A highly sensitive and selective fluorescent probe for fast sensing of endogenous HClO in living cells

Article abstract of DOI:10.1039/c8cc01917g

A new fluorescence probe was developed for HClO detection under physiological conditions, which displayed fast response (t_1/2 < 30 s) and large off-on fluorescence increase (>1000-fold) toward HClO with high sensitivity and selectivity. The probe was successfully applied for the detection of both exogenous and endogenous HClO in living cells. Using the probe-based tool, we could observe the H₂S-induced HClO biogenesis in living cells for the first time.

Full text of DOI:10.1039/c8cc01917g

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ARTICLE TYPE
A highly sensitive and selective fluorescent probe for fast sensing of
endogenous HClO in living cells
Changyu Zhang,‡a Qichang Nie,‡b Ismail Ismail,c Zhen Xi*c and Long Yi*a
Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX
5
DOI: 10.1039/b000000x
cells.11 We therefore hope to investigate the possible crosstalk
A new fluorescence probe was developed for HClO detection
50 between H2S and other ROS such as HClO. Herein, the fast
oxidation of pꢀaminophenyl ether moiety was employed as the
receptor for HClO,5b and a highly sensitive and selective
fluorescence probe 1 for HClO was developed. The probe 1
showed fast response (t1/2 < 30 s) and very low detection limit for
55 HClO (0.65 nM), which was successfully used to observe the
H2Sꢀinduced HClO biogenesis in living cells for the first time.
To realize large fluorescence response, we chose
methoxyfluorescein as fluorophore, which displayed very large
offꢀon from the spirocylized conformation to the πꢀconjugation
60 conformation.12 As shown in Scheme 1, probe 1 was synthesized
from facile twoꢀstep reactions with good isolation yields, and was
in physiological conditions, which displayed fast response (t1/2
< 30 s) and large off-on (> 1000-fold) toward HClO with high
sensitivity and selectivity. The probe was successfully applied
10 for detection of both exogenous and endogenous HClO in
living cells. Using the probe-based tool, we could observe the
H2S-induced HClO biogenesis in living cells for the first time.
Reactive oxygen species (ROS), including hydrogen peroxide
(H2O2), hydroxyl radicals (●OH), peroxyl radicals (ROO●),
15 singlet oxygen (1O2), superoxide anion radicals (O2●ꢀ) and
hypochlorous acid (HOCl), play important roles in various
biological and pathological progresses.1 HClO, one of the most
important ROS, is known to be generated from the reaction
between H2O2 and Clꢀ catalysed by myeloperoxidase (MPO) in
20 vivo.2 As an important microbicidal effector, regulated production
of HOCl is helpful for host innate immunity to cope with
microbial invasion.3 Excessive production of HClO, however, is
closely related to numerous diseases, such as neuron
degeneration, cardiovascular diseases, inflammatory diseases,
25 cystic fibrosis and certain cancers.4 Thus, it is of great value to
develop efficient methods to detect HClO in vivo as well as to
explore its biological functions.
1
well characterized by H NMR, 13C NMR and highꢀresolution
mass spectra (HRMS) (see ESI). As expected, the spirocylized
probe 1 was colorless and nonꢀfluorescent. After reaction with
65 HClO, the spirocyclic structure was unlocked and the green
fluorescence of 2 is released so as to achieve more than 1000ꢀfold
fluorescence enhancement.
Fluorescenceꢀbased methods are highly desirable and sensitive
for in-situ and realꢀtime visualization of HClO in living
30 biological systems. As an ideal method, fluorescent probes were
widely used to detect the biological HClO.5ꢀ9 These fluorescent
probes are majorly based on the specific HClOꢀinduced chemical
reactions. For instance, a deformylation reactionꢀbased probe was
reported for in vivo imaging of HClO.6 Yoon et. al. reported the
35 first fluorescent probe for HClO based on the selective oxidation
of a BꢀH bond.7 Peng et. al. developed an “enhanced PET”ꢀbased
probe for imaging elesclomolꢀinduced HClO in cancer cells.8
Moreover, the oxidation of catechol was also applied for design
of fastꢀresponse HClO probe.9 Though these great successes have
40 been achieved, we still need to develop advanced probes with
high sensitivity, high selectivity and fastꢀresponse properties for
realꢀtime detection of lowꢀconcentration endogenous HClO in
living biological samples. Moreover, such probeꢀbased tools will
further enable exploration of ROS biology, which is still at the
45 infant status.
Scheme 1. Synthesis of probe 1 for HClO detection. (a) pꢀfluoronitrobeꢀ
70 nzene, NaH, Cs2CO3, 86%; (b) SnCl2•2H2O, concd. HCl/H2O2, 49%. Inset:
photos of 1 (20 µM) in the absence or presence of HClO (1 eq.) in PBS
buffer (50 mM, pH 7.4 containing 5% EtOH) under 365 nm UV lamp or
daylight.
With the probe 1 in hand, we examined the absorption and
75 fluorescence spectra of 1 with HClO in buffer (pH 7.4). 1 (20
ꢁM) showed no obvious absorption peak due to separation of the
πꢀconjugation system, while addition of HClO (1 eq.) triggered
new absorption profiles with maxima at around 465 nm (Fig. 1A),
implying fluorophore 2 was produced.13 The reaction products
80 were confirmed by HRMS (Fig. S1), and the colour of the
reaction solution changed from colorless to yellow (scheme 1). 1
(5 ꢁM) was nonꢀfluorescent, while a strong emission at around
We have been interested in development of fluorescent probes
for H2S and biothiols for some time.10 Based on the probeꢀbased
tools, the H2O2ꢀinduced H2S biogenesis was observed in living
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515 nm was observed immediately when HClO (1 eq.) was added
was added (lane 11). These results clearly demonstrated the
(Fig. S2). As shown in Fig. 1B, the reaction of 1 with HClO (1 45 superior selectivity of 1 toward HClO over other analytes.
eq.) almost finished within 5 min with t1/2 < 30 s, implying that 1
was a very fastꢀresponse probe for HClO detection. This property
5
was significant because of the short lifetime of endogenous HClO
in living biological systems. The fluorescence increase at 515 nm
was found to be up to 1046ꢀfold, which is one of the largest offꢀ
on probes for HClO.5ꢀ9 Such large emission response could
provide a high signal to noise ratio for HClO detection in living
10 cells. The result for stability showed that 1 was very stable within
30 min in PBS buffer (Fig. 1B).
Considering the low concentration of HClO in biological
systems, excellent sensitivity for a fluorescent HClO probe is
necessary. To gain detailed information about the sensitivity of 1,
15 the emission spectra was closely monitored by addition of various
low concentrations of HClO into the probe for 10 min (Fig. 1C).
The emissions at 515 nm were linearly related to the
concentrations of HClO from 0.1 to 1.0 µM (Fig. 1D). The
detection limit was determined to be 0.65 ± 0.01 nM using the
20 3σ/k method,14 which is comparable to the most sensitive HClO
probe (0.33 nM).5c Such nanomolar low detection limit should be
beneficial to the detection of endogenous HClO.
Fig. 2. Relative emissions of 1 (5 µM) toward various ions, RSS, RNS
and ROS at 515 nm. Lanes from 1 to 19: Fe2+ (100 µM), Zn2+ (100 µM),
2ꢀ
Mg2+ (100 µM), SO32ꢀ (100 µM), HSO3ꢀ (100 µM), S2O32ꢀ (100 µM), SO4
50 (100 µM), Cys (500 µM), Hcy (500 µM), GSH (5 mM), Na2S (150 µM),
t
ꢀ
●
H2O2 (100 µM), BuOOH (100 µM), O2 (100 µM), NO2ꢀ (100 µM), OH
(100 µM), tBuOO● (100 µM), ONOOꢀ (20 µM), HOCl (5 µM).
Experiments were carried out in PBS buffer (50 mM, pH 7.4 containing
5% EtOH) at room temperature for 10 min.
55
To investigate the effects of pH values on the detection of
HClO, timeꢀdependent fluorescence response of 1 (5 µM) with
HClO (1 eq.) in PBS buffer at different pH values was examined
(Fig. S3). 1 was nonfluorescent at all tested pH values. After
reaction with HClO, the solution of 1 exhibited large turnꢀon for a
60 wide range of pH from 4.5 to 9.5, and the turnꢀon fold at pH 6.5ꢀ
7.5 was higher. For the response rate, 1 reacted with HClO
relatively faster at lower pH values. These results indicated that
the probe 1 was suitable for use at physiological pH. The
cytotoxicity of 1 was also evaluated using RAW 264.7 cells
65 (mouse macrophage cell line) by methyl thiazolyl tetrazolium
(MTT) assay (Fig. S4). 1 did not show much obvious cytotoxicity
at 0.1ꢀ10 µM range within 24 h, implying that 1 should be
suitable for bioimaging of HClO in living cells.
Fig. 1. (A) Timeꢀdependent absorption spectra of 1 (20 µM) toward HClO
25 (20 µM). (B) Timeꢀdependent emissions of 1 (5 µM) at 515 nm in the
presence or absence of HClO (5 µM). (C) Fluorescent spectra of probe 1
(5 µM) with diverse low concentrations of HClO (0.1ꢀ1.0 µM). (D)
Linear relationship (r = 0.9989) between emissions at 515 nm and
concentrations of HClO. All experiments were performed in PBS buffer
30 (50 mM, pH 7.4 containing 5% EtOH) at room temperature.
One of the most important capabilities for a fluorescent probe
to detect HClO is to exhibit distinguishable response toward
HClO over other ROS species and analytes. To evaluate the
35 specific nature of 1 for HClO, it was incubated with various ions,
RSS, RNS and ROS in PBS buffer (pH 7.4) separately, and the
fluorescence increase at 515 nm was recorded (Fig. 2). Among all
70 Fig. 3. Confocal microscopy images of exogenous HClO detection in
living RAW 264.7 cells using 1. Cells were incubated with (A) 1 (5 µM)
alone for 30 min; (B) 1 (5 µM) for 30 min and then NaClO (5 µM) for
another 30 min; (C) 1 (5 µM) for 30 min and then NaClO (20 µM) for
another 30 min. Emissions were collected at green channel (500ꢀ600 nm)
75 with 458 nm excitation. Scale bar, 20 µm.
t
species, only ●OH, BuOO● and ONOOꢀ triggered very slight
fluorescence response. However, the fluorescence increase for
t
40 ●OH (20 eq.), BuOO● (20 eq.) and ONOOꢀ (4 eq.) were only 15,
To explore the biological applicability of 1, we firstly
examined whether 1 could be used to detect intracellular HClO in
living cells. RAW 264.7 cells were incubated with 1 (5 µM) for
80 30 min, washed by PBS buffer, then treated with different
14 and 32 fold, respectively, which is extremely lower than the
response caused by HClO (1 eq., 1046 fold). Specially, no
obvious fluorescence increase was observed when Na2S (30 eq.)
2
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DOI: 10.1039/C8CC01917G
concentrations of NaClO (5 or 20 µM) for another 30 min and
imaged using a confocal fluorescence microscopy immediately
Taken together, these data clearly demonstrated that endogenous
HClO could be generated under the simulation of exogenous H2S
(Fig. 3). Cells incubated with 1 alone showed fairly weak 50 in living cells. Previously, we reported
a dualꢀresponse
fluorescence, while bright green fluorescence was observed upon
addition of 1 eq. NaClO, and more remarkable fluorescence
appeared when treated by 4 eq. NaClO (Fig. 3BꢀC and Fig. S6).
These results indicated that 1 could be applied to detect
intracellular HClO in a concentrationꢀdependent fashion.
fluorescent probe to clarify the H2O2ꢀinduced H2S biogenesis in
living cells.11a Herein, we discovered that the living cells could
generate HClO spontaneously to deal with the reductive pressure
caused by exogenous H2S. We supposed that the redox
55 atomosphere in cells should be balanced to maintain the normal
cell growth. The biobalance proposals include the possible
mutual inducement on the production of both ROS and RSS.15
Our finding in this work should be helpful for further
understanding the intracellular redox homeostasis.
5
60
10
Fig. 4. The relative fluorescence of confocal microscopy images for
detection of LPS/PMAꢀstimulated endogenous HClO in living RAW
264.7 cells using 1. N = 3 fields of cells, error bars are ± sd.
15
For detection of the endogenous HClO using 1, RAW 264.7
cells were stimulated with lipopolysaccharides (LPS, 1 ꢁg/ml) for
12 h followed by phorobol myristate acetate (PMA, 1 ꢁg/ml) for
1 h to generate HClO.6 As shown in Fig. S8B, obvious increase in
fluorescence was observed after cells were stimulated with the
20 LPS/PMA. Ebselen, a ROS scavenger that could decrease the
HClO level, was used in the control experiment. The LPS/PMAꢀ
treated cells displayed fairly weaker fluorescence in the presence
of ebselen (Fig. S8C). The relative emission (Fig. 4) clearly
showcased that stimulation of LPS/PMA could trigger about 2.1ꢀ
Fig. 5. Confocal microscopy images of H2Sꢀinduced endogenous HClO
detection in living RAW 264.7 cells using 1. Cells were incubated with
(A) 1 (5 µM) alone for 30 min; (B) H2S (150 µM) for 1 h, then 1 (5 µM)
25 fold fluorescence increase, which was higher than the ebselenꢀ 65 for 30 min. (C) Cells were pretreated with ebselen (100 µM) for 30 min.
treated control group (about 1.3 fold). Inspired by Peng’s work,8
1 was also used to detect elesclomolꢀinduced HClO in living
cancer cells. As expected, notable increase in fluorescence was
observed after MCFꢀ7 cells were treated with elesclomol (2 µM)
30 for 1 or 2 h (Fig. S9). These results elucidated that 1 could be
used for imaging of endogenous HClO in living cells.
Encouraged with the above results, we further employed the
probe 1 for investigation of possible crosstalk between H2S and
HClO in living cells. To this end, cells were treated with H2S
(D) The mean fluorescence and relative ratio for images AꢀC. Emissions
were collected at green channel (500ꢀ600 nm) with 458 nm excitation.
Scale bar, 20 µm. N = 3 fields of cells, error bars are ± sd.
70
In summary, we report a new fluorescent HClO probe 1, which
displayed very fast response (t1/2 < 30 s) toward HClO (1 eq.) and
possessed high selectivity over other analytes. Due to the ringꢀ
opening reaction of methoxyfluorescein, 1 showed very large
fluorescence offꢀon (1046ꢀfold) upon reaction with HClO. High
35 (150 µM) for 1 h, washed with PBS buffer, and then incubated 75 sensitivity of 1 with a detection limit as low as 0.65 nM was
with 1 (5 µM) for 30 min. To our delight, H2Sꢀtreated cells
exhibited remarkable green fluorescence with the mean intensity
about 3.3ꢀfold higher than that of 1ꢀstained cells (Fig. 5A, B and
D). While the ebselenꢀtreated cells in the control group displayed
beneficial to detection of endogenous HClO in living cells.
Importantly, using the probe 1, we revealed the H2Sꢀinduced
endogenous HClO biogenesis in living cells for the first time,
which could help to understand the intracellular redox
40 much weaker fluorescence, showing around 1.8ꢀfold fluorescence 80 homeostasis. This probe should be an excellent tool for further
increase than that of 1ꢀstained cells (Fig. 5C and D). To further
investigate the H2Sꢀinduced HClO biogenesis, concentrationꢀ
dependent experiments were carried out and the order of
incubation was also studied. We found that H2Sꢀtreated cells all
exploring the biological functions of HClO as well as its crosstalk
with other intracellular molecules.
This work was supported by NSFC (21402007, 21572019), the
45 displayed strong fluorescence when 50ꢀ150 µM H2S was added 85 National Key R&D Program of China (2017YFD0200501), 111
first (Fig. S11). When 1 was added first, H2Sꢀtreated cells also
showed stronger fluorescence than those incubated with 1 alone.
project (B14004).
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Notes and references
aState Key Laboratory of Organic-Inorganic Composites and Beijing Key
Laboratory of Bioprocess, Beijing University of Chemical Technology,
Beijing 100029, China. E-mail: yilong@mail.buct.edu.cn
50
55
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5
bState Key Laboratory of Medicinal Chemical Biology and College of
Pharmacy, Nankai University, Tianjin, 300071, China
cState Key Laboratory of Elemento-Organic Chemistry and Department
of Chemical Biology, National Pesticide Engineering Research Center
(Tianjin), Collaborative Innovation Center of Chemical Science and
10 Engineering, Nankai University, China. Email: zhenxi@nankai.edu.cn
†Electronic Supplementary Information (ESI) available: Experimental
details and supporting figures. See DOI: 10.1039/b000000x/
‡The authors pay equal contributions to this work.
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Coverpicture
A highly sensitive and fastꢀresponse fluorescent probe for HClO
detection was developed and employed to reveal the H2Sꢀinduced
HClO biogenesis in living cells.
5
This journal is © The Royal Society of Chemistry [year]
Journal Name, [year], [vol], 00–00 | 5