Novel triphenylamine-based fluorescent probe for specific detection and bioimaging of OCl?

Article abstract of DOI:10.1016/j.tet.2018.08.010

Hypochlorite (OCl^?) plays important roles both in physiological and pathological processes, the detection of which is of great significance. Herein, a novel fluorescent probe based on the triphenylamine-type schiff base derivative (TPAD) was developed to detect OCl^?. Probe TPAD exhibited specific fluorescence response toward ClO^? with the fluorescence quenching rate up to 80%, and the detection limit was estimated to be 0.8 μM. The sensing mechanism study demonstrated that TPAD reacted with ClO^? via an oxidation process, which was evidenced by MS and NMR characterization. Moreover, owing to the excellent sensing properties and negligible cytotoxicity, TPAD was successfully applied in the bioimaging of OCl^? in living A549 cells.

Full text of DOI:10.1016/j.tet.2018.08.010

Accepted Manuscript
Novel triphenylamine-based fluorescent probe for specific detection and bioimaging of
-
OCl
Yuliang Jiang, Sheng Zhang, Bingxiang Wang, Tao Qian, Can Jin, Shishan Wu, Jian
Shen
PII:
S0040-4020(18)30961-X
10.1016/j.tet.2018.08.010
TET 29730
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 7 June 2018
Revised Date: 18 July 2018
Accepted Date: 10 August 2018
Please cite this article as: Jiang Y, Zhang S, Wang B, Qian T, Jin C, Wu S, Shen J, Novel
-
triphenylamine-based fluorescent probe for specific detection and bioimaging of OCl , Tetrahedron
(2018), doi: 10.1016/j.tet.2018.08.010.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Novel triphenylamine-based fluorescent probe for specific detection
-
and bioimaging of OCl
a
a
a
d
b
c
a,c
Yuliang Jiang , Sheng Zhang , Bingxiang Wang , Tao Qian , Can Jin *, Shishan Wu *, Jian Shen *
a. Jiangsu Collaborative Innovation Center of Biomedical Functional Materials, Nanjing Normal University,
Nanjing 210023, China
b. Institute of Chemical Industry of Forest Products, Chinese Academy of Forestry, Key Laboratory of
Biomass Energy and Material of Jiangsu Province, Nanjing 210042, China
c. School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, China
d. Soochow Institute for Energy and Materials Innovations, College of Physics, Optoelectronics and Energy
&
Collaborative Innovation Center of Suzhou Nano Science and Technology, Soochow University, Suzhou
2
15006, China
Corresponding Authors
Email: envis@163.com (C. Jin), shishanwu@nju.edu.cn (S. Wu), jshen@njnu.edu.cn (J. Shen)
Abstract:
−
Hypochlorite (OCl ) plays important roles both in physiological and pathological
processes, the detection of which is of great significance. Herein, a novel fluorescent
probe based on the triphenylamine-type schiff base derivative (TPAD) was developed
-
-
to detect OCl . Probe TPAD exhibited specific fluorescence response toward ClO with
the fluorescence quenching rate up to 80%, and the detection limit was estimated to
-
be 0.8 μM. The sensing mechanism study demonstrated that TPAD reacted with ClO
via an oxidation process, which was evidenced by MS and NMR characterization.
Moreover, owing to the excellent sensing properties and negligible cytotoxicity, TPAD
−
was successfully applied in the bioimaging of OCl in living A549 cells.
Keywords: Hypochlorite; Detection limit; Triphenylamine; Fluorescence probe; Cell
imaging
Introduction
Reactive oxygen species (ROS) play crucial roles in many physiological and
1-3
pathological processes.
As one of the most important ROS in living body,
−
hypochlorite (OCl ) is generally produced in organisms from the myeloperoxidase
1

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(MPO) mediated peroxidation of chloride ions and hydrogen peroxide, which plays an
4
important role in immune system against inflammation and microorganisms. Owing
to its high reactivity and nonspecificity in physiological conditions, excessive or
−
misplaced production of OCl level could cause harmful chain effects to human
5
6
7
beings, including neuron degeneration, cardiovascular disease, lung injury,
8
9-11
atherosclerosis, and cancers.
Therefore, highly sensitive and selective detection
−
of short-lived OCl in living systems is meaningful and has attracted increasing
12, 13
attention.
Fluorescent sensing strategy has been demonstrated as an ideal tool for OCl
detection in both chemical sensing and biological imaging thanks to its high
−
1
4-18
sensitivity, low cost and technical simplicity.
Recently, a large number of
fluorescent probes have been exploited by utilizing specific recognition units to
−
19
distinguish HOCl/OCl from other ROS species, including p-methoxyphenol,
2
0
21
22, 23
24-26
oxime, selenide, thiol
and other functional groups (e.g. nanomaterial).
Nevertheless, many of these probes might encounter some issues, such as delayed
response,low sensitivity and requiring multi-step synthetic operation. Meanwhile,
−
due to the typical characteristics of OCl is highly reactive, short-lived under
physiological conditions, rapid response and high sensitivity is mostly desirable for
−
real-time monitoring of OCl fluctuation. Thus, it is eagerly desirable but still highly
challenging to design and develop advanced probes with high sensitivity, high
selectivity and rapid-response properties for real-time detection of
−
low-concentration endogenous OCl in living biological systems through simple
synthesis.
Triphenylamine as an ideal molecule matrix has been widely used in the
construction of opto- and electro-active materials owing to its molecular
2
7-29
non-coplanarity, vibronic coupling and high stability.
However, the use of
triphenylamine for fluorescent probes has not received a lot of attention. In order to
explore the application of this matrix in fluorescent probes field, herein, we have
designed and synthesized a novel triphenylamine-type schiff base derivative (TPAD)
-
for the spectic detection of OCl by one-step synthetic process from picolinohydrazide
2

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and 4-(diphenylamino)benzaldehyde (Scheme 1). The probe TPAD exhibited selective,
-
sensitive and rapid response to OCl , which was determined by fluoresence
-
spectroscopy. Furthermore, the bioimaging application of probe TPAD for OCl
detection in living cells was also investigated.
Scheme 1. Synthesis route of TPAD probe
2
. Experimental
2
.1. Chemicals and apparatus
Triphenylamine, picolinohydrazide, hydrogen peroxide, sodium hypochlorite,
and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) were
purchased from Aladdin Chemistry Co. Ltd (Shanghai, China) and used without
further purification. Ethanol and DMSO were analytical pure and distilled before
2-
-
-
-
-
- 1
used. Various testing species including CO , H PO , Cl , H O , OCl , TBuO , HO , O ,
3
2
4
2
2
2
-
-
NO, ONOO and NO were prepared in solutions according to the following methods,
2
-
respectively: T-BuO from Aladdin Co. Ltd was diluted to the required concentration;
-
1
HO was generated from Fenton reaction between Ferrous solution; O was
2
produced via the reaction of H O with NaOCl; NO is emitted from
2
2
-
3
-(Aminopropyl)-1-hydroxy-3-isopropyl-2-oxo-1-triazene (NOC-5); ONOO source was
replaced by the 3-morpholinosydnonimine hydrochloride (SIN-1, 50.0 μM); The
-
source of NO was obtained from NaNO . All other ion salts were purchased from
2
2
Nanjing Chemical Reagent Co., Ltd. (Nanjing, China). Double-distilled water was used
throughout all the experimental solutions. All samples were prepared at room
temperature and were shaken for 1 min before each experiment.
1
13
H NMR and C NMR spectra of samples were recorded on an AVANCE III HD
AN-400 MHz spectrometer (Bruker, Germany) with TMS as an internal standard.
Ultraviolet-Visible (UV-vis) absorption spectra were measured on a Varian Cary 50
3

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spectrophotometer (Agilent, USA) at 1 cm of the light path length. Fluorescence
spectra were recorded on a Varian cary eclipse fluorescence spectrophotometer
(Agilent, USA) with an excitation wavelength of 375 nm. Mass spectroscopy data of
the samples were collected on a LCMS-2020 spectrometer (Shimadzu, Japan).
Element analysis of samples was operated on a PE 2400 elemental analyzer
(PerkinElmer, USA). All fluorescence imaging experiments were conducted using a A1
confocal laser scanning microscope (Nikon, Japan).
2
.2. Synthesis of TPAD
-(diphenylamino)benzaldehyde was prepared according to the reported
4
30
literature. TPAD was conveniently synthesized following the synthetic route
(Scheme 1). 0.273 g 4-(diphenylamino)benzaldehyde (1.0 mmol) was dissolved in 20
mL ethanol solution, then 0.137 g picolinohydrazide (1.0 mmol) was added into the
solution and the mixture was heated at reflux and stirred for 12 h. After the reaction
system was cooled to room temperature, a yellow precipitate was obtained and
collected by filtration, washed with cold ethanol and dried in vacuum to afford probe
1
TPAD as a yellow powder (87% yield, 0.196 g). H NMR (400 MHz, DMSO-d ), δ
6
(ppm): 12.06 (s, 1H), 8.80-8.64 (m, 1H), 8.57 (s, 1H), 8.16-7.97 (m, 2H), 7.72-7.52 (m,
3
2
1
H), 7.36 (t, J = 7.8 Hz, 4H), 7.12 (dd, J = 17.7, J = 7.5 Hz, 6H), 6.98 (d, J = 8.6 Hz,
1 2
1
3
H). C NMR (100 MHz, DMSO-d ), δ (ppm): 159.78 , 150.08, 149.30, 148.74, 147.98,
6
47.01, 137.71, 129.49, 128.94, 126.66, 125.35, 123.92, 122.92, 121.70. MS (ESI)
+
calculated for [M+1] 393.06, found 393.09. Elemental analysis (%) calculated for
1
C H N O: C, 76.51; H, 5.14; N, 14.28; found: C, 76.48; H, 5.12; N, 14.30. ( H NMR,
2
5 20 4
1
3
C NMR and ESI spectra of TPAD could be found in Fig. S1−S3).
.3. UV–vis and fluorescence analysis
2
Double-distilled water and analytical purity DMSO were used to prepared
solvent for the absorption spectra studies. Compound TPAD was dissolved in
DMSO/PBS buffer solution (1:4, V/V, 10 mM PBS buffer, pH 7.4) to afford a stock
solution in 10 mM concentration. Solutions of various testing species were prepared
by dilution of the stock solution with PBS buffer solution. The resulting solutions
4

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were well shaken and incubated for 1 h at room temperature before spectra data
recording.
2
.4. Cytotoxicity studies
The methyl thiazolyl tetrazolium (MTT) assay was used to measure the cytotox-
icity of TPAD in A549 cells. A549 cells were seeded in a 96-well plate (90 μL per well)
overnight and TPAD suspensions with different concentrations (20, 40, 60, 80, and
-
1
1
00 μg mL ) were then added. The cells were cultivated for 24 h, and 20 μL of 1
mg/mL MTT solution was then added to each seeding well. After all cells were
incubated for 4 h, the culture medium was discarded, and 150 μL DMSO solution was
added. The resulting mixture was shaken for 15 min in dark at room temperature,
and its optical density (OD) was measured on a microplate reader (Thermo TY10FC,
USA).
2
.5. Cellular Imaging
Human A549 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM)
supplemented with 10% fetal bovine serum and 1% penicillin in humidified
environment of 5% CO . For living cell imaging experiments of probe TPAD, cells were
2
-
1
incubated with 20 μg mL of probe TPAD (with 20% DMSO, v/v) for 30 min at 37
,
after incubation for 24 h, the cells washed three times with prewarmed PBS, and
then imaged using an excitation wavelength of 488 nm.
3
. Results and discussion
3
.1. pH effect studies
Effect of pH on the fluorescence response behavior of probe TPAD was
investigated with pH ranging from 1.0 to 13.0 in DMSO-PBS solution. As displayed in
Fig. 1, no obvious fluorescence change could be observed for TPAD over a range of
pH from 7.0 to 10.0, demonstrating that probe TPAD could be utilized over a
-
relatively wide pH range. In consideration of the decomposition property of OCl in
strongly acidic conditions and make sure the only ionic species presenting in the
-
31
solution was OCl , physiological pH at 7.4 was chosen for all the following
experiments.
5

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Fig. 1. PL intensity ratio (F/F ) of TPAD at various pH values.
0
3
.2. Selectivity studies
Selectivity experiments were conducted, where TPAD (10 mM) was subjected to
-
-
1
-
-
-
various ROS species (50 µM) including H O , TBuO , HO , O , Cl , ONOO , NO ,
2
2
2
2
-
-
2-
H PO , HCO , CO and OCl , respectively, under the same test conditions. As
-
2
4
3
3
shown in Fig. 2, all the selected ROS did not cause significant fluorescence change
-
-
except for OCl , suggesting that probe TPAD possessed a good selectivity towards OCl
against other competing species.
Fig. 2. Emission intensity ratio (F/F ) of TPAD (10 mM) in aqueous conditions (PBS
0
buffer, 20% DMSO as a co-solvent at pH 7.4) in the presence of various species (50
µM).
3
.3. Photophysical property studies
From UV-vis absorption spectrum in Fig. 3, TPAD exhibited two obvious
absorption bands around 310 nm and 380 nm, which were ascribed to
thetriphenylamine and picolinohydrazide unit, respectively. Upon the addition of OCl
6

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−
,
obvious blue-shift phenomenon appeared, indicating that the conjugated structure
of TPAD was wrecked. Subsquently, fluorescence titration experiments were
-
performed to evaluate the sensing behavior of TPAD to OCl . As shown in Fig. 4a, the
as-prepared TPAD exhibited a characteristic fluorescence emission band at 523 nm
-
under the excitation at 375 nm. Upon the addition of various concentrations of OCl
to TPAD solution, obvious ‘turn-off’ fluorescence quenching response could be
-
observed. The relationship of fluorescence intensities in the presence of OCl can be
2
exhibited in Fig. 4b with a linear equation Y = -0.011X + 0.973, R = 0.992. Moreover,
-
the detection limit (3σ) for OCl was calculated to be as low as 0.8 µM. Interference
experiment was also performed and illustrated in Fig.5 and Fig.S4, which
-
demonstrated that the specific fluorescence response of TPAD towards OCl was not
-
-
1
-
-
-
2
disturbed by the competitive ROS species (H O , TBuO , HO , O , Cl , ONOO , NO ,
2
2
2
-
-
2-
H PO , HCO and CO ).
2
4
3
3
1.0
0.8
0.6
0.4
0.2
0.0
-
TPAD+ClO
TPAD
300
350
400
450
Wavelength / nm
Fig. 3. UV-vis spectra of TPAD and TPAD+ClO-.
Fig. 4. (a) Fluorescence response of TPAD (10 mM, PBS buffer, 20% DMSO as a
7

ACCEPTED MANUSCRIPT
-
co-solvent at pH 7.4) in the presence of increasing concentration of OCl (from up to
-
down the concentration of OCl is 0, 5, 10, 15, 20, 25, 30, 35, 40, 45 and 50 µM). (b)
-
The relationship of F/F versus the concentration of OCl over the range from 0 to 50
0
µM (λ =375 nm).
ex
1.0
0.8
0.6
0.4
0.2
0.0
-
Fig. 5. The competitive selectivity of TPAD for ClO was examined in the presence of
other ROS (50 µM). Red bars represent the addition of a single analyte including 50
-
-
1
-
-
2
-
-
2-
-
μM of ROS (None, TBuO , HO , O , H O , ONOO , NO , Cl , HCO , CO and H PO ,
2
2
2
3
3
2
4
respectively). Black bars represent the subsequent addition of ClO- (50 μM) to the
mixture. All experiments were performed in PBS buffer (10 mM, pH 7.4) with 20%
DMSO.
3
.4. Response time studies
Response behavior of TPAD towards OCl was analyzed by monitoring the
-
fluorescence changes of the reaction mixture. As showed in Fig. 6, in the absence of
-
OCl , TPAD exhibited no visible variations, suggesting that probe TPAD was stable in
-
the assay conditions. Interestingly, upon addition of OCl , dramatic fluorescence
changes were observed and completed within 30 s. Compared with the other
−
reported ClO probes (Table.S1), probe TPAD showed shorter response time, which
-
enables the probe TPAD to facilitate real-time detection of OCl level changing.
8

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Fig. 6. Reaction-time profile of TPAD (10 mM) in the absence and presence 50 µM
−
OCl in aqueous conditions in DMSO–water solution (1 : 4, v/v, 50 mM PBS buffer
solution at pH 7.4). Kinetic studies were performed at ambient temperature.
3
.5. The study on sensing mechanism
In order to study the reaction mechanism between TPAD and OCl , H NMR
−
1
titration experiment through adding NaOCl to a DMSO-d solution of probe TPAD
6
-
was carried out. Upon the addition of OCl species, the signal peak of characteristic
proton (-CH=N) of probe TPAD at 8.56 ppm gradually decreased until disappeared,
while a new signal peak at 9.77 ppm assigned to -CHO proton was observed, which
demonstrated that the addition of hypochlorite triggered the conversion of imine
group to the aldehyde group (Fig. 7). Furthermore, MS analysis in probe TPAD treated
−
with OCl was also performed, where the mass peak at m/z = 273.95 corresponding
to product was observed (Fig. S5). UV-vis absorption spectrum also proved the
reliability of the mechanism indirectly. There is a π-π conjugated structure in probe
TPAD with good fluorescence properties. After the addition of OCl-, the C=N double
bond of TPAD was destroyed and the raw material molecule was released which
results in conjugate degree descent and the blue shift phenomenon for TPAD from
UV-vis spectrum. These results supported the proposed sensing mechanism, which
was shown in Scheme 2.
9

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1
−
Fig.7. Partial H NMR spectra of probe TPAD, probe TPAD + 10 µM OCl and TPAD + 50 µM
−
OCl in DMSO-d .
6
N
N
N
O
ClO-
O
O
H
2
O
N
H
N
N
N
N
N
H
N
OCl
H
N
H
N
OCl
N
ClO-
O
O
H
H
H
O
2
H N
N
H
N
N
CHO
-
Scheme. 2 Proposed sensing mechanism of probe TPAD in response to OCl .
3
.6. Cell imaging
-
Bioimaging application was assessed using TPAD as a fluorescent probe for OCl
detection in living cells. MTT assay was firstly carried out to evaluate the cytotoxicity
of the TPAD to A549 cells. As illustrated in Fig. S6, the viability of A549 cells declined
no less than 8% while the TPAD concentration increasing up to 100 µg/mL, which
indicated that the TPAD concentration in vitro is much higher than that of required
for the imaging of living cells. Furthermore, the bioimaging applicability of TPAD was
evaluated using a Nikon confocal laser scanning microscope. As shown in Fig. 8,
strong green fluorescence in living A549 cells was observed after the cells were
-
incubated with TPAD. Subsequently, after incubated with OCl (50 µM) for 8 h, the
channel green fluorescence was found to be invisible. These results revealed that
-
probe TPAD was able to effectively detect OCl in living cells.
1
0

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Fig. 8. Confocal fluorescence images of A549 cells. (a, c) A549 cells incubated with
-
the TPAD (10 mM) and TPAD + OCl (50 µM); (b) TPAD fluorescence image in the
-
green channel; (d) TPAD+OCl (50 µM) fluorescence image in the green channel (scale
bar: 10 μm).
4
. Conclusions
-
In summary, we have developed a novel probe TPAD for OCl detection by
expediently combining benzaldehyde and picolinate units. The probe TPAD exhibited
-
excellently selective response towards OCl over potentially competing reactive
oxygen species with rapid response time (30 s). In addition, fluorescent imaging
-
analysis showed that TPAD could be used to detect OCl in biological systems with
-
high sensitivity and excellent biocompatibility. The specific detection of OCl provides
novel sight into the reactive-type fluorescent probe and would be of great benefit for
−
exploiting new functions and mechanisms of OCl sensing in living systems.
Acknowledgement
This work was supported the China Postdoctoral Fund Surface Project
(2017M610335), Fundamental Research Funds of Jiangsu Key Lab. of Biomass Energy
and Material (JSBEM-S-201806) and a Project Funded by the Priority Academic
Program Development of Jiangsu Higher Education Institutions (PAPD).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in the online
1
1

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version, at http://dx.doi.org/10.1016/j.tet.2018.XX.XX.
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