G Model
CCLET 4724 No. of Pages 4
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K. Bi et al. / Chinese Chemical Letters xxx (2018) xxx–xxx
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Scheme 1. Probe 1 for fluorescent detection of HSO3
.
Fig. 1. UV–vis absorption (a) and fluorescence spectra (b) of probe 1 (2 Â10À5 mol/
fluorescence emission change. Probe 1 was tested in the buffer
solution of DMF:PBS (3/7, v/v, PBS 0.01 mol/L, pH 7.4), the probe
exhibited high sensitivity and selectivity for HSO3À. In the
maximum of fluorescence emission spectra a blue-shift from
570 nm to 445 nm was observed and the color of the solution
À
L) in the presence or absence of HSO3 (0 À 2 equiv.) in DMF:PBS (3/7, v/v, PBS
0.01 mol/L, pH 7.4). The inset shows color changes (a) and fluorescence changes (b)
À
of probe 1 with HSO3
.
À
Next, with probe 1 in hand, its spectral properties in the
presence or absence of HSO3À in DMF:PBS (3/7, v/v, PBS 0.01 mol/L,
pH 7.4) was evaluated. Probe 1 displayed a broad absorption band
at 467 nm. Upon increasing the concentration of HSO3À, the band
at 467 nm gradually decreased and a new absorption bands at
changed from yellow to blue with the addition of HSO3 to a
physiological aqueous solution. The high sensitivity and sÀelectivity
of probe 1 permitted the detection of exogenous HSO3 in Hela
cells and C. elegans [19,20].
First of all, the materials used in this work were obtained from
commercial suppliers and used without further purification unless
otherwise noted. Flash chromatography was carried out on silica
gel (200 À 300 mesh). 1H NMR spectra was recorded using
500 MHz and 13C NMR was recorded using 101 MHz. Chemical
shifts were expressed in ppm and coupling constants (J) in Hz.
Mass spectrometry was recorded with Xevo TQ-S and Q-TOF mass
spectrometer. UV–vis spectra were recorded on U3010 spectrom-
eter. Fluorescence spectra were recorded on F4500 spectrometer.
Cell imaging was acquired on a Nikon ECLIPSE 90i confocal laser
scanning microscope.
282 nm (ca.185 nm blue-shift) emerged with
a well-defined
isobestic point at 365 nm (Fig. 1a), as well as a noticeable color
change from yellow to colorless (Fig. 1a inset). The significant
colorimetric change was due to the interruption of the
p-
,
À
conjugation by HSO3À. By varying the concentrations of HSO3
the ratio between the fluorescence spectra increment and the
compÀound action was determined at 445 nm with a 1:1 (probe 1:
HSO3
) binding ratio (Fig. S1 in Supporting information).
Subsequently, upon increasing the amount of HSO3À, the fluores-
cence emission band at 570 nm decreased dramatically, which lead
to an increase in the band at 445 nm (Fig. 1b). The fluorescence
intensity of probe 1 Àin solution did not change, until the
concentration of HSO3 reached 2 equiv. resulting in the color
changing from yellow to blue (Fig. 1b inset). This important
colorimetric change confirms the utilization of probe 1 in the
ratiometric detection of HSO3À. By plotting the fluorescence
intensity (445 nm) against the concentration of HSO3À, a good
linear relationship was obtained with the concentration ranging
The probe 1 stock solution (1 mmol/L) was prepared in DMF
[21]. The concentration of probe 1 in a working solution was
2 Â10À5 mol/L. Solutions of various testing species (SO42À, HCO3
,
À
N3À, AcOÀ, SCNÀ, C2O42À, HPO42À, HSO3À, NO2À, FÀ, ClÀ, BrÀ, IÀ, Cys,
Hcy, GSH, S2À, SO32À) were prepared in distilled water (0.01 mol/L).
The resulting solution was placed in a glass and quartz cuvette of
1 cm  1 cm optical path length each time and the UV–vis or
fluorescent spectra titrations were recorded. All spectroscopic
experiments were carried out at room temperature.
from 0 to 5
mmol/L. The limit of detection was calculated as
1.29 mol/L (Fig. S2 in Supporting information).
m
HeLa cells were kindly provided by the Institute of Life Science
at Yunnan University. Cells were cultured in Dulbecco’s modified
Eagle’s medium (DMEM, Invitrogen) containing 10% fetal bovine
serum (FBS) at 37 ꢀC in a 5% CO2 and 95% air incubator. The cells
were incubated for 24 h before use, the cells were incubated with
In order to prove that probe 1 possesses high selectivity towards
HSO3À, we investigated its response with the addition of various
interferential species (probe 1 only, SO42À, HCO3À, N3À, AcOÀ,
SCNÀ, C2O42À, HPO42À, HSO3À, NO2À, FÀ, ClÀ, BrÀ, IÀ, Cys, Hcy, GSH,
S2À, SO32À) in DMF:PBS (3/7, v/v, PBS 0.01 mol/L, pH 7.4). As shown
in Fig. 2a, probe 1 began with ratiometric absorption peaks at
467 nm and 282 nm. When we added 2 equiv. of the various species
to a solution of probe 1, the absorption peak at 282 nm remained
strong. In the case of GSH, an intense absorption peak was
produced at 481 nm. Fig. 2b showed the accumulated data
gathered for the effect the various species has on the 282 nm
band. It was determined that the addition of other anionic species
only gives a weak absorption change compared to the addition of
HSO3À, which possesses a greater effect. We also examined the
10.0 mmol/L probe 1 in a 5% CO2 and 95% air incubator for 30 min.
Then,Àthe cells were incubated with different concentrations of
HSO3 in a 5% CO2 for 2 h, washed twice with PBS. Images were
taken with an Olympus FV1000 confocal microscope with a 40Â
objective lens.
The wild-type strain of C. elegans was acquired from the
Institute of Life Science at Yunnan University. The C. elegans at the
fourth larval stage were incubated in Petri dishes filled with M9
buffer containing 20
incubation, the exposed nematodes were collected with
m
mol/L probe 1 for 1 h at 20 ꢀC. After the
a
ratio of the absorption intensity at 282 nm and 467 nm (Fig. 2c),
centrifugation at the speed of 3000 rpm for 2 min, followed by
three times washing with M9 buffer. The nematodes were next
incubated with increasing concentrations of HSO3À for 2 h at room
temperature.
Precursor 2 was prepared according to a previously reported
method [22]. Probe 1 was readily prepared by the condensation of
compound 3 with precursor 2 in the presence of piperidine and
EtOH, giving probe 1 in moderate yield (Scheme S1 in Supporting
information). The structure of probe 1 was confirmed by 1H NMR
and 13C NMR, and HR-MS analysis. More detailed synthetic
processes and structure characterizations can be found in
Supporting information.
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only with HSO3 showing the largest absorption intensity. ThÀis
result highlights the high selectivity probe 1 has towards HSO3
.
Gratifyingly only when probe 1 was treated with HSO3À, a
significant change in the fluorescence emission at 445 nm was
observed, compared to other species which had a much weaker
effect (Fig. S3 in Supporting information). Even some interfering
species showed certain responses towards HSO3À, their fluores-
cence changes were weak and cannot disturb the sensing of
probe 1. The experimental results showed that probe 1 can be
used to detect sulfites even in the presence of other interfering
species.
Please cite this article in press as: K. Bi, et al., A carbazole-hemicyanine dye based ratiometric fluorescent probe for selective detection of