A ratiometric fluorescent probe for bisulphite anion, employing intramolecular charge transfer

Article abstract of DOI:10.1002/bio.2344

4-(1H-benzimidazol-2-yl)benzaldehyde (1) has been developed as a new ratiometric fluorescent probe for bisulphite, based on the modulation of intramolecular charge transfer (ICT). Upon mixing with bisulphite in aqueous ethanol, an aldehyde-bisulphite adduct was formed and the ICT of the probe was switched off, which resulted in a ratiometric fluorescence response with an enhancement of the ratios of emission intensities at 368 and 498 nm. The detection range of the probe for bisulphite is in the 2.0-200 μmol/L concentration range and the detection limit is 0.4 μmol/L. Probe 1 produces a ratiometric fluorescent response to bisulphite with a marked emission wavelength shift (130 nm) and displays high selectivity for bisulphite over other anions. Copyright

Full text of DOI:10.1002/bio.2344

Research article
Received: 18 September 2011,
Revised: 21 December 2011,
Accepted: 23 December 2011
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/bio.2344
A ratiometric fluorescent probe for bisulphite
anion, employing intramolecular charge
transfer
Gang Wang, Haiping Qi and Xiao-Feng Yang*
ABSTRACT: 4-(1H-benzimidazol-2-yl)benzaldehyde (1) has been developed as a new ratiometric fluorescent probe for bisul-
phite, based on the modulation of intramolecular charge transfer (ICT). Upon mixing with bisulphite in aqueous ethanol,
an aldehyde–bisulphite adduct was formed and the ICT of the probe was switched off, which resulted in a ratiometric fluores-
cence response with an enhancement of the ratios of emission intensities at 368 and 498 nm. The detection range of the probe
for bisulphite is in the 2.0–200 mmol/L concentration range and the detection limit is 0.4 mmol/L. Probe 1 produces a ratiometric
fluorescent response to bisulphite with a marked emission wavelength shift (130nm) and displays high selectivity for bisulphite
over other anions. Copyright © 2012 John Wiley & Sons, Ltd.
Keywords: bisulphite anion; ratiometric fluorescent probe; intramolecular charge transfer; benzimidazole
recent years several probes and chemosensors have been
developed via modulation of the ICT efficiency of the probe upon
Introduction
interaction with an analyte (20–23). One typical way to achieve this
is to directly engage the charge of an analyte with the electron
donating or withdrawing group of the probe in the binding
process (20,21). We envisioned that this key feature of ICT could
be employed in our ratiometric fluorescent probe design.
Bisulphite is an important additive in many food products
because it inhibits development of both enzymatic and non-
enzymatic browning in a variety of processing and storage
situations (1,2). Since it has been discovered that a certain
concentration level can cause asthmatic attacks and allergic
reactions in some individuals, the sulphite content has been
strictly limited in foodstuffs (3,4). Besides, bisulphite is one of
the most widely distributed environmental pollutants, which is
generated and released in industrial processes. Thus, there is a
great demand for fast, sensitive and selective detection of
sulphur dioxide and bisulphite in foods and environmental
samples. In recent years, fluorescent sensing and recognition
of anions by artificial receptors have received intense attention
due to their potential in analytical, environmental applications
and medicine (5–7). Unfortunately, only a few chemosensors and
probes have been developed for bisulphite or sulphur dioxide
(8–16) and most bisulphite probes developed so far are not robust
factors such as instrumental efficiency, environmental conditions
and probe concentration can interfere with the signal output. By
contrast, ratiometric fluorescent probes allow the measurement
of emission intensities at two suitably selected wavelengths, which
should result in the cancellation of variables such as illumination
intensity, photobleaching of the probe and probe concentration,
in the fluorescent signal and increase the dynamic range of fluo-
rescence measurement (17–19).
Herein, we present 4-(1H-benzimidazol-2-yl)benzaldehyde (1)
as a novel ratiometric fluorescent probe for bisulphite. Our
design is based on the selective reaction of bisulphite with
aldehyde to form an aldehyde–bisulphite adduct (Scheme 1)
(24–27). This reaction is frequently employed to purify aldehydes
in organic synthesis, but can also be used to detect bisulphite in
the field of analytical chemistry, because the transformation of
the aldehyde into bisulphite adduct can lead to a change in the
electron acceptor strength (8). Upon mixing with bisulphite, the
aldehyde group in probe 1 was transformed to its corresponding
bisulphite adduct, thus resulting in deconjugation of the electron
donor-accept system, and the ICT photophysical process in probe
1 was blocked. Hence, a decrease of the emission band at 498nm
and an increase of a fluorescence peak around 368 nm were
observed simultaneously. The fluorescence intensity (I) ratio at
368 and 498 nm (I368/I498) increases linearly with bisulphite
concentration in the range 2.0–200 mmol/L. Based on this
mechanism,
a new fluorescent probe for bisulphite was
developed. The proposed probe is simple, fast and shows
excellent selectivity toward bisulphite over other common anions.
However, to our best knowledge, only one ratiometric fluores-
cent probe for bisulphite has been reported thus far (14). Thus,
we decide to develop a fluorescent ratiometric probe for
bisulphite, using intramolecular charge transfer (ICT) as a
signalling mechanism. Generally, an ICT system contains an
electron-donating group and an electron-withdrawing group,
and it undergoes ICT from the donor to the acceptor upon
photo-excitation. In fact, ICT is an effective signalling mechanism
employed in the design of ratiometric fluorescent probes, and in
* Correspondence to: X.-F. Yang, Key Laboratory of Synthetic and Natural
Functional Molecule Chemistry of the Ministry of Education, Institute of
Analytical Science, Department of Chemistry, Northwest University, Xi’an
710069, People’s Republic of China. E-mail: xfyang@nwu.edu.cn
Department of Chemistry, Northwest University, Xi’an, People’s Republic
of China
Luminescence 2012
Copyright © 2012 John Wiley & Sons, Ltd.

G. Wang et al.
ICT Off
ICT On
Procedure
The fluorogenic reaction was performed in a 10 mL volumetric tube. Typically,
to a test tube containing 2.0 mL ethanol, 0.2 mL probe 1 (0.2 mmol/L) and
2.0 mL pH 4.6 HAc-NaAc buffer, different concentrations of bisulphite
were added and the reaction mixture was diluted to 10 mL with water.
The reaction solution was kept at room temperature for 5 min and then
the fluorescence ratio of I368/I498 was measured, with the excitation and
emission wavelengths at 325 and 368/498 nm, respectively.
H
N
H
N
H
-
HSO3
-
CHO
C
SO3
N
N
OH
1
Scheme 1. Design concept of ratiometric fluorescent probe 1 for HSO–3.
Experimental
Results and discussion
Apparatus
Spectral characteristics of probe 1
The fluorescence spectra and relative fluorescence intensity were
measured with a Sanco CRT-970 spectrofluorimeter (Shanghai, China)
with a 10 mm quartz cuvette. The excitation and emission wavelength
bandpasses were both set at 10 nm. The absorption spectra were
measured using a Shimadzu UV-1700 spectrophometer (Tokyo, Japan).
The pH was measured using a Model pHs-3B meter (Shanghai, China).
Infrared spectra (IR) were taken in KBr pellets on a Bruker Tensor 27 FTIR
spectrometer. High-resolution mass spectra were collected using a
Bruker micrOTOF-Q II mass spectrometer (Bruker Daltonics Corp., USA)
in electrospray ionization (ESI) mode. 1H-NMR and 13 C-NMR spectra
were recorded on an INOVA-400 spectrometer (Varian Unity), using
tetramethylsilane (TMS) as the internal standard.
The absorption spectra of compound 1 and PBI in a mixture of
ethanol and acetate buffer solution at pH 4.6 (20 mmol/L) were
recorded and are shown in Figure 1. It was observed that PBI
showed two absorption peaks at 242 and 299 nm, whereas for
compound 1 the two absorption bands shifted to 250 and
318 nm, respectively. An explanation could be that upon intro-
ducing aldehyde group to PBI, an intramolecular donor-accept
electron system was formed, thus making the absorption peak
red-shifted. Furthermore, the fluorescence emission spectra of
1 and PBI were also compared (Figure 2). As expected, the intro-
duction of aldehyde group to PBI leads to the red-shift of
fluorescence emission wavelength because of ICT.
Reagents
Unless otherwise stated, all reagents were purchased from commercial
suppliers and used without further purification. Solvents were purified
and dried by standard methods prior to use. Double-distilled water was
used throughout the experiments. Sodium bisulphite was obtained from
Xi’an Chemical Reagent Co. (Xi’an, China); 2-phenylbenzimidazole (PBI)
was obtained from Alfa Aesar; o-phenylenediamine and terephthalalde-
hyde were both obtained from Shanghai Chemical Reagent Company
(Shanghai, China).
0.20
PBI
probe 1
0.15
0.10
0.05
0.00
Synthesis of 4-(1H-benzimidazol-2-yl)benzaldehyde (1)
To a 100 mL flask, o-phenylenediamine (0.1081 g, 1.0 mmol), terephtha-
laldehyde (0.3353 g, 2.5 mmol) and glacial acetic acid (20 mL) were mixed
and refluxed for 30 min (Scheme 2). The reacting solution was cooled to
room temperature and then adjusted to pH 5.0 with NaHCO3 powder.
The resulting yellow solid was collected by filtration and washed with
water. This yellow solid was further dried and purified by silica gel
column chromatography, using DCM/EtAc (5:1, v/v) as eluent to afford
the desired product as a light-yellow solid (0.09g, 40.5%). IR (KBr, cm–1),
3447, 3054, 2923, 2813, 2721, 1701, 1607, 1573, 1209, 1160, 1113,
1109, 845, 748, 683.1H-NMR (DMSO-d6, 400 MHz), d (ppm): 13.19
(s, 1H, NH), 10.19 (s, 1H, CHO), 8.39 (d, 2H, J= 4.4 Hz), 8.08 (d, 2H, J=7.6Hz),
7.72 (d, 1H, J = 8.0Hz), 7.58 (d, 1H, J= 8.0Hz), 7.26 (t, 2H, J = 7.2Hz); 13 C-
NMR (DMSO-d6, 100MHz), d (ppm): 192.81, 152.02, 143.55, 136.14,
129.80, 126.81, 123.44, 119.7, 111.25; HRMS (ESI) calc. for C14H11N2O
(M+ H)+ 223.0866, found 223.0860.
250
300
350
400
Wavelength (nm)
Figure 1. Absorption spectra of 10mmol/L probe 1 and PBI in 20% v/v ethanol–
water solution buffered at pH 4.6.
1200
1000
a
800
600
400
N
CH3COOH
NH2
CHO
200
0
OHC
CHO
b
+
NH2
N
H
1
N
350
400
450
500
550
600
Wavelength (nm)
N
H
PBI
Figure 2. Fluorescent spectra of PBI (lex = 310 nm), and probe 1 (lex = 325 nm) in
20% v/v ethanol–water solution buffered at pH 4.6 NaAc-HAc. PBI, 2.0 mmol/L;
probe 1, 4.0 mmol/L.
Scheme 2. Synthesis of probe 1 and chemical structure of PBI.
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Luminescence 2012

Ratiometric fluorescent probe for bisulphite anion
Sensing behaviour of probe 1 toward bisulphite
by thin-layer chromatography (TLC) and IR spectrum in the
present study. Upon addition of bisulphite, TLC data of the
reaction mixture indicated that a new low Rf substance was
formed (Rf(1) = 0.29, Rf(1-bisulphite) ꢀ 0, DCM/EtAc (v/v) = 1:1),
indicating the reaction of the aldehyde group with bisulphite.
Moreover, IR spectra of compound 1 and its corresponding
bisulphite addition product were taken in KBr disks, and the
results were shown in Figure 5. It can be observed that upon
reacting with bisulphite, the peak at 1702.62 cm–1, which
corresponds to the characteristic aldehyde group absorption,
disappeared. This indicates that the aldehyde group in probe
1 is indeed involved in the recognition event.
The sensing response of probe 1 toward bisulphite was ex-
amined with absorption and fluorescence spectroscopy. Addi-
tion of different concentrations of bisulphite to a solution of
probe 1 (10 mmol/L) in ethanol–water (20/80, v/v) at pH 4.6
acetate buffer resulted in the decrease of the absorption
band around 320 nm and the formation of a new blue-shifted
absorption band with
a maximum centred at 304 nm
(Figure 3). The changes in the fluorescence spectra of probe
1 in the presence of increasing concentrations of bisulphite
were measured; the results are shown in Figure 4. In the
absence of bisulphite, probe 1 exhibited two typical fluores-
cence peaks at 368 and 498 nm. However, upon addition of
an increasing concentration of bisulphite, the emission band
of probe 1 at 498 nm gradually decreased, with the concom-
itant growth of a new strong emission band at 368 nm due
to the blockage of the ICT process in probe 1. A well-defined
isoemissive point was observed at 447 nm, which may
suggest that only one new species was formed during the
sensing process (Figure 4).
Effect of reaction time
The effect of reaction time on I368/I498 of the system was studied
and the results are shown in Figure 6. It can be seen that I368/I498
of probe 1 itself remained stable; however, upon the addition of
bisulphite, I368/I498 increased quickly with increasing reaction
time, and the signal of the reaction system reached stability after
5 min. Therefore, a 5 min reaction time was selected for the
following experiments.
Based on previous studies (8,26,27), the changes of the
fluorescence spectrum was most likely due to the formation
of bisulphite adduct 1 and this transformation was confirmed
Selectivity studies
0.16
0.12
The selectivity of probe 1 for bisulphite over other anions was
investigated in the present study. The fluorescence spectra of
1 in the presence of 3.5 ꢁ 10–4 mol/L of anions such as S2–,
-
HSO3
0.08
105
a
90
75
60
45
30
0.04
0.00
280
300
320
340
360
380
Wavelength (nm)
Figure 3. UV–vis absorption changes of 1 (10 mmol/L) upon addition of different
concentrations of HSO–3 (0–200 mmol/L) in 20% v/v ethanol–water solution buffered
at pH 4.6 NaAc-HAc.
1800 1600 1400 1200 1000
Wavenumber (cm-1)
800
600
600
b
100
90
80
70
60
HSO3-
400
200
0
350
400
450
500
550
600
1800 1600 1400 1200 1000
Wavenumber (cm-1)
800
600
Wavelength (nm)
Figure 4. Fluorescent spectra (excitation at 325 nm) of 1 (4 mmol/L) toward
different concentrations of HSO–3 [0, 2, 5, 8, 10, 15, 25, 35, 50, 65, 80 mmol/L,
respectively] in ethanol–water solution buffered at pH 4.6 NaAc-HAc.
Figure 5. Partial IR spectra of probe 1 (a) and its corresponding bisulphite
adduct (b).
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G. Wang et al.
7
6
5
4
3
2
1
7
6
5
4
3
2
1
0
b
a
-2
0
2
4
6
8
10 12 14 16 18
Reaction time (min)
1
2
3
4
5
6
7
8
9 10 11 12 13 14
Figure 6. Reaction-time profile of probe 1 (4 mmol/L) in the absence (a) and
presence (b) of HSO–3 (35 mmol/L) in 20% v/v ethanol–water solution buffered at
pH 4.6. The I368/I498 changes were continuously monitored at time intervals.
anions
Figure 8. Ratiometric fluorescence response of 1 (4 mmol/L) to different anions.
Bars represent the ratio of fluorescence intensity collected at 368 and 498 nm
(I368/I498). Filled bars represent the addition of 350 mmol/L of different anions to
the solution of 1 in ethanol–water solution buffered at pH 4.6 NaAc-HAc. Empty
bars represent the subsequent addition of 35 mmol/L HSO–3 to the solution. (1)
HSO–3 only; (2) SO24–; (3) SCN–; (4) NO3–; (5) F–; (6) Cl–; (7) Br–; (8) I–; (9) S2–; (10)
S2O32–, SO42–, SCN–, NO3–, F–, Cl–, Br –, I–, HCO3–, NO2–, benzoate
and citrate were recorded (Figure 7). It can be observed that only
bisulphite gave dramatic changes in fluorescence spectra at the
same conditions. Figure 8 shows the I368/I498 of 1 caused by
other anions, and it could be observed that only bisulphite showed
a significant increase of I368/I498, while other anions did not
cause obvious changes under identical conditions. Furthermore,
to further confirm the high selectivity of the probe to bisulphite,
the competition experiments were carried out by adding
bisulphite to the solution of 1 in the presence of 10 equivalents
of other anions, and no significant variation in I368/I498 intensity
was observed (Figure 8), indicating that 1 could be used for the
selective detection of bisulphite even in the presence of these
common coexistent anions.
HCO3–; (11) NO2–; (12) benzoate; (13) citrate; (14) S2O23–
.
25
R = 0.9975
20
15
10
5
0
Analytical characteristics of 1 for bisulphite
0
5
10
15
20
Under the above conditions, I368/I498 was plotted as a function of
bisulphite concentration and a typical calibration graph was
obtained (Figure 9). The I368/I498 value increased linearly with
bisulphite concentration (C) in the range 2.0–200 mmol/L, with a
correlation coefficient of R = 0.9975 (n = 7). The linear regression
equation was determined to be:
Bisulfite concentration (10-5mol/L)
Figure 9. Plot of emission ratios at 368 and 498 nm (I368:I498) as a function of HSO3–
concentration.
I
368=I498 ¼ 1:11 ꢁ 105Cðmol=LÞ þ 0:68
400
HSO3-
350
According to IUPAC, the detection limit was determined from
three times the standard deviation (SD) of the blank signal (3s)
as 0.4 mmol/L. The relative SD (n = 7) was 3.7% for 35 mmol/L
bisulphite.
300
1only, S2-, SO42-, SCN-, S2O32-
,
NO3-, F-, Cl-, Br-, I-, HCO3-,
NO2-, citrate, benzoate
250
200
150
100
50
Conclusion
In summary, we have developed a benzimidazole-based fluorescent
probe for bisulphite anion, based on the blockage of the ICT process
of probe 1 through a bisulphite-induced addition reaction. Probe 1
features a ratiometric fluorescent response to bisulphite with a
marked emission wavelength shift (130 nm) and displays high
selectivity for bisulphite over other anions. The method proved to
be simple and sensitive and can be broadly applicable to the
detection of bisulphite in an aqueous environment.
0
350
400
450
500
550
600
Wavelength (nm)
Figure 7. Fluorescent emission changes of 1 (4 mmol/L) upon addition of various
anions (35 mmol/L) in 20% v/v ethanol–water solution buffered at pH 4.6 (excitation
at 325 nm).
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Ratiometric fluorescent probe for bisulphite anion
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Acknowledgements
We are grateful for financial support from the Science and
Technology Department (Grant No. SJ08B05) and the Education
Department (Grant No. 09JK787) of Shaanxi Province, China, and
NWU Graduate Experimental Research Funds (Grant No.
09YSY19). G. Wang was supported by the National Science Foun-
dation for Fostering Talents in Basic Research of the National
Natural Science Foundation of China (Grant No. J0830417).
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