A highly selective and sensitive photoinduced electron transfer (PET) based HOCl fluorescent probe in water and its endogenous imaging in living cells

Article abstract of DOI:10.1039/c6cc02603f

A probe based on the phenothiazine-acridine orange conjugate (Ptz-AO) has been designed and synthesized for the sensitive and selective detection of HOCl. Ptz-AO has excellent properties, including pH-independence of fluorescence, high resistance to photobleaching, and response in real time. The value of Ptz-AO was confirmed by exogenous, endogenous and the real-time imaging of HOCl in vitro using a fluorescence microscope.

Full text of DOI:10.1039/c6cc02603f

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COMMUNICATION
Highly Selective and Sensitive Photoinduced Electron Transfer
(PET) Based HOCl Fluorescent Probe in Water and Its Endogenous
Imaging in Living Cells
Received 00th January 20xx,
Accepted 00th January 20xx
Lijuan Liang,a Chang Liu,a,b Xiaojie Jiao,b Liancheng Zhaoa and Xianshun Zengb, *
DOI: 10.1039/x0xx00000x
www.rsc.org/
A probe based on a phenothiazine-acridine orange conjugate (Ptz- physiological and pathological processes of HOCl at
AO) has been designed and synthesized for sensitive and selective cellular/subcellular level due to its excellent temporal and
detection of HOCl. Ptz-AO has excellent properties, including pH- spatial resolution capability in living cells.6 So far various
independence of fluorescence, high resistance to photobleaching, fluorescent probes with different responsive moieties toward
and response in real time. The value of Ptz-AO was confirmed by HOCl have been designed and developed by taking advantage
exogenous, endogenous and real-time imaging of HOCl in vitro of the strong oxidization ability of HOCl.7 Although many of
using a fluorescence microscope.
these probes have been successfully utilized to image HOCl in
cellular environments, few of them could be applied to realꢀ
time detect HOCl at cellular levels. On the other hand, some of
them suffer from low quantum yield, autoxidation, photoꢀ
bleaching, poor water solubility or lack selectivity toward
HOCl over other ROS. As a result, it is challenging and highly
desired to prepare novel realꢀtime responsive probes7s for
investigating the distribution and functions of HOCl at cellular
level.
The reactive oxygen species (ROS) have played important roles
in cell signaling and homeostasis, such as antiinflammation
regulation, pathogen response and so on.1 Among ROS,
hypochlorous acid (HOCl) produced by myeloperoxidase
(MPO) or induced by a number of soluble stimuli has received
special attention for its pivotal antimicrobial nature in the
immune system, which helps inhibit inflammation and regulate
cellular fate.2 In nature, HOCl is a weak acid (pKa = 7.63) and
is highly reactive and shortꢀlived in physiological
It is know that the Nꢀalkylated derivatives of acridine orange
(AO) fluorophore (Chart 1) have several interesting inherent
photophysical natures which are highly expected for its use as
tracers for fluorescence staining caused by the “pushꢀpull”
properties of its internal charge transfer (ICT) excited state,
arising from the charged electronꢀwithdrawing nitrogen atom
environments.3 However, keeping
a
reasonable HOCl
concentration within the physiological environments is
essentially required for numerous cellular functions. Excessive
or uncontrolled production of HOCl can lead to tissue damage
and diseases, including cardiovascular diseases, lung injury
atherosclerosis, osteoarthritis, and cancer.4 Thus, it is urgently
required for the development of highly sensitive and selective
methods for further investigation of the complex contributions
of HOCl to human health.
Me2N
Me2N
S
N
N+
ꢀ
R
Xꢀ
N+
BF4
In recent years, a number of analytical techniques, such as
colorimetric, fluorometric, electrochemical, chromatographic,
and optical imaging methods,5 have been proposed for the
detection of HOCl under different conditions. Of all these
analytical techniques, fluorescence imaging shows some sort of
uniqueness and irreplaceability for the investigation of the
Me2N
PtzꢀAO
Me2N
Nꢀalkylated AO
Chart 1 Structures of N-alkylated acridine orange (AO) and probe Ptz-AO
.
Me2N
Br
a)
b)
c)
S
N
N+
ꢀ
S
NH
BF4
N
a.School of Materials Science and Engineering, Harbin Institute of Technology,
Harbin 150001, China.
S
Me2N
Ptz-AO
b.Tianjin Key Laboratory for Photoelectric Materials and Devices, Tianjin University
of Technology, Tianjin 300384, China. Fax: (+86)22-60215226; Tel: (+86)22-
60216748; E-mail: xshzeng@tjut.edu.cn.
Ptz
1
Scheme 1 Synthesis of the probe Ptz-AO. Reagents and conditions: a) NaH,
1,3-dibromopropane, DMF, r. t., 4 h; b) acridine orange, KI, toluene, reflux,
24 h; c) KBF4, CH2Cl2/EtOH (1:1, v/v), reflux, 3 h.
Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
DOI: 10.1039/x0xx00000x
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Me2N
PET
Me2N
Me2N
PET
PET
HClO
N+
N+
O
S
N
N
N+
+
S
N
S
+
ꢀ
ꢀ
ꢀ
BF4
BF4
BF4
Me2N
Me2N
Ptz-AO
Me2N
Ptz-AO cation
OPtz-AO
Scheme 2 Proposed sensing mechanism of the probe Ptz-AO towards HOCl.
within the chromophore and the two electronꢀdonating amino
moieties. The merits for its use in biological applications are
that the intense absorption band at ~490 nm meets perfectly the
discrete excitation of the argon laser at 488 nm and emits at ~
540 nm while also possessing high quantum yields and good
photostability in varieties of organic and aqueous media. It has
been widely used in cell staining of DNA in apoptosis studies.8
Herein, we report a photoinduced electron transfer (PET) based
Fig. 1 Fluorescent titration spectra of Ptz-AO (5 μM) in the presence of
different concentrations of NaClO in H2O; Inset: the fluorescence at 540 nm
of Ptz-AO (5 μM) as a function of the NaClO concentration. λex = 475 nm,
Slit = 5 nm, 5 nm.
HOCl fluorescent probe PtzꢀAO,
a propylene tethered
conjugate of phenothiazine (Ptz) and acridine orange (AO) dye
(Chart 1). The probe shows high selectivity, high sensitivity
(detection limit of 2.7 nM) and fast response (within 5 seconds)
towards HOCl in water. The marvellous sensing properties of
the probe PtzꢀAO enable its use in living cells for the realꢀtime
monitoring of HOCl in INSꢀ1 βꢀislet cells and RAW264.7
macrophage cells.
Fig. 2 Change ratio (F - F0)/F0 of fluorescence intensity of Ptz-AO (5 μM)
upon the addition of 2.0 equiv. other ions and anions in H2O; histogram
representing the fluorescence enhancement of the probe in the presence
of other ions and ROS. 1: O21; 2: AcO-; 3: Al3+; 4: Ca2+; 5: Cl-; 6: Cu2+; 7: Fe2+; 8:
-
Fe3+; 9: H2O2; 10: ClO-; 11: Hg2+; 12: K+; 13: Na+; 14: NO2 ; 15: ·OH; 16:
The synthetic processes of the target probe were shown in
.
Scheme 1. The compound
1
was prepared in 65% yield by the ONOO- ; 17: TBHP; 18: Zn2+; 19: NO; 20: O2.-. λex = 475 nm, Slit = 5 nm, 5
nm.
reaction of phenothiazine with 1,3ꢀdibromopropane in sodium
hydride (60% dispersing in mineral oil) in dry DMF at room
temperature. Then, PtzꢀAO was facilely obtained in 58% yield
calculated as 8.95 µM (R = 0.9988) (Fig. S2, ESI†).9 From the
changes in ClOꢀꢀdependent fluorescence intensity, the detection
limit of PtzꢀAO was estimated to be 2.7 nM (Fig. S3, ESI†).9b
To explore the selectivity of the probe towards HOCl among
other reactive oxygen species ROS, we simultaneously
evaluated the response of PtzꢀAO to other ions and ROS in
by the reaction of acridine orange with
1 in the presence
catalytic amount of KI. The anion exchange was achieved by
refluxing the CH2Cl2/EtOH (1:1, v/v) solutions PtzꢀAO with
excess KBF4. The chemical structure of PtzꢀAO was confirmed
by 1H NMR, 13C NMR, and HRMS spectra.
water. As shown in Fig. 2, the addition of 2 equiv. of other
The emission properties of the probe PtzꢀAO and those in the
presence of an incremental amount of HOCl were initially
elucidated in H2O (Fig. 1). The probe showed weak
fluorescence emission (Φ = 0.011) at 540 nm in water due to
the strong electron donating property of Ptz moiety which
quenched the fluorescence emission of AO via the photoꢀ
induced electron transfer (PET) process. As can be seen from
Fig. 1, the fluorescence titration of the probe with NaClO led to
a rapid increase of the fluorescence emission intensity at 540
nm due to the HOClꢀpromoted oxidation of the electron donatꢀ
ing Ptz moiety to Ptz sulfoxide (OPtzꢀAO) and Ptz cation (Ptzꢀ
AO cation)10 which prevented the PET process from Ptz to AO
(Scheme 2). Highꢀresolution mass spectra (HRMS) of PtzꢀAO,
and the UVꢀVis spectra and HRMS of a model compound Nꢀ
ethylphenothiazine provided reliable evidence for the oxidation
of the probe (Fig. S1aꢀ1c, ESI†). When the ratio of
[NaClO]total/[PtzꢀAO] reached 5 : 1, higher [NaClO]total did not
lead to further emission enhancement as shown in the inset of
Fig. 1. Over a 75ꢀfold enhancement of fluorescence intensity (Φ
= 0.823) was observed under saturated conditions. The increase
of the fluorescence emission at 540 nm followed the sigmoidal
curves and the fluorescence turnꢀon constant (Kturnꢀon) was
.
ROS, such as O2 , H2O2, ONOOꢀ , NO, O2.-, and tꢀBuOOH, and
1
other ions, such as AcOꢀ, Clꢀ, NO3 , NO2 , Al3+, Ca2+, Cu2+,
Fe2+, Fe3+, K+, Na+, and Zn2+ had no obvious effect on the
fluorescence emission, only ·OH responded with a slight
increase (ca. 2.0ꢀfold enhancement) in the fluorescent intensity.
In contrast, the addition of NaClO resulted in a significant
enhancement (over 26ꢀfold) of the fluorescence intensity
positioned at 540 nm. Thus, PtzꢀAO can function as highly
selective fluorescence probe for the HOCl.
ꢀ
ꢀ
To further explore the utility of the probe as a fluorescent
chemosensor for HOCl, the competition experiments were
subsequently conducted in which PtzꢀAO (5 ꢁM) was first
mixed with 2 equiv. of other ions and ROS, and then 2 equiv. of
NaClO was added. As shown in Fig. S4, no strong interference
was observed in the presence of other ions and ROS. The
excellent selectivity towards HOCl among other ions and ROS
suggested that PtzꢀAO had the potential to detect HOCl in
complex biological environment.
At the same time, fluorescence changes of PtzꢀAO as a
function of pH were measured in the presence and absence of
NaClO in H2O. The probe showed weak fluorescence signal
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over a pH range of 1ꢀ12 (Fig. S5, ESI†). However, it showed HOCl, macrophages RAW 264.7 were successively preꢀtreated
very strong fluorescence emission upon the addition of 4.0 with bacterial cell wall lipopolysaccharide (LPS) and phorbol
equiv. NaClO from pH 5.0 to pH 11.0. Thus, the results
indicated that the probe Ptz-AO was capable of detecting
hypochlorite and hypochlorous acid in the pH range 5-11
because the pKa of HOCl is 7.6.3
Finally, the timeꢀdependent fluorescence response and the
photostability of the probe under excitation at 475 nm were
measured in the absence and presence of NaClO. As shown in
Error! Reference source not found., a weak fluorescence
signal was observed with the probe alone. Upon the addition of
NaClO (0.75 equiv.), however, the fluorescence intensity
increased immediately and reached the maximum within 5 s.
Then the fluorescence intensity was stable under continuous
Fig. 3 Fluorescence image of INS-1 β-islet cells incubated with the probe Ptz-AO
wavelength 475 nm laser excitation from 5 s to 300 s. The
results suggested that the probe was photostable. Meanwhile,
the fast response to ClOꢀ allowed it to be applied to realꢀtime
detecting HOCl in water phase.
(3 µM). a) fluorescence and brightfield image after staining for 20 min; b)
fluorescence and brightfield image after incubated with ClO- (6 µM) for 20 min.
In light of the above desirable fluorescence properties of Ptzꢀ
AO as fluorescence probe for the detection of intracellular
HOCl, it was then applied to detect exogenous HOCl in INSꢀ1
cells, a murine βꢀislet cell line (Fig. ). As shown in Fig. a, when
the INSꢀ1 cells were incubated with the probe PtzꢀAO (3 µM)
for 20 min in the growth medium and then washed with DPBS,
a weak fluorescence signal of the probe was present in the cells
with an excitation of green light (488 nm). After treatment with
NaClO (6 ꢁM) for 20 min, however, intense fluorescence signal
of the probe was emerged within the cells (Fig. b). By
comparison with the brightfield image, we found that the
fluorescence signals were only located in the intracellular area,
demonstrating that the probe PtzꢀAO was able to detect
exogenous HOCl in cells.
Fig. 4 Time-dependent fluorescence image of INS-1 β-islet cells incubated with
the probe Ptz-AO (1 µM). Fluorescence (A1) and brightfield (A2) image after
staining for 20 min. Fluorescence (B1-F1) and brightfield (B2-F2) image after
incubated with ClO- (2 µM) for 2, 3, 4, 5, 15 min.
Due to the highly reactive and shortꢀlived properties of
HOCl in physiological environments, we then investigated the
timeꢀdependent fluorescence response of the probe in living
cells to elucidate the realꢀtime responsive properties of the
probe. When INSꢀ1 βꢀislet cells were incubated with probe (1
µM) for 20 min in PBS at 37 oC, no notable fluorescence
responses were observed (Fig. A). Upon the addition of NaClO
(2 µM), an obvious fluorescence signal was observed within 2
min. Meanwhile, a significant increase of the fluorescence
signals was observed with the incubated time (Fig. BꢀF). The
integrated optical densities (IOD) of the imagings with the
incubated time indicated that there was a rapid increase of the
fluorescence signals from 2 min to 5 min after the addition of
NaClO (Fig. S7, ESI†). The time-dependent fluorescence
response of the probe (1 µM) in RAW 264.7 cells also showed a
very fast response. There was a marked increase of the
fluorescence signals after the addition of NaClO within 2 min
(Fig. S8, ESI†). Therefore, the probe Ptz-AO was very effective
for HOCl detection and should be suitable for practical
application in vitro.
Finally, we investigated whether the probe PtzꢀAO was
capable of detecting intracellular endogenous HOCl produced
by resting tissue macrophages under mimic inflammatory
conditions. To elicit the elevated production of endogenous
Fig. 5 Fluorescence image of RAW264.7 cells incubated with the probe Ptz-
AO (1 µM). a) fluorescence and brightfield image after staining for 20 min;
b) fluorescence and brightfield image after stimulated by LPS and PMA for
20 min; c) fluorescence and brightfield image after stimulated by NAC for
30 min, and then stimulated by LPS and PMA for 20 min.
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12ꢀmyristate 13ꢀacetate (PMA).11 With LPS/ PMA
stimulation, prominent enhanced fluorescence signals were
observed from the living RAW 264.7 cells compared with the
controls (Fig. a and 5b), indicating the activation of the probe
PtzꢀAO under mimic inflammatory conditions. When Nꢀ
acetylcysteine (NAC), a general antioxidant,12 was used to treat
the cells together with LPS/PMA elicitation, no obvious
fluorescence was observed after incubation with the probe Ptzꢀ
AO (Fig. c). The result indicated that NAC scavenges
endogenously generated HOCl from RAW 264.7 cells and
effectively inhibited the activation of the probe PtzꢀAO. These
results clearly demonstrated that the probe PtzꢀAO can
efficiently detect HOCl produced in stressed cells.
In conclusion, we have designed and synthesized a novel
fluorescence probe PtzꢀAO by the reaction of Nꢀ(3ꢀbromoꢀ
propyl)phenothiazine with acridine orange. PtzꢀAO functions as
a highly HOClꢀselective fluorescent probe with a fast (within 5
s) and sensitive response, and PtzꢀAO exhibits excellent
properties as HOClꢀspecific fluorescent probe for biological
applications, including pHꢀindependence and tolerance to
photobleaching during excitation laser irradiation. With the
probe PtzꢀAO, we successfully conducted exogenous,
endogenous and realꢀtime imaging of HOCl by means of
fluorescence microscopy. We anticipate that this probe should
prove useful for investigation of the wide range of biological
functions of HOCl.
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