A colorimetric and fluorogenic probe for bisulfite using benzopyrylium as the recognition unit

Article abstract of DOI:10.1002/bio.3316

A coumarin–benzopyrylium (CB) platform has been developed for the colorimetric and fluorogenic detection of bisulfite. The proposed probe utilizes coumarin as the fluorophore and positively charged benzopyrylium as the reaction site. The method employs the nucleophilic addition of bisulfite to the benzopyrylium moiety of CB to inactivate the electron-deficient oxonium ion. The driving force for photo-induced electron transfer is considerably diminished, thereby promoting the emission intensity of the coumarin fluorophore. The fluorescence intensity at 510?nm is linear with bisulfite concentration over a range of 0.2–7.5?μM with a detection limit of 42?nM (3δ). CB shows a rapid response (within 30?s) and high selectivity and sensitivity for bisulfite. Preliminary studies show that CB has great potential for bisulfite detection in real samples and in living cells.

Full text of DOI:10.1002/bio.3316

Received: 14 January 2017
DOI: 10.1002/bio.3316
Revised: 16 February 2017
Accepted: 20 February 2017
R E S E A R C H A R T I C L E
A colorimetric and fluorogenic probe for bisulfite using
benzopyrylium as the recognition unit
1,2
1
1
2
|
|
|
Yun Zhang
Xiangwen Zhang
Xiao‐Feng Yang
Juan Zhang
1
Key Laboratory of Synthetic and Natural
Functional Molecule Chemistry of Ministry of
Education, College of Chemistry & Materials
Science, Northwest University, Xi'an, People's
Republic of China
Abstract
A coumarin–benzopyrylium (CB) platform has been developed for the colorimetric and
fluorogenic detection of bisulfite. The proposed probe utilizes coumarin as the fluorophore and
positively charged benzopyrylium as the reaction site. The method employs the nucleophilic addi-
tion of bisulfite to the benzopyrylium moiety of CB to inactivate the electron‐deficient oxonium
ion. The driving force for photo‐induced electron transfer is considerably diminished, thereby
promoting the emission intensity of the coumarin fluorophore. The fluorescence intensity at
2
Chemical Research Institute, School of
Chemical Engineering, Northwest University,
Xi'an, People's Republic of China
Correspondence
Xiao‐Feng Yang, Key Laboratory of Synthetic
and Natural Functional Molecule Chemistry of
Ministry of Education, College of Chemistry &
Materials Science, Northwest University, Xi'an
5
10 nm is linear with bisulfite concentration over a range of 0.2–7.5 μM with a detection limit
of 42 nM (3δ). CB shows a rapid response (within 30 s) and high selectivity and sensitivity for
bisulfite. Preliminary studies show that CB has great potential for bisulfite detection in real
samples and in living cells.
7
10069, People's Republic of China.
Email: xfyang@nwu.edu.cn
Juan Zhang, Chemical Research Institute,
School of Chemical Engineering, Northwest
University, Xi'an, People's Republic of China.
Email: zjuannn@163.com
KEYWORDS
benzopyrylium, bisulfite, coumarin, fluorescent probe
Funding Information
National Natural Science Foundation of China,
Grant/Award Number: 21475105; Scientific
and Technological Projects of Shaanxi
Province, Grant/Award Number: 2014k10–10;
NFFTBS, Grant/Award Number: J1210057
1
|
INTRODUCTION
1
However, excessive intake of these derivatives destroys vitamin B ,
causes neuron damage, and induces asthmatic attacks and allergic
[
7,8]
Sulfur dioxide (SO
2
) is a harmful gas and is soluble in aqueous solu-
reactions in some individuals.
Therefore, it is essential to develop
2−
−
tion at neutral pH, producing sulfite (SO
3
) and bisulfite (HSO
is known to be involved in various physiological
and pathological processes, including regulating cardiovascular func-
3
)
reliable analytic techniques for the selective, rapid and sensitive
detection of bisulfite in foods and in living cells.
[
1]
(
1: 3, M/M). SO
2
In recent years, the use of fluorescent probes has been shown to
be a powerful technique for sensing anions because of their good
selectivity, high sensitivity, simplicity, real‐time detection and in vivo
fluorescence imaging. Several fluorescent probes for bisulfite have
been constructed based on the nucleophilic reaction with
[2,3]
tion and increasing anti‐oxidative capacity.
However, an abnormal
level of bisulfite is associated with lung cancer, cardiovascular dis-
ease, neurological disorders and voltage‐gated sodium and potassium
[4,5]
channel respiratory diseases.
In addition, moderate sulfur dioxide
[
9–13]
[14,15]
derivatives have been in widespread use for many years as a general
food additive to prevent the degradation and prolong the storage
period of food, medicine and beverages, because of their preserva-
aldehyde,
deprotection of levulinate,
Michael addition reac-
[
16–22]
tion,
etc. Although a few fluorescent probes for bisulfite have
been reported, most have some limitations such as a long response
time and low sensitivity, and suffer interference from reactive sulfur
species, which limit their application in biological systems. Therefore,
the development of fluorescent probes for bisulfite with new reaction
mechanisms is highly desirable.
[
6]
tive, antioxidant, antimicrobial and bacteriostatic properties.
Abbreviations used: BSA, Bovine serum albumin; DMSO, Dimethyl sulfoxide;
ESI, Electrospray ionization; GSH, glutathione; HRMS, High‐resolution mass
spectra; MTT, 3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide;
NIR, Near‐infrared; NMR, Nuclear magnetic resonance; PBS, Phosphate‐buffered
saline; PET, Photo‐induced electron transfer; UV, Ultraviolet; Vis, Visible.
Recently, benzopyrylium‐based dyes have received great attention
due to their good photochemical properties, such as high molar
Luminescence. 2017;1–7.
wileyonlinelibrary.com/journal/bio
Copyright © 2017 John Wiley & Sons, Ltd.
1

2
ZHANG ET AL.
extinction coefficients, large fluorescence quantum yields, and long
was used throughout all experiments. Column chromatography was
conducted on silica gel (200–300 mesh, Qingdao Ocean). Human
hepatocellular carcinoma SMMC‐7721 cells were obtained from Cell
Engineering Research Centre and Department of Cell Biology of
Fourth Military Medical University (China). Crystal sugar and red sugar
were purchased from local supermarket.
[23–25]
excitation and emission wavelengths.
More significantly, the
benzopyrylium unit can act as a guest receptor, as its C‐2 and C‐4 atoms
[26]
are both liable to attack by nucleophiles,
thus altering the large π‐
conjugated system of the dye and thereby affording remarkable blue
shifts in the optical spectra of the sensing system. Based on this mech-
[27]
[28–30]
[31]
[32]
anism, fluorescent probes for Cys/Hcy,
H
2
S,
have been reported.
Here, a colorimetric and fluorogenic probe for bisulfite was devel-
SO
2
,
HClO,
A Shimadzu RF‐5301 spectrofluorimeter with a 10 mm standard
quartz cuvette was used to record the fluorescence spectra and rela-
tive fluorescence intensity. A Shimadzu UV‐2550 spectrophotometer
2
+ [33]
[34]
2
[35]
Hg
,
H
2
O
and peroxynitrite
1
oped using the benzopyrylium unit as the recognition site. The method
employs the nucleophilic addition of bisulfite to the benzopyrylium
moiety of CB to inactivate the electron deficient oxonium ion. There-
fore, the driving force for photo‐induced electron transfer (PET) would
be considerably diminished thereby promoting the emission intensity
of the coumarin fluorophore (Scheme 1). The fluorescence intensity
at 510 nm is linear with the bisulfite concentration over a range of
was used to determine the UV/Vis spectra. H NMR (400 MHz) and
13
C NMR (100 MHz) spectra were recorded on an INOVA‐400 Spec-
trometer (Varian Unity) with tetramethylsilane as the internal standard.
The ESI high‐resolution mass spectra (HRMS) were obtained on a
Bruker micrOTOF‐Q II mass spectrometer (Bruker Daltonics Corp.,
USA). A Sartorius PB‐10 pH meter was used to measure the pH of all
solutions. Fluorescence imaging was acquired with an Olympus
FV1000 confocal laser scanning microscope (Japan).
0
.2–7.5 μM with a detection limit of 42 nM. The proposed probe
shows excellent selectivity toward bisulfite over other common anions
and biothiols, and has been successfully used to detect bisulfite in real
samples and in living cells.
2
.2
|
Synthesis of the probe (Scheme 2)
2
.2.1
|
6‐(N,N‐diethylamino)‐1,2,3,4‐tetrahydroxantilium
perchlorate (1)
2
2
|
EXPERIMENTAL
To a solution of cyclohexanone (0.33 ml, 3.2 mmol) in concentrated
2
H SO
4
(5
ml)
at
0°C
was
added
4‐diethylamino‐2‐
.1
|
Materials and instrumentation
hydroxybenzaldehyde (0.309 g, 1.6 mmol) in portions with vigorous
[
36]
Unless otherwise stated, all solvents and other reagents were obtained
from the commercial suppliers and used as received. Distilled water
stirring.
The reaction mixture was stirred at 90°C for 1.5 h, cooled
to room temperature and poured onto ice (100 g). Perchloric acid
SCHEME 1 Proposed sensing mechanism of
probe CB for the detection of bisulfite
SCHEME 2 Synthesis of probe CB

ZHANG ET AL.
3
(
70%, 3 ml) was added to the above solution, and the resulting precip-
2.5
|
Cell cultures and fluorescence imaging
itate was filtered off, washed with cold water and dried under vacuum
to afford 1 as a red solid (0.35 g, 61.5% yield). Compound 1 was used
for the next step without further purification.
SMMC‐7721 cells were cultured in culture media (supplemented with
0% fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml strepto-
mycin) at 37°C under an atmosphere of 5% CO and 95% humidity
1
2
for 24 h. SMMC‐7721 cells were seeded in a six‐well plate in culture
media. Before imaging, the cells were incubated with probe CB
2.2.2
|
3‐chloro‐3‐(7‐(diethylamino)‐2‐oxo‐2H‐chromen‐3‐
(
5.0 μM) in PBS (containing 1 mg/ml BSA) at room temperature for
yl) acrylaldehyde (3)
[
37]
30 min, and then incubated with NaHSO (50 μM) for another
3
Compound 3 was synthesized according to the reported protocol.
1
15 min. After washing with PBS, the fluorescence images were
acquired with an Olympus FV1000 confocal laser fluorescence micro-
scope. In the control experiment, cells were treated with probe CB
6
H NMR (400 MHz, DMSO‐d ) δ (ppm): 10.26 (d, J = 6.8 Hz, 1H),
8
6
1
.36 (s, 1H), 7.66 (d, J = 6.8 Hz, 1H), 7.39 (d, J = 9.2 Hz, 1H),
.64 (d, J = 8.4 Hz, 1H), 6.47 (s, 1H), 3.46 (q, J = 6.8 Hz, 4H),
.24 (t, J = 6.8 Hz, 6H).
(
5.0 μM) in PBS (containing 1 mg/ml BSA) at room temperature for
0 min and then rinsed three times with PBS.
3
2.2.3
|
Coumarin‐benzopyrylium dyad (CB)
3
|
RESULTS AND DISCUSSION
Development of the probe
Compounds 1 (0.1 g, 0.39 mmol) and 3 (0.179 g, 0.58 mmol) were dis-
solved in acetic acid (5 ml). The reaction mixture was stirred at 95°C for
3.1
|
2
h. After cooling to room temperature, the reaction mixture was
added dropwise to diethyl ether (10 ml), and the resulting precipitate
was filtered off and washed with diethyl ether to give the crude prod-
uct, which was further purified by silica gel column chromatography
In this study, 6‐(N,N‐diethylamino)‐1,2,3,4‐tetrahydroxantilium (1) and
‐chloro‐3‐(7‐(diethylamino)‐2‐oxo‐2H‐chromen‐3‐yl) acrylaldehyde
3) were fused to afford dye CB, which is a hybrid of coumarin and
benzopyrylium. To our surprise, although CB showed near‐infrared
NIR) absorption at 652 nm, almost no fluorescence emission was
3
(
(
CH
2
Cl
2
/CH
3
CN = 5: 1, v/v) to afford CB as a blue–green solid
1
(
125 mg, 50% yield); H NMR (400 MHz, DMSO‐d
6
): δ 8.47 (s, 2H),
(
8
1
6
.34 (d, J = 12 Hz, 1H), 7.94 (d, J = 10.8 Hz, 1H), 7.86 (d, J = 9.6 Hz,
observed in the coumarin band or in the NIR region. We speculated
that no emission of CB is possibly a result of C = C bond isomeriza-
tion‐reduced quenching. Moreover, emission from the coumarin
fluorophore cannot be observed, which might be due to an efficient
PET process from its coumarin moiety to the benzopyrylium group
H), 7.67 (d, J = 9.6 Hz, 1H), 7.46 (d, J = 9.2 Hz, 1H), 7.21 (s, 1H),
.81 (d, J = 9.2 Hz, 1H), 6.60 (s, 1H), 3.74 (q, J = 6.4 Hz, 4H), 3.49
(
(
(
q, J = 6.4 Hz, 4H), 2.86 (t, J = 6.0 Hz, 2H), 2.79 (t, J = 5.6 Hz, 2H), 1.90
13
m, 2H), 1.26 (t, J = 6.8 Hz, 6H), 1.15 (t, J = 6.8 Hz, 6H); C NMR
100 MHz, DMSO‐d ): δ 160.6, 158.2, 155.8, 155.6, 152.1, 147.3,
43.2, 136.5, 131.9, 131.1, 130.8, 129.5, 124.2, 122.6, 119.2, 118.4,
12.8, 110.2, 108.3, 95.5, 95.2, 45.6, 44.4, 27.1, 25.8, 20.5, 12.5. HRMS
6
[39]
upon photo‐excitation.
1
1
It has been reported that bisulfite can react with α,β‐unsaturated
[40]
compounds efficiently in aqueous solution.
We thus envisioned that
+
+
(
2 3
ESI): [M] m/z 543.2425, calcd for C33H36ClN O 543.2414.
nucleophilic bisulfite would attack the benzopyrylium moiety of probe
CB to inactivate the electron‐deficient oxonium ion, and the driving
force for PET would be considerably diminished, thereby promoting
fluorescence of the coumarin fluorophore. On the other hand, nucleo-
philic attack of the benzopyrylium moiety by bisulfite would interrupt
the π‐conjugation of CB, thus colorimetric method might also be appli-
cable for bisulfite assay. Therefore, it is possible to design a
chemosensing system for bisulfite based on this coumarin–
benzopyrylium dyad.
2
.3
|
General procedure for the spectral
measurements
The stock solution of probe CB (1.0 mM) was prepared in acetonitrile,
4
and 0.1% HClO was added to stabilize it. Stock solutions of bisulfite
were prepared freshly before the measurements. Unless otherwise
specified, all the fluorescence measurements were made as follows.
In a 5 ml test tube, 25 μl probe CB (1.0 mM), 0.5 ml phosphate buffer
(
0.2 M, pH 7.4), 1.5 ml ethanol, and different amounts of bisulfite were
3
.2
|
Optical response of CB toward bisulfite
added. The final volume was adjusted to 5 ml with water. The resulting
solution was shaken well and kept at room temperature (25°C) for
The sensing behavior of probe CB toward bisulfite was investigated
with absorption and fluorescence spectroscopy in ethanol/phosphate
buffer (30: 70 v/v, 20 mM, pH 7.4) at room temperature (25°C). As
shown in Figure 1(a), CB itself shows two absorption peaks at 652
and 440 nm, which can be assigned to the absorption bands of CB
and coumarin, respectively. Upon addition of increasing concentra-
tions of bisulfite (0–20.0 μM) to the solution of probe CB (5.0 μM),
the absorption peak at 652 nm decreased distinctly, while the band
at 440 nm underwent a noticeable increment, accompanied by a red‐
shift of absorption maxima to 450 nm simultaneously. A well‐defined
isobestic point was observed at 503 nm, indicating the formation of
1
min, and then its absorption and fluorescence spectra were recorded.
2
.4
|
Cytotoxicity assay
A standard 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bro-
mide (MTT) assay was conducted to evaluate the cytotoxicity of CB
to SMMC‐7721 cells. The detailed approaches were the same as we
described previously, and the absorbance values of the wells were
[
38]
measured at 490 nm.

4
ZHANG ET AL.
FIGURE 1 UV/vis (a) absorption and (b) fluorescence spectra (λex = 460 nm) of probe CB (5.0 μM) upon treating with increasing concentrations of
bisulfite (0–20 μM) in ethanol/phosphate buffer (30: 70 v/v, 20 mM, pH 7.4) for 1 min. (Inset) Photographs of probe CB before and after addition of
bisulfite (10 μM) under (a) visible and (b) 365 nm UV light
a new species upon mixing CB with bisulfite. The decrease and increase
1 mg/ml BSA (Figures S2 and S3), which indicates that the proposed
probe is suitable for bisulfite assays in biological systems.
in absorption at 652 and 450 nm may contribute to the attack of nucle-
−
ophile HSO
3
on the positively charged benzopyrylium moiety of CB.
Meanwhile, a significant color change from cyan to yellow was observed,
which could be easily observed by the naked eye, indicating that CB
can be used as a visual probe for colorimetric detection of bisulfite.
Next, the fluorescence response of probe CB toward bisulfite
was examined under the same conditions (Figure 1b). Probe CB is
essentially non‐fluorescent in pH 7.4 phosphate buffer solution
3
.3
|
Kinetic studies
Time‐dependent fluorescence changes of probe CB in the absence and
presence of bisulfite was investigated in phosphate buffer (pH 7.4,
20 mM) containing 30% ethanol. As described in Figure 3, the back-
ground fluorescence of probe CB in the absence of bisulfite remained
unchanged under identical conditions, indicating that probe CB had
good stability. However, upon the addition of bisulfite (10 μM), the
fluorescence intensity at 510 nm increased promptly and the sensing
reaction could be completed within 30 s, indicative of the fast reaction
kinetics. Indeed, the response time of the current probe is superior to
most of the previously reported bisulfite probes. This provides the
probe with an advantage in real‐time intracellular imaging of endoge-
(
20 mM) containing 30% ethanol at 25°C. By contrast, a new emis-
sion band centered at 510 nm can be easily observed with the addi-
tion of bisulfite. The emission intensity at 510 nm increased
−
progressively until the concentration of HSO
3
reached 20.0 μM
(
37‐fold increase). In addition, it was observed that the fluorescence
emission maximum of the sensing system is red‐shifted by ~24 nm
compared with compound 2 (Figure S1), which can be explained by
−
3
the fact that the ‘C = C’ double bonds in CB–SO
extend the conju-
2
nous SO in biological systems.
gation system of coumarin. The fluorescence intensity at 510 nm was
linearly proportional to the concentration of bisulfite over a range of
3
.4
|
Selectivity studies
0
.2–7.5 μM, and a typical calibration graph was obtained, as shown
in Figure 2. The detection limit was calculated to be 42 nM (3δ). In
To investigate the selectivity of the probe for bisulfite over other
relevant species, CB (5.0 μM) was treated with various analytes. As
illustrated in Figure 4, only bisulfite induces a dramatic fluorescence
−
addition, probe CB is also can be used to detect HSO
3
in pure
aqueous media (phosphate buffer, 20 mM, pH 7.4) containing
FIGURE 3 Time‐dependent fluorescence intensity changes of probe
CB (5 μM) in the absence and presence of bisulfite (10 μM) in
ethanol/phosphate buffer (30: 70 v/v, 20 mM, pH 7.4). λex/λem = 460/
510 nm
FIGURE
2
Calibration curve of the fluorescence intensity as a
function of bisulfite concentration when using probe CB (5.0 μM).
ex/λem = 460/510 nm
λ

ZHANG ET AL.
5
observed for CB over a wide pH range from 3.0 to 7.4, indicating that
the probe was very stable in a wide pH range. However, increased
fluorescence intensity at 510 nm was observed when the pH was ≥8.
−
This is due to the nucleophilic addition of OH on the 2‐position of
[
41]
the positively charged benzopyrylium moiety of probe CB.
Upon
treatment with bisulfite, changes in fluorescence intensity increased
with pH over a range of 3.0–5.0. It was observed that the probe
showed substantial responses to bisulfite over a pH range of 5.0–7.4.
When the pH exceeded 7.4, the fluorescence intensity at 510 nm
−
decreased sharply, indicating that the CB–SO
3
adduct is unstable
and would decompose in the alkaline environment. The above results
show that probe CB is stable and can be used for bisulfite detection
at physiological pH.
FIGURE 4 Fluorescence responses of CB (5 μM) in the presence of
various analytes in ethanol/phosphate buffer (30: 70 v/v, 20 mM,
3
.6
|
Mechanism studies
−
−
pH 7.4) for 1 min. 1, Probe CB only; 2, HSO
3
(10.0 μM); 3, HS
It has been reported that the C‐4 atoms of the benzopyrylium unit are
[26]
−
−
(
50.0 μM); 4, GSH (1 mM); 5, Cys (100 μM); 6, Hcy (10 μM); 7, ClO
4
;
prone to attack by nucleophiles.
Thus, we hypothesized that nucle-
2−
2−
2−
−
−
−
8
1
, SO
4
; 9, CO
3
; 10, S
2
O
3
; 11,SCN ; 12, NO
3
−
; 13, NO
2
; 14, AC ;
−
−
−
−
−
2−
ophilic attack of HSO
3
toward the benzopyrylium C‐4 atom disturbs
5, F ; 16, Cl ; 17, Br ; 18, I ; 19, C
2
O
4
; 20, N
3
; 21, H
2
O
2
(50 μM of
the π‐conjugation of probe CB and recovery of the coumarin moiety
each). λex/λem = 460/510 nm
1
fluorescence. To verify the above proposed sensing mechanism,
H
NMR titration analysis and high‐resolution mass spectroscopy were
−
2−
2−
2−
enhancement, whereas other anions (HS ; SO
4
; CO
3
; S
2 3
O ;
1
carried out. The H NMR titration experiment was conducted for a
−
−
−
2
−
−
−
−
−
2−
−
3
SCN ; NO
3
; NO
; AC ; F ; Cl ; Br ; I ; C
2
O
4
; N
; 50.0 μM),
mixture of NaHSO
tion. In the absence of NaHSO
can be ascribed to the proton on the benzopyrylium moiety (H
allene proton (H ), and the proton on the coumarin moiety (H ) were
observed. Upon addition of NaHSO to the solution of CB, however,
it was observed that the proton (H ) at 9.04 ppm disappeared with
the concomitant appearance of a new peak at 4.90 ppm (H ), which
) adja-
3
and probe CB in DMSO‐d
, three protons at δ = 9.04 ppm, which
), the
6 2
/D O (2: 1 v/v) solu-
−
4
reactive oxygen species (ClO
; H
2
O
2
; 50.0 μM) as well as several
3
biothiols (GSH, 1 mM; Cys and Hcy, 100 μM) showed almost no
changes in fluorescent emission under identical conditions. High con-
centrations of GSH exhibited a moderate increase in fluorescence
intensity, which might be due to their direct nucleophilic attack on
the C‐4 atoms of the benzopyrylium unit. All these investigations
clearly indicate that probe CB exhibited good selectivity toward
bisulfite.
1
2
3
3
1
4
was assigned to the corresponding saturated –CH– proton (H
4
−
cent to the SO
3
group (Figure 6). Furthermore, the mass spectroscopy
analysis of probe CB treated with NaHSO
3
was carried out in ethanol/
−
3
2
H O (1: 1 v/v) to confirm formation the CB–SO
adduct. A prominent
3
.5
|
Effect of pH
peak at m/z 623.1971 is clearly observed, which is identical to the
−
−
The effect of pH on the fluorescence response of probe CB toward
bisulfite was investigated. As shown in Figure 5, in the absence of
bisulfite, only negligible changes in fluorescence intensity were
theoretical molecular weight of the CB–SO3 adduct (calcd. for [M]
623.1983) (Figure S4). These data are strongly support the proposed
sensing mechanism shown in Scheme 1.
FIGURE 5 Fluorescence intensity of probe CB (5 μM) in the absence
1
and presence of NaHSO
60/510 nm
3
(2 equiv.) at various pH values. λex/λem
=
FIGURE 6 Partial H NMR spectra of probe CB in the absence (upper)
4
3 6 2
and presence (lower) of NaHSO (10 equiv.) in DMSO‐d /D O (2: 1 v/v)

6
ZHANG ET AL.
3
.7
|
Detection of bisulfite in food samples
which proves that probe CB is suitable for the detection of bisul-
fite in real food samples.
We investigated the application potential of probe CB to detect
bisulfite in food samples. Sugar sample solutions were prepared
by dissolving 2.5 g of crystal or red sugar with water and diluting
to 10 ml. The samples were then analyzed according to the
procedure described above. Furthermore, aliquots of the sugar
3
.8
|
Fluorescence imaging of probe CB in living cells
Encouraged by the above results, we further investigated potential
−
samples were spiked with different concentrations of NaHSO
3
,
applications of probe CB for sensing HSO3 in living cells. First, the
and the resulting samples were analyzed according to the proce-
dure described above. Good recoveries ranging from 96% to
MTT assay was conducted to examine the activity of cells and the
cytotoxicity of probe CB over a concentration range of 0–35 μM. It
was observed that when the cells were treated with probe CB
1
05% were observed for these bisulfite spiked samples (Table 1).
TABLE 1 Determination of bisulfite in realistic samples
a
−
b
Sample
HSO
3
level (μM)
NaHSO
3
added (μM)
NaHSO
3
found (μM)
Recovery (%)
Red sugar
1.02
–
2
3
–
2
3
–
–
105
96
–
1
3.12
2
3.90
–
Crystal sugar
2.22
1
2
4.26
5.19
102
99
a
The concentration of sugar was 2.5 g/10 ml.
b
Average values were calculated based on three measurements.
FIGURE 7 Confocal fluorescence images of bisulfite in SMMC‐7721 cells with probe CB. Cells were incubated with probe CB (5 μM) for 30 min at
room temperature, then washed and subjected to different treatments; (a, b) no bisulfite; (c, d) cells treated bisulfite (50 μM) for 15 min; (a) and (c)
show the corresponding bright‐field images of (b) and (d). The emission was collected at 500–600 nm with excitation at 488 nm. Scale bar, 30 μm

ZHANG ET AL.
7
(
35 μM) for 24 h, the cell viability was ~86% (Figure S6), which indi-
[16] Q. Wang, W. J. Wang, S. Q. Li, J. Jiang, D. X. Li, Y. Feng, H. T.
Sheng, X. M. Meng, M. Z. Zhu, X. Wang, Dyes Pigm. 2016, 134, 297.
cates that probe CB exhibits minimal cytotoxicity toward living cells.
For cellular imaging studies, SMMC‐7721 cells were incubated with
CB (5 μM) for 30 min at room temperature, and a very dim fluores-
cence was observed. By contrast, when SMMC‐7721 cells were
stained with probe CB (5 μM) for 30 min and then incubated with
bisulfite (50 μM) for 15 min at room temperature, the strong green
fluorescence was easily observed (Figure 7). All the investigations
indicate that CB has good cell permeability and is also suitable for
bisulfite detection in living cells.
[
17] H. Y. Zhang, Z. J. Huang, G. Q. Feng, Anal. Chim. Acta 2016, 920, 72.
18] Y. Q. Sun, J. Liu, J. Y. Zhang, T. Yang, W. Guo, Chem. Commun. 2013,
[
4
9, 2637.
19] M. Y. Wu, K. Li, C. Y. Li, J. T. Hou, X. Q. Yu, Chem. Commun. 2014, 50,
83.
[
[
1
20] L. Tan, W. Y. Lin, S. S. Zhu, L. Yuan, K. B. Zheng, Org. Biomol. Chem.
2014, 12, 4637.
[21] W. Xu, C. L. Teoh, J. J. Peng, D. D. Su, L. Yuan, Y. T. Chang, Biomaterials
015, 56, 1.
2
[
[
[
22] J. C. Xu, J. Pan, X. M. Jiang, C. Q. Qin, L. T. Zeng, H. Zhang, J. F. Zhang,
Biosens. Bioelectron. 2016, 77, 725.
4
|
CONCLUSION
23] P. Czerney, U. W. Grummt, W. G. Günther, J. Prakt Chem. 1998, 340,
2
14.
24] P. Czerney, G. Graneβ, E. Birckner, F. Vollmer, W. G. Rettig, J. Photoch.
Photobio. A 1995, 89, 31.
In summary, a colorimetric and fluorogenic probe for bisulfite was
developed based on the coumarin–benzopyrylium platform. The gener-
ation of the fluorescence can be ascribed to the π‐conjugation interrup-
tion of positively charged benzopyrylium moiety of probe CB by the
nucleophilic addition of bisulfite. The proposed probe shows a fast
response time (~ 30 s), high sensitivity, favorable selectivity, as well as
a low detection limit for bisulfite (42 nM). Furthermore, preliminary
studies show that probe CB can detect bisulfite in real samples and image
living cells. Thus, we believe that this sensing platform can be broadly
[
[
25] L. Yuan, W. Y. Lin, H. Chen, Biomaterials 2013, 34, 9566.
26] R. Gavara, C. A. T. Laia, A. J. Parola, F. Pina, Chem. – Eur. J. 2010, 16,
7
760.
[
27] H. M. Lv, X. F. Yang, Y. G. Zhong, Y. Guo, Z. Li, H. Li, Anal. Chem. 2014,
86, 1800.
[28] J. Liu, Y. Q. Sun, J. Y. Zhang, T. Yang, J. B. Cao, L. S. Zhang, W. Guo,
Chem. – Eur. J. 2013, 19, 4717.
[
29] H. D. Li, Q. C. Yao, J. L. Fan, N. Jiang, J. Y. Wang, J. Xia, X. J. Peng,
Chem. Commun. 2015, 51, 16225.
2
applied for the detection of SO derivatives in a variety of real samples.
[
30] L. Yuan, F. P. Jin, Z. B. Zeng, C. B. Liu, S. L. Luo, J. S. Wu, Chem. Sci.
2
015, 6, 2360.
ACKNOWLEDGEMENTS
[31] Y. H. Chen, X. Wang, X. F. Yang, Y. G. Zhong, Z. Li, H. Li, Sensor Actuat.
B‐Chem. 2015, 206, 268.
This research was supported by the National Natural Science
Foundation of China (no. 21475105) and the Scientific and
Technological Projects of Shaanxi Province (no. 2014 k10‐10). X.W.
Zhang was supported by NFFTBS (no. J1210057).
[
32] S. S. Ding, Q. Zhang, S. H. Xue, G. Q. Feng, Analyst 2015, 140, 4687.
[
33] J. Liu, Y. Q. Sun, P. Wang, J. Y. Zhang, W. Guo, Analyst 2013, 138,
2
654.
34] B. Dong, X. Song, X. Kong, C. Wang, Y. Tang, Y. Liu, W. Lin, Adv. Mater.
016, 28, 8755.
[
2
REFERENCES
[35] D. Cheng, Y. Pan, L. Wang, Z. Zeng, L. Yuan, X. Zhang, Y.‐T. Chang,
J. Am. Chem. Soc. 2017, 139, 285.
[
[
[
[
1] G. Li, N. Sang, Ecotoxicol. Environ. Saf. 2009, 72, 236.
[
[
[
[
36] L. Yuan, W. Y. Lin, Y. T. Yang, H. Chen, J. Am. Chem. Soc. 2012, 134,
2] G. M. Sapers, Food Technol. 1993, 47, 75.
1
200.
3] Z. Q. Meng, H. F. Zhang, Inhalation Toxicol. 2007, 19, 979.
37] T. Debnath, J. Dana, P. Maity, H. Lobo, G. S. Shankarling, H. N. Ghosh,
4] A. A. T. Mahmoud, G. M. Hassan, A. M. S. Hassan, A. K. M. A. Latif,
Chem. – Eur. J. 2015, 21, 5704.
M. F. Ramadan, Eur. J. Integr. Med. 2015, 7, 234.
38] L. H. Geng, X. F. Yang, Y. G. Zhong, Z. Li, H. Li, Dyes Pigm. 2015, 120,
[5] N. Sang, Y. Yun, H. Y. Li, L. Hou, M. Han, G. K. Li, Toxicol. Sci. 2010,
2
13.
39] W. Q. Chen, Q. Fang, D. L. Yang, X. Z. Song, J. Foley, Anal. Chem. 2015,
7, 609.
114, 226.
[
[
[
[
6] H. Filik, G. Çetintaş, Food Anal. Methods 2012, 5, 1362.
7] S. L. Taylor, N. A. Higle, R. K. Bush, Adv. Food Res. 1986, 30, 1.
8] S. J. Stohs, M. J. S. Miller, Food Chem. Toxicol. 2014, 63, 240.
8
[
[
40] M. Morton, H. Landfield, J. Am. Chem. Soc. 1952, 74, 3523.
41] F. Pina, M. J. Melo, C. A. T. Laia, A. J. Parola, J. C. Lima, Chem. Soc. Rev.
2012, 41, 869.
9] X. Liu, Q. W. Yang, W. Q. Chen, L. N. Mo, S. Chen, J. Kang, X. Z. Song,
Org. Biomol. Chem. 2015, 13, 8663.
[
[
[
[
10] S. L. Yu, X. L. Yang, Z. L. Shao, Y. Feng, X. G. Xi, R. Shao, Q. X. Guo,
SUPPORTING INFORMATION
X. M. Meng, Sensor Actuat. B‐Chem. 2016, 235, 362.
11] X. H. Cheng, H. Z. Jia, J. Feng, J. G. Qin, Z. Li, J. Mater. Chem. B 2013, 1,
Additional Supporting Information may be found online in the
supporting information tab for this article.
4
110.
12] X. H. Chen, H. Z. Jia, J. Feng, J. G. Qin, Z. Li, Sensor Actuat. B‐Chem.
013, 184, 274.
2
13] Y. T. Yang, F. J. Huo, J. J. Zhang, Z. H. Xie, J. B. Chao, C. X. Yin,
H. B. Tong, D. S. Liu, S. Jin, F. Q. Cheng, X. X. Yan, Sensor Actuat.
B‐Chem. 2012, 166, 665.
How to cite this article: Zhang Y, Zhang X, Yang X‐F, Zhang J.
A
colorimetric and fluorogenic probe for bisulfite using
benzopyrylium as the recognition unit. Luminescence.
017;0:1–7. https://doi.org/10.1002/bio.3316
[14] H. Y. Zhang, S. H. Xue, G. Q. Feng, Sensor Actuat. B‐Chem. 2016, 231,
2
752.
[15] X. Ma, C. X. Liu, Q. L. Shan, G. H. Wei, D. B. Wei, Y. G. Du, Sensor
Actuat. B‐Chem. 2013, 188, 1196.