A highly sensitive and selective turn-on fluorescent probe for hypochlorous acid monitoring

Article abstract of DOI:10.1039/c4ra14579h

A novel turn-on fluorescent probe 3 for monitoring hypochlorous acid was investigated, which showed high selectivity and sensitivity to hypochlorous acid over other ROS and was loaded into cell media to image HClO.

Full text of DOI:10.1039/c4ra14579h

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A highly sensitive and selective “turn-on”
fluorescent probe for hypochlorous acid
monitoring†
Cite this: RSC Adv., 2015, 5, 18275
Received 15th November 2014
Accepted 28th January 2015
*
Ye-Xin Liao, Mao-Die Wang, Kun Li, Zhao-Xuan Yang, Ji-Ting Hou, Ming-Yu Wu,
*
Yan-Hong Liu and Xiao-Qi Yu
DOI: 10.1039/c4ra14579h
www.rsc.org/advances
A novel “turn-on” fluorescent probe 3 for monitoring hypochlorous electron-rich group (such as anilino group, pyrrole ring and
acid was investigated, which showed high selectivity and sensitivity to phenyl group) oxidation,13 double bond oxidation14 and
hypochlorous acid over other ROS and was loaded into cell media to others.15 With the unclear mechanism of HClO/ClOꢀ in bio-
image HClO.
logical processes and the signicance of monitoring HClO/ClOꢀ
in industry, a further investigation into more selective and
sensitive HClO/ClOꢀ probes is desperately needed.
Reactive oxygen species (ROS, such as HClO and H2O2), play
important roles in many biological processes. Excessive ROS
conduce oxidative stress (OS), which will cause irreversible
damage to living cells or tissues.1 Hypochlorite acid (HClO), or
its anionic form the hypochlorite anion (ClOꢀ), generated by the
myeloperoxidase (MPO)-mediated reaction between H2O2 and
Clꢀ in vivo,2 is a highly reactive species. HClO/ClOꢀ is an
essential microbicidal agent in the immune defenceꢀsystem,
such as in leukocytes.3 On the other hand, HClO/ClO readily
reacts with proteins, lipids and nucleic acids by oxidation or
chlorination,4 resulting in some related diseases such as
inammatory diseases, cardiovascular disease, neurodegener-
ative diseases, and cancer.5 However, the exact role that HClO/
ClOꢀ plays in biological processes is still not clearly known yet.6
In addition, HClO/ClOꢀ is also used as a bleaching and
microbicidal agent in industry or in the healthcare services,7
such as for purifying drinking water and for preparing the
famous “84” disinfectant. Excess HClO/ClOꢀ residue in
drinking water is harmful to plants, animals and human
beings.
Electron-rich groups can induce an “enhanced photoin-
duced electron transfer (PET)” process to a uorophore and
quench its uorescence, which is a more effective way to quench
the uorescence rather than through the use of single atom
electron donors like N, S or Se, resulting in a weaker back-
ground, as Peng and co-workers reported.13f As an electron-rich
group, the anilino group was demonstrated to be an excellent
quencher in “off–on” type uorescent probes for ROS.16 Thus,
the anilino group was introduced to our target probe named 3
(Scheme 1) as a modulator to quench the uorescence of the
uorophore 1-(benzo[d]thiazol-2-yl)naphthalen-2-ol (compound
1) through an enhanced PET process, giving a non-uorescent
compound. We hoped that HClO/ClOꢀ could react with probe
With some excellent advantages such as high temporal–
spatial resolution, high selectivity and sensitivity,8 uorescent
probes have become outstanding tools in modern analytical
technology. Many uorescent probes for monitoring HClO/
ClOꢀ have been designed and developed based on some specic
selective reactions, which include oxime oxidation,9 sulfur or
selenium oxidation,10 dehydrogenation,11 lactam oxidation,12
Key Laboratory of Green Chemistry and Technology (Ministry of Education), College of
Chemistry, Sichuan University, Chengdu, 610064, P. R. China. E-mail: kli@scu.edu.cn;
xqyu@scu.edu.cn
† Electronic supplementary information (ESI) available: 1H NMR, 13C NMR and
Scheme 1 The synthetic processes of probe 3 and the reaction
between 3 and HClO. (a) Na2S2O5, DMF, 120 ꢁC; (b) 4-chloroni-
trobenzene, 60% NaH, DMF, 90 ꢁC; (c) H2, Pd/C, MeOH, r.t. overnight.
HRMS for all of the compounds. See DOI: 10.1039/c4ra14579h
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3 by oxidizing the anilino group to release compound 1, and to
manifest a uorescence “turn-on” phenomenon as a signal
conversion. As expected, aer reacting with HClO/ClOꢀ, probe 3
was oxidized and compound 1 (Scheme 1) was generated, along
with a dramatic uorescence enhancement.
The synthetic processes of the target probe 3 were shown in
Scheme 1. Starting with 2-aminothiophenol and 2-hydroxyl-1-
naphthaldhyde, compound 1 was obtained by using Na2S2O5
as a catalyst. Aer compound 1 reacted with 1-uoro-4-
nitrobenzene, compound
2 was generated, followed by
hydrogen reduction catalyzed by Pd/C. Finally, the target probe
1
3 was synthesized, and was characterized using H NMR, 13C
NMR and high-resolution mass spectroscopy (HRMS).
Fig. 1 The HPLC results for 1 (green line), 3 (red line) and the reaction
between 3 and HClO/ClOꢀ (blue line).
In order to determine the optimal detecting condition, we
rst studied the uorescence intensity in different solvents. As
shown in Fig. S1,† in solvents like H2O, DMSO, DMF, THF,
MeCN, MeOH and EtOH, no obvious uorescence changes were
observed upon addition of 5 mM of 3 with 50 mM of HClO/ClOꢀ.
On the other hand, considering that cetyltrimethyl ammonium
bromide (CTAB, critical micelle concentration 0.9 mM in
water17), has been successfully applied in some sensing appli-
cations to disperse insoluble compounds in water and to
promote the reaction,18 CTAB was introduced in order to
disperse probe 3 to form a stable sensing solution. As expected,
when 1 mM of CTAB was added, the uorescence intensity was
enhanced 100 fold aer 5 mM of 3 reacted with 50 mM of HClO/
ClOꢀ in water. Hence, water with CTAB (1 mM) was chosen as
the detecting media in the following experiments.
Subsequently, the absorbance and uorescence properties of
3 before and aer dealing with HClO/ClOꢀ were studied. In the
absorbance spectrum of 3, there are two side peaks at 295 nm
and 335 nm. Besides, 3 exhibits extremely weak uorescence in
DMSO/HEPES buffer (1 : 99, v/v, 1 mM CTAB, pH 7.40, 20 mM
HEPES) with a quantum yield of 0.005. Aer 3 reacted with
HClO/ClOꢀ, a wide absorbance peak around 410 nm appeared
(see Fig. S2a†). We also found a similar peak in the absorbance
spectrum of compound 1. A dramatic enhancement was
observed in the uorescence spectra centred at 465 nm aer 3
was mixed with HClO/ClOꢀ, which tted well with the emission
spectrum of 1 when the spectra were normalized (Fig. S2b†),
nally yielding a blue emission with a quantum yield of 0.212.
In addition, the HPLC results for the reaction between 3 and
HClO/ClOꢀ showed that a new peak appeared at 3.83 min, as
shown in Fig. 1, while there was a similar peak at the same
retention time in the HPLC spectrum of compound 1. Mean-
while, an [M ꢀ H]ꢀ peak for compound 1 (calculated as
276.0489) was also found at m/z 276.0483 in the high resolution
mass spectrum (HRMS) for the reaction between 3 and HClO/
ClOꢀ (Fig. S3†). These results indicated that compound 1 was
generated during the interaction between 3 and HClO/ClOꢀ.
Next, the response of 3 towards HClO/ClOꢀ was investigated
value increased from 5.26 to 6.99, the uorescence intensity at
465 nm enhanced gradually, until nally reaching a maximum.
Moreover, the effect of the pH value on compound 1 showed
that 1 also exhibited relatively weak uorescence when it was in
acidic conditions (pH < 5, Fig. S4†), and we attributed this
phenomenon to the pH effect of 1, in some degree. When the
pH value was above 7.40, the uorescence intensity at 465 nm
decreased with an increase in the pH value. ClOꢀ should be the
dominating species at a high pH value.12g As HClO is a more
powerful oxidizing agent than ClOꢀ, we considered that ClOꢀ
was not efficient enough to oxidize probe 3. So we suspected
that probe 3 reacted with HClO rather than ClOꢀ. Meanwhile, 3
exhibited no uorescence across the whole pH test range (pH 3–
12), as shown in Fig. S4.†
With the determination of these reaction conditions, the
spectral responses of 3 in the presence of HClO were then
recorded. Absorption spectra of 3 upon the addition of different
concentrations of HClO showed that the absorption peak cen-
tred at 410 nm arises gradually (Fig. 2a). The uorescence
responses of probe 3 (5 mM) upon titration with HClO in Fig. 2b
exhibited that the uorescence intensity increased to
a
maximum with the addition of 35 mM of HClO, and was then
Fig. 2 (a) UV spectra of 3 (20 mM) reacted with different equivalents of
HClO in DMSO–water (2 : 98, v/v) buffered by 20 mM HEPES at pH
at different pH values (Fig. S4†). As can be seen, in the solutions 7.40. The different equivalents included: 0, 1, 3, 5, 7, 9, 10 and 12. (b)
containing probe 3 and HClO/ClOꢀ, almost no uorescence at
The fluorescence responses of 3 (5 mM) to different concentrations of
HClO. The concentrations of HClO included: 5 mM, 10 mM, 15 mM, 20
mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 60 mM, 75 mM, 85 mM
and 100 mM. The reaction solutions were kept at ambient temperature
465 nm was found when the pH value was below 5.26. The
proposed mechanism suggests that the amino group of probe 3
might be protonated at a low pH value, which would block the
oxidation of the amino group by HClO/ClOꢀ. When the pH different concentrations of HClO.
for 30 min. Inset: scatter diagram of relative fluorescence intensity in
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quenched up until the addition of 50 mM of HClO, when an
equilibrium was nally reached. We subsequently set 50 mM as
the test concentration of HClO in the following experiments.
Additionally, it was shown that the uorescence intensity of the
reaction solution at 465 nm was linearly proportional (R2
¼
0.9994, Fig. S5†) to the concentrations of HClO from 0 to 20 mM,
and the detection limit was as low as 26.2 nM. Time-dependent
uorescence responses of the reaction of probe 3 and HClO
showed that the reaction completed within 15 min (Fig. S6†).
Thus, we set 30 min as the reaction time in the following
investigations to ensure a completed reaction.
Furthermore, we did an investigation on the selectivity of
probe 3 for different ROS (Fig. 3). Among the ROS, 3 only
responded to HClO and a slight enhancement with ONOOꢀ.
Some evidence shows that a normal concentration of steady-
state ONOOꢀ is at a nanomolar level, when HClO is at a
micromolar level in physiological conditions. The concentra-
tion level of ONOOꢀ wouldn’t be high enough in comparison
with the level of HClO.12d Therefore, we consider 3 to be a highly
selective probe for HClO over other ROS. Addtionally, some
Fig. 4 Fluorescence images of HeLa cells incubated with 5 mM 3 (with
1 mM of CTAB) for 30 min in the absence (a and b) and for another 20
min in the presence (c and d) of 50 mM NaClO. (b) and (d) are the merge
windows.
reactive sulfur species (RSS), including GSH, Cys, Hcy and S2ꢀ
,
which exist in an abundant amount in vivo, were incubated with
probe 3 at high concentration levels. However, no remarkable
uorescence responses for these test reaction systems were
recorded.
Finally, probe 3 was loaded into HeLa cells to image HClO.
As seen in Fig. 4, when HeLa cells were incubated with only
probe 3 (with 1 mM CTAB) for 30 min in PBS at 37 ꢁC, no notable
uorescence responses were observed upon excitation at 405
nm (Fig. 4a). Aer dealing with HClO for another 20 min, the
Acknowledgements
This work was nancially supported by the National Program on
Key Basic Research Project of China (973 Program,
2012CB720603 and 2013CB328900), the National Science
Foundation of China (nos 21232005, 21472131, J1310008 and
J1103315), and the Specialized Research Fund for the Doctoral
Program of Higher Education in China (20120181130006).
cells pretreated with
enhancement (Fig. 4c). Herein, we successfully applied probe 3
for HClO imaging in cell media.
In conclusion, we report the novel, easily available, “turn-on”
uorescent probe 3 based on the mechanism of enhanced PET,
which exhibited excellent selectivity and a high sensitivity
toward HClO with a detection limit of 26.2 nM. Moreover, 3 was
successfully applied for the detection of HClO in HeLa cells.
3 showed remarkable uorescence
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