Benzimidazole-based turn-on fluorescence probe developed for highly specific and ultrasensitive detection of hypochlorite ions in living cells

Article abstract of DOI:10.1002/bio.4063

Hypochlorite (ClO^?), as one of the active oxygen species (ROS), plays an essential role in the cellular defence system and organism immunity. In this paper, we successfully synthesized a new ‘turn-on’ fluorescent probe BMF based on benzimidazole and characterized it by spectroscopic methods. The designed probe can quickly respond to ClO^? with the obvious colour change from pink to colourless. Notably, the probe BMF exhibited almost no fluorescence, but showed strong fluorescence after adding ClO^?, including an excellent fluorescence turn-on effect. The fluorescence turn-on phenomenon of BMF was attributed to the strong oxidation of ClO^?, which severed the connecting double bond and disrupted the intramolecular charge transfer (ICT) system, plus light-induced electron transfer effect between the fluorophore and the recognition group was discontinued. In addition, the cytotoxicity assay showed that the probe had lower cytotoxicity. Based on these advantages, we demonstrated that probe BMF might be a good candidate for detecting ClO^? in biological systems.

Full text of DOI:10.1002/bio.4063

Received: 21 March 2021
DOI: 10.1002/bio.4063
Accepted: 22 April 2021
R E S E A R C H A R T I C L E
Benzimidazole-based turn-on fluorescence probe developed
for highly specific and ultrasensitive detection of hypochlorite
ions in living cells
Yi Yang1,2 | Siyao Cheng2 | Wei Dong2
1Outpatient Department, First Affiliated
Abstract
Hospital of Nanjing Medical University,
Hypochlorite (ClOÀ), as one of the active oxygen species (ROS), plays an essential role
Nanjing, China
2School of Chemical Engineering, Nanjing
in the cellular defence system and organism immunity. In this paper, we successfully
University of Science and Technology, Nanjing,
China
synthesized a new ‘turn-on’ fluorescent probe BMF based on benzimidazole and
characterized it by spectroscopic methods. The designed probe can quickly respond
to ClOÀ with the obvious colour change from pink to colourless. Notably, the probe
BMF exhibited almost no fluorescence, but showed strong fluorescence after adding
ClOÀ, including an excellent fluorescence turn-on effect. The fluorescence turn-on
phenomenon of BMF was attributed to the strong oxidation of ClOÀ, which severed
the connecting double bond and disrupted the intramolecular charge transfer (ICT)
system, plus light-induced electron transfer effect between the fluorophore and the
recognition group was discontinued. In addition, the cytotoxicity assay showed that
the probe had lower cytotoxicity. Based on these advantages, we demonstrated that
probe BMF might be a good candidate for detecting ClOÀ in biological systems.
Correspondence
Wei Dong, School of Chemical Engineering,
Nanjing University of Science and Technology,
Nanjing, 210094, China.
Email: weidong@njust.edu.cn
Funding information
National Natural Science Foundation of China
(NSFC), Grant/Award Number: 51573078
K E Y W O R D S
benzimidazole, cell imaging, fluorescent probes, ICT change
1
|
INTRODUCTION
To date, various monitoring methods have been successfully
applied to the determination of ClOÀ, including potentiometric, elec-
Reactive oxygen species (ROS) are significant substances in the
human body, and mainly include hydroxide ions (OHÀ), oxygen (O2),
hydrogen dioxide (H2O2), nitrite (NO2À), and hypochlorite (ClOÀ).
Hypochlorite (ClOÀ), as active oxygen species, also plays an impor-
tant role in functioning of the human body as it is produced biologi-
cally in organisms by the catalysis of chloride ions (ClÀ) and H2O2
through the enzyme myeloperoxidase.[1,2] ClOÀ plays an essential
role in various physiological and pathological processes, including
preventing infection and destroying pathogens.[3–5] Therefore,
abnormal ClOÀ concentration levels can lead to oxidative stress, tis-
sue lesion, and many relative risks related to immune deficiency,
myocardial injury, nerve damage, kidney disease, cardiovascular dis-
ease, and tumours.[6–11] Therefore, designing and developing effi-
cient, as well as convenient, monitoring methods that can detect
ClOÀ is significant.
trochemical, chemiluminescence, and fluorescence analyses and
methods.[12–16] Fluorescence imaging technology is considered a
promising method for monitoring biological species in living cells due
to its incomparable advantages of efficiency, simple processing, and
high spatial and temporal resolution.[17–28] A highly fluorescent prod-
uct is produced by the specific reaction between the identifying group
and HClO/ClOÀ, which is the design strategy of the HClO/ClOÀ fluo-
rescent probe.[29–31] Although some fluorescent probes of ClOÀ
already exist, some of these have disadvantages of long, time-
consuming methodology, high detection limits, and the inability to be
detected in living cells, which limits their application in detection of
ClOÀ.[32–37] In addition, some probes show the typical ‘turn off’ fea-
ture, which makes visual detection of ClOÀ inconvenient.[38] There-
fore, the development of ‘turn-on’ fluorescent probes is of great
significance in the field of bioimaging.
Luminescence. 2021;1–8.
wileyonlinelibrary.com/journal/bio
© 2021 John Wiley & Sons, Ltd.
1

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YANG ET AL.
Here, we prepared a benzimidazole–furfural conjugate probe
2.3
|
Synthesis of probe BMF
BMF, which is displayed in Scheme 1, as a turn-on fluorescent
probe to detect ClOÀ in dimethyl sulphoxide (DMSO)–H2O solution
(1:1, v/v). The furfural moiety is the fluorophore part, and the benz-
imidazole moiety is the recognition group part containing the electron
donor. There is a photo-induced electron transfer effect between the
fluorophore and the recognition group that inhibits the occurrence of
fluorescence. The colour of the BMF solution was observed to change
from pink to colourless under natural light, in that the intramolecular
charge transfer (ICT) and photo-induced electron transfer (PET) mech-
anisms were interrupted due to the oxidation effect of ClOÀ. Spectro-
scopic experiments showed that BMF gave an excellent performance
for selective and sensitive detection of ClOÀ in DMSO–H2O solution
(1:1, v/v). In addition, the cytotoxicity assay showed that the probe
had low cytotoxicity, in addition the cell imaging test showed that the
probe could be used with excellent biocompatibility to detect endoge-
nous ClOÀ in L929 fibroblast cells.
Synthesis steps were as follows. p-Nitroaniline (4.1 g), hydrochloric
acid (8 ml) and water (16 ml) were added into a round-bottomed flask
and heated, then the solution was stirred to precipitate crystals under
ice bath conditions. Sodium nitrite (6 g) in water (9 ml) was added
dropwise into a hydrochloric acid solution of p-nitroaniline and left to
react for 30 min after addition. Furaldehyde (2.9 g), acetone (20 ml)
and water (40 ml) were added to the filtrate, which was stirred in an
ice-water bath. Copper chloride (0.8 g) was dissolved in a small
amount of water, then the copper chloride solution was dripped into
the filtrate and stirring continuously at room temperature for 8 h.
After the reaction finished, a brick-red precipitate was formed, filtered
using suction, and recrystallized with dimethyl formamide (DMF) to
obtain the 5-(4-nitrophenyl)-2-furaldehyde (desired product). The
5-(4-nitrophenyl)-2-furaldehyde (1 mmol) and 2-acetonitrile ben-
zothiazole (1 mmol) were added to the ethanol solution with
triethylamine as a catalyst. The reaction was carried out for 5 h and
thin-layer chromatography was used to monitor the reaction product.
When the reaction was completed, the product was solidified from
the reaction mixture and purified using column chromatography to
get a red solid powder (BMF).
2
|
EXPERIMENTAL
| Chemicals
2.1
All reagents and solvents used in this work were purchased from com-
mercial suppliers and used without further purification. 4-Nitroaniline,
sodium nitrite, and furfural were purchased from Sinopharm Chemical
Reagent Co., Ltd, Shanghai, China, or Nanjing Chemical Reagent Co.,
Ltd, Nanjing, China.
2.4
|
General procedures for spectroscopic tests
All spectroscopic tests are carried out in DMSO/PBS solution (1:1,
v/v). Stock solutions for other analyses (Cys, Tyr, Arg, Gly, Met, Phe,
Leu, Lys, Thr, Try, Val, Glu, Asp, Pro, Hyd, Ser, Al3+, Ca2+, Cu2+, Fe2+
,
2À
Ni2+, Mg2+, Na+, K+, NH4+, FÀ, ClÀ, BrÀ, IÀ, AcOÀ, SO42À, CO3
,
2.2
|
Apparatus
NO3À, NO, ONOOÀ, H2O2, •OH and ClOÀ) were prepared at 50 mM
using deionized water. The fluorescence spectra were recorded using
a fluorescence spectrometer.
Fluorescence spectra from our samples were measured using a
Shimadzu RF-5301 fluorescence spectrophotometer. pH values for
our samples were measured using a Sartorius (PB-10) digital pH meter
(LeiCi, Shanghai, China). The morphology of the samples was investi-
gated using scanning electron microscopy (NanoSEM 450 microscope).
1H NMR spectra were characterized on a Mercury-400BB spectrome-
ter. Cell viability was determined using the Thermo Scientific Multi-
skan MK3 spectrophotometer. Cell images were taken with a confocal
fluorescence microscope.
2.5
|
Cytotoxicity assay
The methyl thiazolyl tetrazolium (MTT; 10 μM) assay was used to
measure the cytotoxicity of BMF with L929 cells.[39] Here, 96-well
plates were used to seed L929 cells at a density of 7 Â 103 cells per
well and cells were incubated for 24 h at 37ꢀC in a 5% CO2 in air
SCHEME 1 Synthesis of probe BMF

YANG ET AL.
3
incubator. Specified concentrations of probes BMF (0, 0.01, 0.1, 1.0,
5.0 and 10 μM) were then added to the cells and all experiments for
different concentrations were repeated three times. The above cells
were cultured under identical conditions, and MTT solution was
added within the same hour. Then, PBS buffer was incorporated into
each well to eliminate unreacted MTT and the resulting formazan
crystal in L929 cells was dissolved using DMSO. Absorbance at
545 nm was recorded using a Thermo Scientific Multiskan MK3
spectrophotometer.
diazonium salt, which was then reacted with furan-2-carbaldehyde
using the Sandmeyer synthesis method to generate the furfural
derivative part. Then, the furfural derivative part was reacted with
2-(benzothiazol-2-yl)acetonitrile to generate probe BMF, which is
connected by a vinyl group. The linkage C=C moiety could enable
the conjugated structure of molecule to be more extended and the
capability for electron transfer stronger. The furfural moiety is the
fluorophore part, while the benzimidazole moiety is the recognition
group part containing the electron donor. There was a PET effect
between the fluorophore and the recognition group, which inhibited
the occurrence of the fluorescence. The structural conformation of
BMF was identified using 1H NMR and mass spectrometry (MS) at
high resolution.
2.6
|
Cell culture and imaging
L929 cells were incubated in Dulbecco’s modified Eagle’s medium
supplemented with 10% fetal bovine serum, 100 μg/ml penicillin and
100 μg/ml streptomycin at 37ꢀC in a 5% CO2 in air incubator.[40] Then
24-well plates were used to seed L929 cells for 24 h to obtain a
monolayer of cells. The prepared probe BMF (5 μM) was put into
wells to incubate for 1 h, followed by the addition of NaClO solution
(0, 5 and 10 μM). Then, the combination process between probe and
ClOÀ was conducted for 30 min. The results of tests were photo-
graphed using the red channel (λem = 570–620 nm) of a confocal fluo-
rescence microscopy.
3.2
probe BMF
|
Optimization conditions of ClOÀ probing for
The pH of the environment where a probe is located is an important
factor that affects a probe’s sensitivity and selectivity for detecting
ClOÀ.[41,42] Figure 1a shows the effects of acid solutions on the fluo-
rescence intensity of BMF and BMF–ClOÀ. The fluorescence intensity
of BMF was stable at pH 3.0–9.0, while the fluorescence intensity of
BMF–ClOÀ did not show any obvious change at pH 3.0–11.0. It was
indicated that the acidity of the solution had little effect on the detec-
tion of ClOÀ. Therefore, pH 7.1 (common use) was selected as the
optimized condition for further experiments. Based on the optimized
condition, colour changes of probe BMF for monitoring ClOÀ were
observed in the DMSO/PBS buffer (v/v, 1:1, pH 7.1). From Figure 1b,
it was seen that, after addition of ClOÀ, the colour of BMF solution
changed from pink to colourless under natural light and changed from
pink to bright blue fluorescence under an ultraviolet (UV) light lamp
with 365 nm wavelength.
3
|
RESULTS AND DISCUSSION
3.1
|
Designment and preparation of fluorescence
probe BMF
The method for synthesizing probe BMF through the aldol conden-
sation mechanism is sketched out in Scheme 1. First of all, a diazoti-
zation reaction was used to make the 4-nitroaniline generate
FIGURE 1 (a) Fluorescence intensity of probe BMF and probe BMF with ClOÀ at 513 nm by pH values according to fluorescence titration
(pH 3.0–13.0). (b) Colour changes of probe BMF after adding ClOÀ under natural light or UV lamp

4
YANG ET AL.
3.3
|
Specificity and selectivity of probe BMF for
and sodium hypochlorite were added to DMSO-d6 solution sepa-
rately, and then sodium hypochlorite solution was added dropwise to
the BMF solution. As shown in Figure 3b, in the presence of ClOÀ,
the original proton signals of ethylene at 7.63 ppm gradually
decreased to extinction and a new aldehyde proton signals at
9.62 ppm appeared at the same time, indicating that the ClOÀ had
oxidized and broken the ethylene double bond. As the ethylene dou-
ble bond was broken, the original conjugated system of probe was
destroyed and the original proton signals from probe BMF moved
upfield dramatically. The reaction mechanism of BMF and ClOÀ was
verified by MS spectra (Figure 4), in which two peaks at 176.08 m/z
and 217.04 m/z were seen. SEM images of the BMF powder are
shown in Figure 5. Figure 5a,b demonstrates the stick-like structure
with clear shape boundaries and relatively smooth surfaces; the stick-
shaped powder structures were c. 5 μm in diameter and 50 μm in
length. After ClOÀ addition (Figure 5c,d) the powder became needle-
like with a length of c. 10 μm, and seemed to aggregate into clusters.
These results demonstrated that the probe reaction with ClOÀ would
lead to changes in the conjugation structure of the probe and produce
the main fluorophore 5-(4-nitrophenyl)-2-furaldehyde.
detecting ClOÀ
To study the ability of probe BMF to specifically recognize anions, we
conducted experiments using a fluorescence spectrometer and results
are shown in Figure 2a. In the fluorescence spectrum, fluorescence
intensity at 513 nm did not demonstrate any obvious change in the
presence of other species including Cys, Tyr, Arg, Gly, Met, Phe, Leu,
Lys, Thr, Try, Val, Glu, Asp, Pro, Hyd, Ser, Al3+, Ca2+, Cu2+, Fe2+, Ni2
+, Mg2+, Na+, K+, NH4+, FÀ, ClÀ, BrÀ, IÀ, AcOÀ, SO42À, CO32À, NO3
NO, ONOOÀ, H2O2, or •OH at 0.5 nM concentration, except for
ClOÀ. As is shown in Figure 2b, there were no observable changes in
fluorescence intensity with respect to the coexistence of ClOÀ and
other substances through various contrast experiments. This indicated
that BMF had excellent anti-interference capacity for ClOÀ following
the addition of other common competing ions and that BMF was sta-
ble over a certain pH range, so that it could be applied in biological
cells for detecting ClOÀ.
À
,
3.4
|
Sensing mechanism and binding behaviour of
The Gaussian 09 program at the B3LYP/6–31 G(d) level was
used to complete the binding mechanism of BMF and the oxidant.
Density-functional theory optimal configurations for BMF and
BMF–ClOÀ are shown in Figure 6. The highest occupied orbit of
BMF was uniformly distributed on the furfural moiety and the
probe with ClOÀ
The reaction mechanism between BMF and ClOÀ was investigated
through 1H NMR titration experiments and MS methods. Probe BMF
FIGURE 2 (a) Fluorescence response of probe BMF with various analytes in DMSO/PBS buffer (1:1, v/v). (b) Fluorescence intensity of BMF
with ClOÀ at 513 nm in the presence of other substances at 0.5 nM (1, none; 2, Cys; 3, Tyr; 4, Arg; 5, Gly; 6, Met; 7, Phe; 8, Leu; 9, Lys; 10, Thr;
11, Try; 12, Val; 13, Glu; 14, Asp; 15, Pro; 16, Hyd; 17, Ser; 18, Al3+; 19, Ca2+; 20, Cu2+; 21, Fe2+; 22, Ni2+; 23, Mg2+; 24, Na+; 25, K+;
26, NH4+; 27, FÀ; 28, ClÀ; 29, BrÀ; 30, IÀ; 31, AcOÀ; 32, SO42À; 33, CO32À; 34, NO3À; 35, NO; 36, ONOOÀ; 37, H2O2; 38, •OH; and 39, ClOÀ) in
DMSO/PBS (1:1, v/v). Data were obtained 30 min after addition of the analyte. λex = 469 nm. Slit widths: Wex = 5 nm, Wem = 10 nm

YANG ET AL.
5
FIGURE 3 (a) Oxidation reaction of BMF solution with added ClOÀ solution. (b) 1H NMR spectra of probe BMF in DMSO-d6 without ClOÀ
and with presence of 1.0 equiv and 2.0 equiv ClOÀ respectively
FIGURE 4 (a) MS of probe BMF in DMSO-d6 without the presence of ClOÀ. (b) MS of probe BMF in DMSO-d6 with the presence of ClOÀ
benzothiazole moiety because of integral π-conjugation and vibrant
ICT effect. With the existence of ClOÀ, the π-conjugate state of
BMF was broken in that the C=C bond was oxidized by ClOÀ and
ICT process was interrupted, which indicated that the electronic
cloud of BMF–ClOÀ was located in the furfural moiety and ben-
zothiazole moiety. In addition, BMF decomposed into furfural and
benzothiazole derivative during the reaction. The band gap for the
furfural part (5.411 eV) notably exceeded the band gap for BMF
(2.853 eV), which demonstrated that the furfural part could make
probe BMF specific for ClOÀ, and the band gap of the ben-
zothiazole part also exceeded the band gap for BMF. Therefore, we
could conclude that BMF detected ClOÀ through a nucleophilic
addition reaction and then decomposition.
3.5
L929 cells
|
Biocompatibility and bioimaging features in
To extend the utility value of the probe BMF in the field of biology,
biocompatibility of the probe was tested in L929 cells.[43,44] Standard
MTT assay is an essential assessment criteria to investigate potential
toxicity. As shown in Figure 7, the survival rate of cells exceeded 80%,
even if the concentration of probe BMF reached 10 μM. Therefore,
probe BMF demonstrated the potential for application for probing
ClOÀ in living cells. As shown in Figure 8, probe BMF could enter cells
and diffuse throughout the cytoplasm. After incubating cells for
30 min at 37ꢀC in the presence of BMF (5 μM), they displayed a
completely black colour with no red fluorescence. As the

6
YANG ET AL.
FIGURE 5 Scanning electron microscope (SEM) images of the BMF powder and BMF–ClOÀ powder. (a) BMF (5 μm); (b) BMF (10 μm);
(c) BMF–ClOÀ (5 μm); and (d) BMF–ClOÀ (10 μm)
FIGURE 6 Energy diagrams of optimized configurations, the highest and lowest occupied orbits of BMF and decomposition products of
BMF–ClOÀ. A, BMF; B, furfural derivative; C, benzothiazole derivative

YANG ET AL.
7
concentration of ClOÀ increased, red fluorescence from the L929 cells
began to appear and became stronger and stronger until strong red
fluorescence was produced. These results demonstrated that probe
BMF had good cell permeability and could be used for ClOÀ detection
in biological cells.
4
|
CONCLUSION
In summary, we have successfully prepared a ‘turn-on’ fluorescent
probe BMF. Probe BMF displayed high selectivity for ClOÀ rather
than other ROS and analytes because of the presence of a unique rec-
ognition unit of an ethylene double bond group. In addition, the probe
solution could quickly respond to ClOÀ with an obvious colour change
from pink to colourless under natural light. Notably, probe BMF pro-
duced almost no fluorescence, but exhibited a strong fluorescence
enhancement at 513 nm after addition of ClOÀ, showing an excellent
fluorescence turn-on effect. Furthermore, cytotoxicity tests and
FIGURE 7 Cell cytotoxicity assay of different concentrations of
probe BMF solution in L929 cells. 0 μM, contrast
FIGURE 8 Confocal fluorescence microscopy photographs of NaClO detection in L929 cells with probe BMF. L929 cells treated with 5 μM
BMF (a–c), then further incubated with 5 μM ClOÀ (d–f) and 10 μM ClOÀ (g–i). Fluorescence images from left to right: red channel and merged
images (red channel, λem = 580–630 nm)

8
YANG ET AL.
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living organisms due to the above results.
ACKNOWLEDGEMENTS
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CONFLICT OF INTEREST
The authors have declared no conflict of interest.
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Luminescence 2021, 1. https://doi.org/10.1002/bio.4063
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