A mitochondria-targeted fluorescent probe based on coumarin-pyridine derivatives for hypochlorite imaging in living cells and zebrafish

Article abstract of DOI:10.1039/c9tb01948k

Hypochlorite plays a critical role in various physiological processes and is involved in many diseases. Thus, real-time, rapid, and accurate monitoring of hypochlorite has important medical and physiological significance. Herein, a novel coumarin-pyridine derivative (CPD) probe was designed and synthesized, which exhibited fantastic advantages, such as a rapid response (within 10 s), naked eye recognition, large Stokes shift (185 nm), dual-channel detection, and high selectivity and sensitivity toward OCl^- (detection limit 0.012 μM, S/N = 3). Furthermore, the current CPD probe was successfully used to image OCl^- in the mitochondria of both A549 cells and zebrafish, which further demonstrated its suitability for practical applications in biological systems.

Full text of DOI:10.1039/c9tb01948k

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Materials Chemistry B
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PAPER
A mitochondria-targeted fluorescent probe based
on coumarin–pyridine derivatives for hypochlorite
imaging in living cells and zebrafish†
Cite this: J. Mater. Chem. B, 2019,
7, 7332
Xiuli Zhong, Qing Yang, Yingshuang Chen, Yuliang Jiang, * Bingxiang Wang* and
Jian Shen*
Hypochlorite plays a critical role in various physiological processes and is involved in many diseases.
Thus, real-time, rapid, and accurate monitoring of hypochlorite has important medical and physiological
significance. Herein, a novel coumarin–pyridine derivative (CPD) probe was designed and synthesized,
which exhibited fantastic advantages, such as a rapid response (within 10 s), naked eye recognition, large
Stokes shift (185 nm), dual-channel detection, and high selectivity and sensitivity toward OClÀ (detection
limit 0.012 mM, S/N = 3). Furthermore, the current CPD probe was successfully used to image OClÀ in
the mitochondria of both A549 cells and zebrafish, which further demonstrated its suitability for practical
applications in biological systems.
Received 8th September 2019,
Accepted 14th October 2019
DOI: 10.1039/c9tb01948k
rsc.li/materials-b
extremely susceptible to environmental factors and thus
limited their applications. Therefore, developing novel probes
Introduction
As one of the most important reactive oxygen species, hypo- without the above-mentioned defects is still an urgent challenge.
chlorite participates in many physiological processes in living Studies have shown that molecules based on an excited-state
organisms and abnormal concentration levels have been shown intramolecular proton transfer (ESIPT) mechanism can be used
to be involved in many diseases.1–5 Therefore, an accurate to construct multi-channel probes accompanied by large Stokes
quantitative and qualitative hypochlorite assay can provide a shifts.22–25 Thus, exploring such probes should be of utmost
more detailed understanding of its complex biological effects, priority. So far, many of these probes have been developed based
which may also improve disease prevention and diagnosis.
on benzoxazole,26 benzothiazoles,27 imidazo[1,5-a]pyridine,28 and
Fluorimetry has been evaluated as an attractive and versatile flavonoid dyes,29 which often require multi-step complex post-
tool for the detection of biomarkers due to its simple method of processing. As such, the development of probes with simple
use, rapid response and non-invasive detection. As such, the synthesis and high yields based on ESIPT has great practical
methods in which it is applied have expanded, being used as value. The mitochondria, as the main source of ROS produc-
chemical sensors and for bioimaging.6–9 Therefore, the search tion, play an important role in life-sustaining activities,
for probes with excellent performance has become a popular however the build-up of ROS in mitochondria can result in cell
avenue of research. In recent years, a great quantity of OClÀ death and various diseases.30–32 Therefore, the development of
fluorescent probes have been developed, due to the efforts of mitochondria-targeted probes for OClÀ detection in live cells
chemical researchers,10–16 which opens a new era for OClÀ and animals will help further our knowledge of the chemical
probes. Until now, various fluorescence probes have been and biological properties of OClÀ in mitochondria.
developed and applied to the highly selective and sensitive
Based on the above considerations, in this paper, a coumarin
detection of OClÀ in biological systems,17–21 which promoted derivative and pyridine cation were selected as starting materials
medical research tremendously. However, some of the developed for the construction of a novel, mitochondria-targeted fluorescent
probes had inherent defects, such as single-channel detection, probe (CPD) for the selective detection of OClÀ and imaging both
long response time, and small Stokes shifts, which made them in vitro and in vivo. As a recognition group, the carbon–carbon
double bonds interact with OClÀ creating negative oxygen ions
that interact with an adjacent hydroxyl group of the benzene ring
Jiangsu Collaborative Innovation Center of Biomedical Functional Materials,
of the parent molecule, thereby initiating the ESIPT process.
Jiangsu Key Laboratory of Bio-functional Materials, School of Chemistry and
A dual-channel signal for the detection of OClÀ with a large
Materials Science, Nanjing Normal University, Nanjing 210023, P. R. China.
Stokes shift was observed, in conformity with our expectations.
E-mail: 07205@njnu.edu.cn, Wangbingxiang@njnu.edu.cn, jshen@njnu.edu.cn
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c9tb01948k Furthermore, CPD was successfully applied for imaging, as well as
7332 | J. Mater. Chem. B, 2019, 7, 7332--7337
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to monitor mitochondrial OClÀ in mitochondria of living cells Cellular imaging
and zebrafish.
A549 cells were incubated in Dulbecco’s modified Eagle’s
medium (DMEM) supplemented with 10% fetal bovine serum,
in a humidified environment containing 5% CO2, and were left
to adhere for 24 h. For fluorescence imaging experiments, the
cells were divided into four samples; one was incubated with
the CPD probe (10 mM) as a control, and the three other
samples were further incubated with 5, 20, and 40 mM OClÀ
for 40 min at 37 1C, respectively. Cells were then imaged after
being washed with PBS buffer. Fluorescence images were
captured using a Nikon A1 laser-scanning confocal microscope
at an excitation wavelength of 405 and 488 nm.
Experimental section
Materials and methods
7-Hydroxy-4-methyl-2H-chromen-2-one, iodomethane, 4-methyl-
pyridine, sodium hypochlorite, piperidine and hexamethylene-
tetramine were purchased from Adamas-betas (Shanghai, China).
Some other testing species such as Ag+, Co2+, H2S, ONOOÀ,
GSH, HCO3À, Cys, CO32À, HSO3À, NO2À, NO3À, HSO4À, OHÀ,
H2PO4À, H2O2, and HOCl were purchased from Shanghai
Experiment Reagent Co. Ltd (Shanghai, China). All the reagents
were of analytical grade and used as received.
Fluorescence imaging in zebrafish
Ultraviolet-Visible (UV-vis) absorption was recorded on
a Lambda 650s spectrophotometer (PerkinElmer, USA) and
fluorescence spectra were recorded on an F-7100 fluorescence
spectrophotometer (Hitachi, Japan). 1H NMR and 13C NMR
experiments were performed in d6-DMSO on an AVANCE III
HD AN-400 MHz spectrometer (Bruker, Germany). Mass spectra
were recorded on a LCMS-2020 spectrometer (Shimadzu,
Japan). High resolution mass spectroscopy data were obtained
on a high resolution Orbitrap Fusion Lumos (Thermo Fisher
Scientific, USA). Fluorescence imaging experiments were per-
formed by using an A1 confocal laser scanning microscope
(Nikon, Japan).
Zebrafish studies were approved by the Animal Ethics and
Welfare Committee of NNU and were conducted in compliance
with the European guidelines for the care and use of laboratory
animals. Zebrafish were provided by Shanghai FishBio Co., Ltd.
In order to image the endogenous HOCl in zebrafish, 3 day-old
zebrafish were incubated in E3 media containing 10 mM CPD
probe at 28 1C for 60 min and then washed thrice with the
culture medium to eliminate residual CPD. This group was
used as a negative control group. For the experimental group,
zebrafish were pretreated with CPD for 30 min and subsequently
incubated with OClÀ (5, 20, and 40 mM) for another 60 min.
Following that, they were washed with PBS and then imaged.
Fluorescence imaging experiments were carried out by using the
Nikon A1 confocal laser scanning microscope. The excitation
wavelengths were 405 and 488 nm.
Synthesis of probe CPD
The synthetic route is illustrated in Scheme 1. Compound 2 was
prepared according to the previous ref. 33. The 1H NMR
spectrum of compound 2 is shown in Fig. S1 (ESI†). The
synthesis of probe CPD can be described as follows: compound
2 (0.16 g, 0.8 mmol) and 1,4-dimethyl pyridine-1-ium iodide
(0.38 g, 1.6 mmol) were dissolved in anhydrous ethanol (15 ml).
A drop of piperidine was added and the mixture was refluxed
overnight. A red solid was precipitated after reaction was
accomplished, with 83.4% yield. The chemical structure of
CPD was characterized by 1H NMR, 13C NMR, and HRMS
Results and discussion
To better understand the role of hypochlorite at the mito-
chondrial level, a novel organelle-targeted fluorescent probe
for in vitro and in vivo detection and imaging is urgently
needed. Some factors, including mitochondria-targeting ability
and real-time high sensitivity and selective detection, should
be considered. Inspired by previous work,33,34 coumarin deri-
vatives were selected as a matrix, in which a pyridine cation was
introduced via the Knoevenagel reaction. As such, mitochondria-
targeting and OClÀ recognition groups were simultaneously
obtained. Unlike previous reports, no aldehyde group was formed
after the interaction of OClÀ and ‘‘CQC’’.35–37 Instead, CPD–OClÀ
containing oxygen anions interact with an o-hydroxyl group
and initiate the ESIPT process, thereby enabling dual-channel
detection, which exhibited excellent performance for the detection
and imaging of OClÀ in mitochondria of A549 cells and zebrafish.
The routes of CPD synthesis are illustrated in Scheme 1.
1
(Fig. S2–S4, ESI†). H NMR (400 MHz, DMSO-d6) d 8.62 (d, J =
6.5 Hz, 2H), 8.15 (s, 2H), 7.98 (d, J = 6.6 Hz, 2H), 7.38 (d, J =
9.1 Hz, 1H), 6.53 (d, J = 9.0 Hz, 1H), 5.85 (s, 1H), 4.18 (s, 3H),
2.31 (s, 3H); 13C NMR (100 MHz, DMSO-d6): d (ppm) 169.6,
160.8, 155.3, 154.9, 154.7, 144.9, 133.0, 128.5, 127.9, 123.3,
122.9, 117.1, 110.0, 108.5, 107.1, 46.9, 18.9; HR-MS (ESI) calcu-
+
lated for C18H16NO3 (M+), 294.1157; found, 294.1118.
We first assessed the ultraviolet absorption spectrum of CPD
either with or without OClÀ in a DMSO–PBS buffer solution
(10.0 mM, pH = 7.4, 1 : 5 (v/v)) at 25 1C. As shown in Fig. 1, free
probe CPD exhibited two absorption peaks with maxima at 371
and 542 nm. Upon the addition of OClÀ (40 mM) to the CPD
solution, the intensity of the 371 nm absorption peak increased
slightly, with a micro-red shift, while the other peak (542 nm)
Scheme 1 Synthesis of probe CPD.
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intensities and [OClÀ] can be described with a linear equation
as follows: Y = 48.86X + 585.59, R2 = 0.994, with the OClÀ
detection limit (3s) calculated to be 0.012 mM (Fig. 2b), while at
525 nm, a logarithmic equation Y = 362.77 ln(X) + 1015.7,
R2 = 0.942, can describe the relationship between [I] and [OClÀ]
(Fig. 2c). Moreover, we investigated the relationship between
OClÀ concentration and I425nm/I525nm ratio, as shown in Fig. 2d.
In the concentration range of 2–40 mM, the ratio of I425nm/I525nm
showed a good linear relationship described as Y = 0.017X+
0.423, R2 = 0.997. These data fully confirmed that the CPD
Fig. 1 UV-vis spectral changes of probe CPD (10.0 mM) upon addition of
OClÀ (40 mM). The inset shows color changes.
probe could effectively detect OClÀ with
relationship.
a good linear
exhibited a slight blue shift to 531 nm. This indicated that the
structure of the CPD probe system changed after hypochlorite
was added, providing a basis for the accurate detection of
hypochlorite. Meanwhile, the pink solution transformed into a
colorless solution, which was observable to the naked eye (Fig. 1,
inset), suggesting that CPD facilitates visual OClÀ detection.
Subsequently, fluorescence spectra were measured under
the same conditions. Free CPD showed a wide peak at about
430 nm with excitation at 340 nm. However, two emission
bands at 435 and 525 nm appeared, with significant fluores-
cence enhancement, after 40 mM OClÀ was added. Moreover,
the color of the solution changed from blue to green (Fig. S5,
ESI†). The Stokes shift of CPD was calculated to be 185 nm,
which can effectively avoid self-absorption and thereby have
more valuable applications in living organisms. Fluorescence
titration spectroscopy was further performed and the spectra
are displayed in Fig. 2a. Upon addition of OClÀ (0–40 mM), both
fluorescence peaks (435 and 525 nm) showed an obvious
fluorescence turn-on phenomenon but with different rates.
Subsequently, we studied the enhancement of these two optical
windows. At 435 nm, the relationship between fluorescence
When considering applications in complex biological systems,
the CPD probe must possess high selectivity. To evaluate the
selectivity of CPD, various potential interfering species (40 mM),
2À
including Ag+, Co2+, H2S, ONOOÀ, GSH, HCO3À, Cys, CO3
,
HSO3À, NO2À, NO3À, HSO4À, OHÀ, H2PO4À, H2O2, and OClÀ,
were assessed under the same test conditions. As illustrated in
Fig. 3, only OClÀ caused an obvious fluorescence enhancement
with dual emission peaks, while the other interfering substances
showed no obvious change in fluorescence and the same
phenomenon can be seen in DMF solution (Fig. S6, ESI†). This
demonstrated that CPD can be used as a reliable probe to monitor
OClÀ with high selectivity.
The time-dependent fluorescence response of the probe CPD
toward OClÀ was used to evaluate its capability for real-time
monitoring in a biological system. Herein, we took the fluores-
cence intensity at 435 nm as an example. We further researched
the time-dependent fluorescence signaling of CPD towards
OClÀ, as is illustrated in Fig. 4a. Free CPD probe exhibited
stable weak fluorescence when 40 mM OClÀ was added, with the
emission intensity of CPD at 435 nm increasing rapidly and
maintaining stability for less than 10 s. Compared to previous
reports38–43 (Table S1, ESI†), the CPD probe showed excellent
reliability for real-time detection of OClÀ in biological systems,
owing to its extremely short half-life in organisms.44 In addition,
to apply the probe in living systems, the probe needs to have a
suitable pH range. Thus, the working pH range of the CPD probe
for detection of OClÀ was also investigated by evaluating the
fluorescence changes at 435 nm. As shown in Fig. 4b, both CPD
and CPD–OClÀ showed stable fluorescence in the pH range of
7–10, indicating that the CPD probe can detect OClÀ in biological
and environmental samples.
Fig. 2 (a) Fluorescence spectral changes of probe CPD (10 mM) upon
addition of OClÀ (0–40 mM). lex = 340 nm, and inset shows color changes;
(b) the relationship of I435nm versus the concentration of OClÀ over the
range from 0 to 40 mM; (c) the relationship of I525nm versus the concen-
tration of OClÀ over the range from 0 to 40 mM; (d) the relationship of
I
435nm/I525nm versus the concentration of OClÀ over the range from 0 to
Fig. 3 Emission intensity of CPD under aqueous conditions (DMSO/PBS
buffer solution 1 : 4, v/v, 10 mM, and pH 7.4) in the presence of various
species (40 mM).
40 mM (from down to up, the concentration of OClÀ is 0.5, 1, 2, 4, 6, 8, 10,
20, 30 and 40 mM).
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Fig. 4 (a) The changes of fluorescence intensities of probe CPD over time
in the presence of OClÀ; (b) fluorescence intensity changes (at 435 nm) of
the probe CPD (10 mM) with and without OClÀ (40 mM) under different pH
conditions.
The carbon–carbon double bond is a typical hypochlorite
recognition group that has been widely applied in earlier
studies. According to our experimental results, we believe that
the carbon–carbon double bond did not break, resulting in the
Fig. 5 Confocal fluorescence images of A549 cells. Fluorescence images
with green channel (a1–d1), blue channel (a2–d2) and merge (a3–d3) of
corresponding aldehyde, during the interaction between the A549 cells pretreated with 10 mM probe CPD for 30 min and then followed
by incubation with 5, 20, and 40 mM OClÀ for another 30 min, respectively.
The cells were excited with a light laser at 405 and 488 nm. Scale bar:
probe and hypochlorite, which is different from previous
reports. Thus, the reaction mechanism can be described as
follows: hypochlorite was added to the carbon–carbon double
10 mm.
bond to form negative oxygen ions that could interact with the
hydroxyl group on the adjacent benzene ring, leading to the
appearance of the ESIPT phenomenon, which presents as dual
fluorescence emission peaks. The results of the ESI mass
spectrometry (Fig. S7, ESI†) showed a peak at m/z 345.3 (calcd:
345.5) that is assigned to product A, as illustrated in Scheme 2,
which further confirms our hypothesis.
of ROS production. Fluorescence imaging of mitochondria
could provide the most direct and convenient method for the
understanding of the physiological and pathological processes
related to mitochondrial function. As such, whether or not the
Encouraged by the excellent photophysical properties of
CPD toward OClÀ, we further investigated the practical utilities
of the CPD probe for fluorescent imaging of intracellular OClÀ
in living cells. The MTT method was first used to assess the
cytotoxicity of CPD in A549 cells and the results are shown in
Fig. S8 (ESI†). CPD showed low cytotoxicity toward A549 cells, with
over 92% cell viability after 24 h incubation at a concentration of
10 mM. This indicated that CPD is suitable for applications in
biosystems. A549 cells incubated with CPD (10 mM, 30 min at
37 1C) only displayed a very faint fluorescence in the green and blue
channels. In contrast, after treatment with various concentrations
(5, 20, and 40 mM) of OClÀ for another 30 min, both the green and
blue channels exhibited a gradual enhancement of fluorescence, as
shown in Fig. 5. This demonstrated that the CPD probe is a
sensitive fluorescent probe for the specific recognition and imaging
of OClÀ in the cellular environment.
As one of the most important organelles, mitochondria
serve as the powerhouse of cells and are the main source
Fig. 6 Intracellular co-localization fluorescence imaging of CPD, CPD–
OClÀ and MitoTracker Green FM in live A549 cells. Cells incubated with
CPD (10 mM) and CPD–OClÀ (10 mM–40 mM) for 30 min and MitoTracker
Green FM (200 nM) for 10 min at 37 1C. (a) Bright-field and (b) confocal
images of CPD in the green channel (lex = 405 nm); (c) fluorescence
imaging of MitoTracker Green FM in the green channel (lex = 488 nm);
(d) the merged image of (b) and (c); (e) bright-field and (f) confocal images
of CPD–OClÀ in the blue channel (lex = 405 nm); (g) fluorescence imaging
of MitoTracker Green FM in the green channel (lex = 488 nm); (h) the
merged image of (f) and (g); (i and j) the normalized intensity profile of the
regions of interest across A549 cells. Scale bar = 10 mm.
Scheme 2 Proposed mechanism of probe CPD for highly selective sensing
of OClÀ.
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Conflicts of interest
There are no conflicts to declare.
Acknowledgements
This work was supported by the China Postdoctoral Fund
Surface Project (2017M610335) and a Project Funded by the
Priority Academic Program Development of Jiangsu Higher
Education Institutions (PAPD).
Fig. 7 Fluorescence imaging of CPD response to OClÀ in zebrafish:
(a1–d1) green channel of zebrafish fed with various concentrations of OClÀ
(0, 5, 20 and 40 mM); (a2–d2) blue channel of zebrafish fed CPD with
various concentrations of OClÀ (0, 5, 20 and 40 mM); (a3–d3) the merged
images of (a1–d1) and (a2–d2); all scale bars = 100 mm.
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