetric and ratiometric fluorescence detec-
tion of benzoyl peroxide in food and living cells, J. Mater. Chem. B 7 (2019)
5775–5781.
[33] J. Xu, Y. Zhang, L. Zeng, J. Liu, J.M. Kinsella, R. Sheng, A simple naphthalene-based
fluorescent probe for high selective detection of formaldehyde in toffees and HeLa
cells via aza-cope reaction, Talanta 160 (2016) 645–652.
[34] S. Garrod, M.E. Bollard, A.W. Nicholls, S.C. Connor, J. Connelly, J.K. Nicholson, E.
Holmes, Integrated metabonomic analysis of the multiorgan effects of hydrazine
toxicity in the rat, Chem. Res. Toxicol. 18 (2005) 115–122.
[32] Z. Ye, C. Duan, R. Sheng, J. Xu, H. Wang, L. Zeng, A novel colorimetric and ratiometric
fluorescent probe for visualizing SO2 derivatives in environment and living cells,
Talanta 176 (2018) 389–396.
8