An ESIPT-based colorimetric and fluorescent probe with large Stokes shift for the sensitive detection of hypochlorous acid and its bioimaging in cells

Article abstract of DOI:10.1039/d0nj05807f

Hypochlorous acid (HOCl), with a low physiological concentration, plays a vital role in killing the pathogens and anti-inflammation in the human immune system. Deviation from the normal concentration is directly related to the inflammation-associated diseases and even cancer. Thus, it is necessary to sensitively detect the tiny concentration changes of HOCl. However, few of the reported probes could practically work well due to small Stokes shift, high detection of limits, and low SNR. In this study, HBT (2-(2′-Hydroxyphenyl)benzothiazole) was used as the candidate for the ESIPT-based fluorophore and connected to pyridinevia“CC” bonds. The “CC” bonds are the reaction site for HOCl and are activated by the strong electron-withdrawing CN group located on the double-bond carbon atom, resulting in a high sensitivity towards HOCl. Thereby, the probe's emission shifted effectively to the red channel avoiding the interference from biological autofluorescence. The excellent response of the probeJBDtowards HOCl enables it to detect endogenous hypochlorous acid in the cells and provides an ideal molecular tool for exploring the mechanism of hypochlorous acid in oxidative stress and its immune role.

Full text of DOI:10.1039/d0nj05807f

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An ESIPT-based colorimetric and fluorescent
probe with large Stokes shift for the sensitive
detection of hypochlorous acid and its bioimaging
in cells†
Cite this: New J. Chem., 2021,
45, 4724
c
ab
Haixian Ren,ab Fangjun Huo
and Caixia Yin
*
Hypochlorous acid (HOCl), with a low physiological concentration, plays a vital role in killing the
pathogens and anti-inflammation in the human immune system. Deviation from the normal
concentration is directly related to the inflammation-associated diseases and even cancer. Thus, it is
necessary to sensitively detect the tiny concentration changes of HOCl. However, few of the reported
probes could practically work well due to small Stokes shift, high detection of limits, and low SNR. In
this study, HBT (2-(20-Hydroxyphenyl)benzothiazole) was used as the candidate for the ESIPT-based
fluorophore and connected to pyridine via ‘‘CQC’’ bonds. The ‘‘CQC’’ bonds are the reaction site for
HOCl and are activated by the strong electron-withdrawing CN group located on the double-bond
carbon atom, resulting in a high sensitivity towards HOCl. Thereby, the probe’s emission shifted
effectively to the red channel avoiding the interference from biological autofluorescence. The excellent
response of the probe JBD towards HOCl enables it to detect endogenous hypochlorous acid in the
cells and provides an ideal molecular tool for exploring the mechanism of hypochlorous acid in
oxidative stress and its immune role.
Received 28th November 2020,
Accepted 6th February 2021
DOI: 10.1039/d0nj05807f
rsc.li/njc
been employed to design fluorescent probes, including PET,
ICT, FRET and ESIPT.18–20 In particular, the ESIPT process that
1. Introduction
Hypochlorous acid (HOCl), a biologically active oxygen species, takes place rapidly along with a large Stokes shift21,22 and
can be generated via the oxidation of chloride ions by hydrogen improves the SNR (Signal to Noise Ratio) by avoiding internal
peroxide under the catalysis of MPO.1,2 Although it is present at filtering and self-absorption effects.23–25 It has been employed
low concentrations in organisms, HOCl plays a vital role in to design the probes for many important ROS including hydrogen
regulating the physiological processes, such as antibacterial peroxide,26 superoxide,27 and HOCl.28 However, the reported
activity.3–7 Abnormal levels of intracellular HOCl are associated ESIPT-based HOCl probes28–31 come with short emission that
with numerous diseases, including cancer and neurodegenera- limits the practical applications due to severe biological autofluor-
tive diseases.8–10 Therefore, the sensitive monitoring of the tiny escence interference. Thus, a important field could be shifted
changes in the concentration of HOCl in cells is helpful for the towards the ESIPT-based HOCl probes with red, even NIR
prevention and timely treatment of the related diseases; there- emission.32
fore, qualified detecting tools for HOCl are urgently needed.
Given a low physiological concentration of HOCl,33 a competent
Fluorescent probes have aroused great interest and atten- HOCl probe needs to have low detection limits and ultra-high
tion because of their highly sensitive, non-destructive and sensitivity, which require the perfect synergy of fluorophores and
multilevel fluorescence imaging.11–17 Many mechanisms have reaction sites. It is known that the ‘‘CQC’’ bonds have the ability of
reacting with hypochlorous acid through the natural reducibility
reaction.34,35 In this study, the HBT group was used as the ESIPT
a Department of Chemistry, Xinzhou Teachers University, Xinzhou 034000, China.
fluorophore and connected to the pyridine group through the
‘‘CQC’’ bonds to construct the probe JBD (Scheme 1), in which
the reaction site ‘‘CQC’’ bonds were activated by the strong
electron-withdrawing CN group located on the double-bond carbon
atom, resulting in a high sensitivity towards HOCl. Thereby,
the probe’s emission was shifted to the red channel, effectively
avoiding the interference caused by biological autofluorescence.36,37
E-mail: yincx@sxu.edu.cn; Fax: +86-351-7011022; Tel: +86-351-7011022
b Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of
Education, Institute of Molecular Science, Shanxi University, Taiyuan 030006,
China
c Research Institute of Applied Chemistry, Shanxi University, Taiyuan 030006,
China
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
d0nj05807f
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HOCl to the probe, the absorption at both peaks decreased with
the change in color of the solution from light red to colorless
(Fig. 1a). Fluorescent spectra of JBD were examined in different
solvents (Fig. 1b). The probe JBD showed strong emission at
long wavelengths in acetonitrile and dichloromethane, but
relatively weak ones in DMSO and protic solvents such as
ethanol or methanol. This is because protic solvents are unfavorable
for the ESIPT process. Therefore, we chose acetonitrile as the solvent
to examine the fluorescent response of JBD towards HOCl.
Excited by visible light (400 nm), probe JBD exhibited double
emission: an enol-form one at 469 nm and a keto-form one at
607 nm, along with a large Stokes shift 207 nm, indicating that
the ESIPT process occurred on probe JBD (Fig. 1c). After adding
hypochlorous acid to the probe, the double emissions were
turned off, which could possibly be caused by the strong
electron-withdrawing carboxyl group (see details in Section 3.6).
Scheme 1 The design and synthesis of the probe JBD.
The probe JBD stood out through the large Stokes shift, low
detection limit and high SNR for HOCl, realizing the sensitive
detection of endogenous HOCl in cells.
2 Experimental section
2.1. Synthesis and characterization
As shown in Scheme 1, 4-pyridylacetonitrile (0.24 g, 2 mmol) and
3-(benzo[d]thiazol-2-yl)-2-hydroxy-5-methylbenzaldehyde (0.54 g,
2 mmol) were dissolved in 30 mL CH3CH2OH, along with 50 mL
piperidine. The resulting mixture was stirred at 78 1C for 2 h.
After the reaction completed, the product was filtered, washed
and dried to obtain a pure yellow solid. (0.64 g, 86%). It was
characterized by NMR and further assigned as chemical shifts
(atomic code). The atomic codes are shown in Fig. S1 (ESI†).
1H NMR (600 MHz, CDCl3) d 13.43 (H12), 8.72 (H11, 10), 8.44 (H7),
8.23 (H1), 8.01 (H4), 7.95 (H2), 7.73 (H8,9), 7.65 (H3), 7.55 (H5),
7.47 (H6), 2.46 (H13). 13C NMR (151 MHz, CDCl3) d 168.70 (C7),
155.44 (C13), 151.43 (C1), 150.43 (C14), 142.25 (C18,19), 139.40 (C16),
132.57 (C6), 131.92 (C10), 131.75 (C9), 128.97 (C11), 127.03 (C3),
126.02 (C4), 122.25 (C8), 122.00 (C5), 121.69 (C2), 120.09 (C17,20),
117.18 (C21), 116.97 (C12), 108.83 (C15), 20.71 (C22). HR-MS [JBD]+:
m/z Calcd 370.1009, Found 370.10069. (Fig. S1, ESI†).
3.2. Sensitivity of JBD for HOCl
To investigate the probe’s response towards HOCl in depth, we
tracked the probe’s emission at 607 nm by adding different
concentrations of HOCl to the probe (Fig. 1d and 2). The
induced emission at 607 nm decreased as the concentration
of added HOCl increased and reached a balanced point upon
the addition of 10 mM HOCl to the probe. Further, the emission
intensity linearly changed with the concentration of added
HOCl in the 0–9.5 mM range. The detection limit (LOD) was
0.067 mM as calculated by the general 3s/k method, suitable for
sensitively monitoring the HOCl concentration in the biological
system (Fig. 3).
3.3. pH effect
2.2. General measurements
Next, we evaluated the pH interference of the probe JBD for HOCl
detection. The probe had stable optical properties in the pH range
of 5–10, and showed a relatively strong signal at pH 7–8.
JBD (2 mM) was prepared in CH3CN as the stock solution. HOCl
and other competing species were dissolved in deionized water to
prepare the stock solutions, respectively. All the detection experi-
ments were measured in a CH3CN/PBS system (30%acetonitrile in
PBS, pH 7.4). The fluorescent signal was monitored by adding an
analyte sample into a CH3CN/PBS system containing 10 mM JBD.
2.3. Imaging experiments
HeLa cells were cultured in DMEM media containing 10% fetal
bovine serum, 100 U mLꢀ1 penicillin and 100 mg mLꢀ1 strepto-
mycin at 37 1C in a 5% CO2-contained atmosphere. The cells
stained with JBD (10 mM, 30 min) were incubated with different
concentrations of HOCl or lipopolysaccharide (LPS: a substance
that stimulates cells to produce HOCl)38 for 30 min. After
washing cells with pH 7.4 PBS, cells were monitored by a
fluorescent microscope.
3. Results and discussion
Fig. 1 Spectroscopic responses of probe JBD to HOCl. (a) UV-Vis spectra
of JBD (20 mM) upon adding HOCl; (b) fluorescent spectra of JBD in
different solvents; (c) fluorescent spectra of JBD in the absence or
presence of HOCl in the CH3CN/PBS system; (d) fluorescent spectral
responses of JBD (10 mM) towards different concentration of HOCl.
3.1. Spectrum evaluation
In order to confirm our hypothesis, the spectral response of the
probe JBD to HOCl was investigated. The UV-Vis absorption of
JBD itself peaked at 303 nm and 493 nm. With the addition of
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Scheme 2 The reaction mechanism between JBD and HOCl.
species including reducing agents, reactive oxygen species,
sulfur-containing species and inorganic salts (Fig. S2, ESI†).
The signal changes caused by these competing species were
negligible. Hence, the probe JBD could selectively monitor
HOCl under complex physiological conditions.
Fig. 2 Working curve of JBD for HOCl (All data are the average value
obtained from three independent experiments).
3.6. Proposed mechanism
To further confirm the reaction mechanism of JBD towards
HOCl, mass spectrometry was conducted. As shown in Fig. S3
(ESI†), when JBD was treated with HOCl, both peaks at m/z
284.03853 [M ꢀ H]ꢀ and at m/z 124.03932 [M ꢀ H]+ were found,
which corresponded to 3-(benzo[d]thiazol-2-yl)-2-hydroxy-5-
methyl benzoate and isonicotinic acid (calcd: 284.0387, 124.
0393) as the oxidation product of JBD by HOCl (Scheme 2).
HBT-COOH exhibited a dark state likely due to strong electron-
withdrawing effects from the carboxyl group. Therefore, HOCl
could turn the JBD emission off.
Fig. 3 Fluorescent intensity at 607 nm of the probe itself and the probe-
HOCl system at different pH levels. lex = 400 nm; slit, 5 nm/5 nm.
3.7 Cellular imaging
To carry out the detection of HOCl with JBD in biological
systems, cytotoxicity is an important parameter that needed
to be effectively controlled. Cell cytotoxicity experiments of JBD
were then carried out by the MTT-8 assay. Fig. S4 (ESI†) shows
that after co-incubation with 0–50.0 mM JBD for 12–24 h, the
cell viability remained above 80%; therefore, probe JBD was
proved to possess relatively low toxicity.
As shown in Fig. 5, the strong red fluorescence was observed
when the cells were incubated only with the probe JBD. However,
the fluorescence signals in the red channel showed a significant
concentration-dependent decrease after the JBD-treated cells were
incubated with different concentrations of HOCl for 10 min.
Therefore, the probe JBD could sensitively detect exogenous HOCl
in living cells.
Therefore, the probe was found suitable for detecting hypochlor-
ous acid in the physiological system.
3.4. Time-dependent fluorescent intensity of JBD after adding
HOCl
As described in Fig. 4, after adding HOCl to the probe, the
emission signals at 607 nm leveled off within 26 s and reached
one-fifteenth of that of the probe. This excellent fast way could
be helpful to monitor the minor change of HOCl (its deriva-
tives) inside the cells.
3.5. Selectivity evaluation
Afterwards, the selectivity of JBD towards HOCl was determined
by examining the probe’s response towards biologically related
Fig. 4 The time-dependent fluorescence intensity of the probe (10 mM) at
607 nm after adding equivalent HOCl, lex = 400 nm, slit width: 5 nm/5 nm.
Fig. 5 HOCl concentration-dependent images of JBD-treated HeLa cells.
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transformation plan of Qinghai Province (2020-GX-101) and
Scientific Instrument Center of Shanxi University (201512).
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