Dual-site fluorescent probe for highly selective and sensitive detection of sulfite and biothiols

Article abstract of DOI:10.1016/j.cclet.2017.11.011

A dual-site fluorescent probe with double bond and aldehyde as reactive sites, was designed for the selective detection of sulfite and biothiols. Sulfite reacts with conjugate bond selectively, while Cys responses with aldehyde and GSH occurs substitution reaction. Different interactions cause different absorption and fluorescence responses. Moreover, it could be further applied in imaging in living cells.

Full text of DOI:10.1016/j.cclet.2017.11.011

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CCLET 4326 No. of Pages 3
Chinese Chemical Letters xxx (2017) xxx–xxx
Contents lists available at ScienceDirect
Chinese Chemical Letters
journal homepage: www.elsevier.com/locate/cclet
Communication
Dual-site fluorescent probe for highly selective and sensitive detection
of sulfite and biothiols
*
*
Mengyang Li, Pengcheng Cui, Kun Li , Jiahui Feng, Mingming Zou, Xiaoqi Yu
Key Laboratory of Green Chemistry and Technology, Ministry of Education, College of Chemistry, Sichuan University, Chengdu 610064, China
A R T I C L E I N F O
A B S T R A C T
Article history:
A dual-site fluorescent probe with double bond and aldehyde as reactive sites, was designed for the
selective detection of sulfite and biothiols. Sulfite reacts with conjugate bond selectively, while Cys
responses with aldehyde and GSH occurs substitution reaction. Different interactions cause different
absorption and fluorescence responses. Moreover, it could be further applied in imaging in living cells.
© 2017 Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of Medical Sciences.
Published by Elsevier B.V. All rights reserved.
Received 17 August 2017
Received in revised form 8 November 2017
Accepted 9 November 2017
Available online xxx
Keywords:
Dual-site
Fluorescent probe
Sulfite
Biothiols
Cell imaging
As
a
potential gasotransmitter [1], SO2 exhibits unique
substitution but not rearrangement is a common used method to
differentiate GSH from Cys/Hcy [20–24].
bioactivities such as vasodilation [2]. The abnormal endogenous
SO2 level can induce toxicological effects leading to cancer,
cardiovascular diseases and neurological disorders [3]. Thus, the
monitoring of cellular concentration of it is of great significance. As
a readily water soluble compound (40 L SO2 (gas) in 1 L H2O), SO2
Besides these single site responding probes, dual site probes are
emerging recent years. As early as 2013, Guo group [25] designed a
three sites chlorinated coumarin-hemicyanine fluorescent probe
for selective detection of Cys and GSH from two emission channels.
More recently, Yin et al. [26] developed a coumarin-based and two
responding sites probe to distinguish Cys and sulfite from different
emission channels. More recently, You and their co-wokers also
synthesized a two-channel responding probe utilizing malononi-
trile moiety to detect SO2/ClOꢀ [27]. With the multi-response
probe’s properties in mind, here we reformed our previously
synthesized SO2 detection probe [28], by adding an aldehyde group
on the carbazole moiety to selectively recognize SO2 and biothiols.
The probe Cz-Bz-CHO could be easily synthesized with the
.
forms a very stable hydrated complex (SO2 H2O); Nevertheless,
many studies on SO2 fluorescent probes are centered on the
detection of bisulfite/sulfite, but not the molecular SO2 (SO2 + H2O
Ð HSO3ꢀ + OHꢀ; HSO3ꢀ Ð SO32ꢀ + Hꢀ). In the last few years,
fluorescent probes based on nucleophilic addition to aldehydes/
ketones [4,5] and double bond [6–10] had been developed for its
detection.
On the other hand, biothiols, such as cysteine (Cys), homocys-
teine (Hcy) and glutathione (GSH) are a kind of sulfhydryl-
containing small molecular amino acids that play crucial roles in
biological systems. The detection of them are usually performed by
the nucleophilicity of the sulfhydryl group [11,12]. A numerous of
reaction-based probes have been developed for either detection or
discrimination them [13–17], including probes containing alde-
hydes. With shorter distance between ꢀSH and ꢀNH2 comparing
to GSH, Cys and Hcy can react with aldehydes to form
thiazolidines/thiazinanes, which is the most frequently used
method to distinguish Cys/Hcy from GSH [18,19]. Besides, aromatic
condensation of carbazole and benzoindole (Scheme
1 and
Scheme S1 in Supporting information). The fast reaction process
makes it possible to real-time image and distinguish the
relationship between SO2 and biothiols.
With the probe in hand, the spectral properties of Cz-Bz-CHO
with NaHSO3 and three common biothiols were first studied.
Interestingly, Cz-Bz-CHO itself possesses two emissions at 445 nm
and 590 nm with the excitation at 350/497 nm respectively, and
can be used as a two channels imaging probe. When NaHSO3
within 2 equiv. in PBS/DMSO (2/1, v/v, pH 7.4) was added, a gradual
increase of fluorescence intensity at 445 nm was observed in less
than 4 min (Fig. S2b in Supporting information) and the emission
at 590 nm disappeared, with a color change from pale yellow to
colorless (Fig. 1b). Accompanying the fluorescence changes, the
* Corresponding authors.
E-mail addresses: kli@scu.edu.cn (K. Li), xqyu@scu.edu.cn (X. Yu).
https://doi.org/10.1016/j.cclet.2017.11.011
1001-8417/© 2017 Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of Medical Sciences. Published by Elsevier B.V. All rights reserved.
Please cite this article in press as: M. Li, et al., Dual-site fluorescent probe for highly selective and sensitive detection of sulfite and biothiols,
Chin. Chem. Lett. (2017), https://doi.org/10.1016/j.cclet.2017.11.011

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CCLET 4326 No. of Pages 3
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M. Li et al. / Chinese Chemical Letters xxx (2017) xxx–xxx
Scheme 1. Proposed sensing mechanism of the dual-site probe.
Fig. 3. The selectivity of 10 mmol/L Cz-Bz-CHO toward various amino acids (10
equiv.). (1) blank, (2) Ala, (3) Val, (4) ASP, (5) Glu, (6) Arg, (7) Lys, (8) Tyr, (9) His, (10)
Phe, (11) Ser, (12) Cys, (13) Hcy, (14) GSH. lex = 497 nm.
maximum absorption peak at 497 nm in the UV–vis spectra
decreased and the absorption at 295 nm increased (Fig. 1a). With
the ratio property in mind (I445/I590), the detection limit was
estimated to be 59.9 nmol/L (Fig. S2a in Supporting information).
Besides sulfite, Cz-Bz-CHO can also response with biothiols as
we designed. The addition of Cys and GSH into a system of Cz-Bz-
CHO in PBS/DMSO (2/1, v/v, pH 7.4) induced a fluorescence
enhancement at 590 nm with excitation at 497 nm (Fig. 2b and
Fig. S1b in Supporting information). We could also get the
consistent outcome from the UV–vis spectra (Fig. 2a, Fig. S1a in
Supporting information), in which the absorption peak at 497 nm
increased. The reaction can complete within 10 min and be stable
even 90 min later, as shown in Figs. S3b and 4b (Supporting
information), implying the excellent property of Cz-Bz-CHO in
distinguishing sulfite and biothiols. The detection limit of Cz-Bz-
Fig. 1. UV–vis absorption (a) and fluorescence spectra (b) of 10 mmol/L Cz-Bz-CHO
in the presence of 0–30 mmol/L NaHSO3 in PBS/DMSO (2/1, v/v, pH 7.4) system.
Inset: Corresponding fluorescent color change under the irradiation of UV lamp
(left: probe; right: probe + NaHSO3). lex = 350 nm.
CHO toward Cys and GSH was calculated to be 8.18
7.22 mol/L respectively (Figs. S3a and 4a in Supporting informa-
tion).
mmol/L and
m
To evaluate the effect of various analytes on Cz-Bz-CHO, Na2S
and various amino acids were discussed. As a nucleophile, S2ꢀ
reacted with aldehyde group first, generating an unstable
intermediate product, which induced fluorescence and absorption
enhancement at 590 nm and 497 nm respectively (Fig. S5 in
Fig. 2. UV–vis absorption (a) and fluorescence spectra (b) of 10
mmol/L Cz-Bz-CHO
in the presence of 0–100 mol/L Cys in PBS/DMSO (2/1, v/v, pH 7.4) system. Inset:
m
Corresponding fluorescent color change under the irradiation of UV lamp (left:
probe; right: probe + Cys). lex = 497 nm.
Fig. 4. Confocal images of Cz-Bz-CHO towards biothiols and sulfite. (a) Fluorescence image of HeLa cells pre-treated with NEM (0.5 mmol/L) for 20 min, and incubated with
Cz-Bz-CHO (5 mol/L) for 10 min; (b) Fluorescence image of (a) from the blue channel; (c) Overlay of (a) and (b); (d) Bright field of (a). (e) Fluorescence image of HeLa cells
incubated with Cz-Bz-CHO (5 mol/L) for 10 min from the orange channel; (f) Fluorescence image of (e) from the blue channel; (g) Overlay of (e) and (f); (h) Bright field of (e).
Excited at 488 nm for the orange channel (550–680 nm); Excited at 405 nm for the blue channel (420–500 nm). Scale bar: 20 m (a–d), 10 m (e–h).
m
m
m
m
Please cite this article in press as: M. Li, et al., Dual-site fluorescent probe for highly selective and sensitive detection of sulfite and biothiols,
Chin. Chem. Lett. (2017), https://doi.org/10.1016/j.cclet.2017.11.011

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CCLET 4326 No. of Pages 3
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3
Supporting information). After a few minutes, aldehyde group
recovered and double bond was attacked, the emission at 590 nm
decreased while at 445 nm increased, as shown in Fig. S6
(Supporting information). These phenomenon can be captured
by 1H NMR titration experiment (Fig. S7 in Supporting informa-
tion). When Na2S was added, original aldehyde hydrogen signal at
10.04 ppm splited, indicating that aldehyde was involved in the
reaction. One hour later, the aldehyde hydrogen signal recovered.
This two-step reaction, which took more than 1 h to be completed,
proceeded so slowly, making the interference induced by Na2S
negligible (Fig. S8 in Supporting information). Besides, other amino
acids also had no interference (Fig. 3).
can provide some useful information for the further probes design
and analytes detection.
Acknowledgment
This work was financially supported by the National Natural
Science Foundation of China (Nos. 21572147, 21232005 and
J1103315).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at https://doi.org/10.1016/j.cclet.2017.11.011.
To verify the proposed mechanism, 1H NMR titration
experiments between Cz-Bz-CHO and NaHSO3/Cys/GSH were
conducted. As shown in Fig. S9 (Supporting information), in a
mixture of DMSO-d6 and D2O (4:1, v/v) solution, the proton
signals at 8.68 and 7.83 ppm, which belong to the double bound
disappeared after the addition of NaHSO3 and new signals
appeared at 5.19 and 3.76 ppm. However, aldehyde hydrogen
signal persisted throughout. In contrast, Cys induced the
disappearance of aldehyde hydrogen signal at 10.04 ppm and
the appearance of new resonances centered at 5.68 and
5.57 ppm, which were assigned to the methane protons of the
thiazolidine diastereomers [29]. Although GSH caused the same
fluorescence enhancement with Cys, we did not know the exact
mechanism currently.
Considering the capability of the probe to distinguish NaHSO3
and biothiols under physiological conditions (pH 7.4), the potential
application of Cz-Bz-CHO for fluorescence imaging of NaHSO3 and
biothiols in living cells was explored. HeLa cells were previously
treated with NEM (N-ethylmaleimide, a common intracellular thiol
scavenger), then Cz-Bz-CHO for NaHSO3 imaging was conducted.
As shown in Fig. 4, fluorescence from the blue channel was only
seen. When incubated with probe without previous NEM
treatment, fluorescence from the origin channel revealed and
blue channel grew brighter, indicating potential relationships
between reactive sulfur species. These results indicated the
application of the probe for cellar imaging of NaHSO3 and biothiols
from blue channel and red channel respectively.
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In summary, we have developed a dual-site fluorescent probe
for the selective detecting of NaHSO3 and biothiols based on the
different fluorescent responses caused by different active site
reaction. NaHSO3 reacts with conjugate bond specifically, while
Cys responses with aldehyde. The probe displays excellent
selectivity and sensitivity, and could be applied in imaging of
biothiols in live cells successfully. We anticipate the current probe
Please cite this article in press as: M. Li, et al., Dual-site fluorescent probe for highly selective and sensitive detection of sulfite and biothiols,
Chin. Chem. Lett. (2017), https://doi.org/10.1016/j.cclet.2017.11.011