A fluorescent probe for rapid detection of thiols and imaging of thiols reducing repair and H2O2 oxidative stress cycles in living cells

Article abstract of DOI:10.1039/c2cc36839k

A diselenide containing fluorescent probe based on a fluorescein scaffold for thiols was developed. The fluorescent probe exhibited rapid response, high selectivity and reversibility. Confocal fluorescence microscopy was used to visualize the redox change

Full text of DOI:10.1039/c2cc36839k

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A fluorescent probe for rapid detection of thiols and
imaging of thiols reducing repair and H2O2 oxidative
stress cycles in living cells†
Cite this: Chem. Commun.,
2013, 49, 391
Received 20th September 2012,
Accepted 30th October 2012
Zhangrong Lou,z Peng Li,z Xiaofei Sun, Songqiu Yang, Bingshuai Wang and
Keli Han*
DOI: 10.1039/c2cc36839k
www.rsc.org/chemcomm
A diselenide containing fluorescent probe based on a fluorescein imaging is less invasive, more sensitive and more convenient.11
scaffold for thiols was developed. The fluorescent probe exhibited Numerous fluorescent probes for thiols based on Michael addition,
rapid response, high selectivity and reversibility. Confocal fluores- aldehyde cyclization, fluorophore deprotection, and thiol-disulfide
cence microscopy was used to visualize the redox changes exchange protocols have been reported.2,12 However, very few of
mediated by thiols and reactive oxygen species in living HeLa cells. these probes react with thiols rapidly enough to achieve real-time
detection in vivo. And most of these probes can only respond to
Intracellular thiols play an essential role in maintaining redox thiols statically. Research has shown that fluorescent probes which
homeostasis by regulating the redox status between reduced free are capable of responding to thiols and ROS reversibly offer better
thiols and oxidized disulfides.1 Glutathione (GSH), which has been chances of visualizing redox signaling, stress, and thiols repair.13
identified as the most abundant non-protein thiol, is considered to Real-time methods for measuring thiols in vivo based on redox-
be the main player in combating oxidative stress and regulating the sensitive green fluorescent protein have been reported.1,14 However,
redox environment of the internal cellular compartments.1,2 Intra- such techniques require gene manipulation while redox-responsive
cellular thiol levels can change dynamically in response to oxidative probes based on small molecules are scarce.13,15
stress,3 and the balance between reactive oxygen species (ROS) and
Glutathione peroxidase (GPx) is a selenoenzyme that protects
thiols governs many essential biological processes. Moreover, various organisms from oxidative damage by catalyzing
modest alterations in the redox equilibrium would lead to major ROS reduction in the presence of GSH.16 To clarify the catalytic
consequences, such as damaging high-order structures of protein mechanism of GPx and develop antioxidant drugs, researchers
or enzyme activity due to the presence of a cysteine in the active have synthesized many organoselenium compounds as GPx
site.1 Converging evidence has suggested that a decrease in the mimics, in which monoselenides, diselenides, and ebselen analogs
concentration of GSH induced by excessive oxidative stress is are the major ones.16,17 Steinmann et al.18 recently reported that the
associated with various diseases, including autism spectrum cleavage of diselenide bonds by thiols is 5 orders of magnitude
disorders,4 diabetes mellitus,5 neurodegenerative diseases6 and faster than that of disulfide bonds. Building on these results, we
cardiovascular diseases.7 On the other hand, an abnormal increase designed and synthesized a diselenide-based fluorescent probe
in GSH would induce reductive stress, which could lead to similar (FSeSeF, Scheme 1) for the rapid detection of thiols and redox
deleterious effects,8 such as cardiomyopathy,9 mitochondrial oxida- changes mediated by thiols and ROS in living cells.
tion and cytotoxicity.10
FSeSeF is a weakly fluorescent molecule that contains a
Further development of intracellular thiol detection methods diselenide bond as a fast, reversible recognition center for
is necessary to understand the complicated roles of thiols in redox thiols and two fluoresceins as signal reporters. Once FSeSeF
biology and elucidate the pathogenesis of various disorders in is cleaved by GSH, both the selenenyl sulfide FSeSG and the
living systems. Compared with other approaches, fluorescence
State Key Laboratory of Molecular Reaction Dynamics, Dalian Institute of Chemical
Physics (DICP), Chinese Academy of Sciences (CAS), 457 Zhongshan Road, Dalian
116023, P. R. China. E-mail: klhan@dicp.ac.cn; Fax: +86-411-84675584;
Tel: +86-411-84379293
† Electronic supplementary information (ESI) available: Additional fluorescent
confocal microscopy images, fluorescence spectra assays, details of the synthesis,
1H NMR, 13C NMR, 77Se NMR, MS spectra of compounds in this communication.
Scheme 1 Structure of FSeSeF and the nucleophilic attack by GSH at Se–Se bond
See DOI: 10.1039/c2cc36839k
with the generation of selenenyl sulfide FSeSG and selenol FSeH.
‡ These authors contributed equally.
c
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Chem. Commun., 2013, 49, 391--393 391

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Fig. 2 (a) Fluorescence responses of FSeSeF to various analytes. Bars represent the
ratio of intensity (F/F0), where F and F0 are the emission intensities at 514 nm in the
presence and absence of substrates, respectively. 1, GSH (20.0 mM); 2, dithiothreitol
(20.0 mM); 3, cysteine (20.0 mM); 4, H2O2 (2.0 mM); 5, L-histidine (2.0 mM); 6,
aspartic acid (2.0 mM); 7, glycine (2.0 mM); 8, alanine (2.0 mM); 9, ascorbic acid
(2.0 mM). (b) Time course of the response of FSeSeF (1 mM) to GSH (100 mM). The
kinetic curve was obtained using a stopped-flow spectrophotometer at 25 1C and
pH 7.40. Fluorescence intensity was collected at 514 nm (lex = 488 nm).
Fig. 1 Changes in the absorption (a) and fluorescence (b) spectra of FSeSeF with
different concentrations of GSH (0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.5,
3.0, 3.5, 4.0, 6.0, 8.0, 10.0, 12.0, 100.0 mM).
selenol FSeH containing one fluorescein are released and
strong fluorescence is emitted. The reaction has been detected with
API-ES mass spectroscopy and 77Se NMR (ESI†). Furthermore, in
congruence with the ping–pong mechanism of the GPx activity of
diselenide compounds,16,17,19 FSeSG and FSeH can transform back to different concentrations of GSH gave the estimated value for the
into FSeSeF in response to H2O2, thus providing a reversible readout rate constant (kobsd) of 8.4 ꢀ 105 Mꢁ1 sꢁ1 at pH 7.4 and 25 1C
of fluorescence for redox changes mediated by thiols and ROS.
(Fig. S5, ESI†). Considering the thiolate anion, the rate constant k
The spectroscopic properties of FSeSeF were assessed under for the reaction of FSeSeF with GSH was 2.0 ꢀ 107 Mꢁ1 sꢁ1 at
simulated physiological conditions (20 mM PBS, pH 7.40). pH 7.4 and 25 1C (ESI†),12a,c,23 which was 3–4 orders of magnitude
Because of the relatively high cytosolic concentration of GSH in higher than the previously reported rates for thiol quantification
living cells, we selected GSH to represent thiols in a series of fluorescent probes.12a,c This speedy response could contribute to
measurements. The maximum absorption intensity of FSeSeF at monitoring the changes in thiols in situ.24 Moreover, kinetic assays
490 nm changed gradually with the increasing GSH concentration of the probe indicated that the probe was stable to light and air
(Fig. 1a). This phenomenon implied that there was a ground-state both in the absence and presence of GSH within 1 h under the
intramolecular dimer complex between the two fluorescein scaf- experimental conditions (Fig. S6, ESI†). Based on these results, a
folds in FSeSeF. The hypochromism generated in the dimer would quick and stable response of FSeSeF to GSH had been verified.
likely reduce the absorption intensity of FSeSeF.20 Fig. 1b shows
Having demonstrated that the probe can detect GSH selectively
the changing tendency of the emission spectra of FSeSeF and rapidly, we wondered whether FSeSeF could respond to GSH
depended on the amount of GSH with an excitation wavelength reversibly in the presence of ROS. A reversible assay of the probe for
of 470 nm. As proposed, when FSeSeF reacted with GSH, a GSH was carried out with the addition of a variety of ROS. As shown
significant increase in fluorescence intensity was observed (emis- in Fig. S7a (ESI†), with the addition of H2O2, the emission intensity
sion images are shown in Fig. S1, ESI†), and the changes in the at 514 nm was reduced gradually. Furthermore, the result of
corresponding quantum yield with the increase in GSH are shown treating the solution with 2 equiv. of GSH revealed that the
in Fig. S2 (ESI†). The weak fluorescence of FSeSeF can be attributed fluorescence of the probe can be recovered and the reversible
to fluorescence self-quenching of the two identical fluorophores in oxidation–reduction cycles can be repeated at least three times.
close proximity.21 The detection limit for GSH was determined to Similar results were obtained for other ROS (Fig. S7b, ESI†). In
be 2.25 ꢀ 10ꢁ7 M under the experimental conditions (Fig. S3, general, the results confirmed that the FSeSeF could respond to
ESI†).22 In addition, fluorescence titration indicated that the thiols and ROS reversibly.
fluorescence intensity of FSeSeF was linearly proportional to the
concentration of GSH in the range of 0–2.0 mM (Fig. S2, ESI†).
Subsequently, we applied FSeSeF to HeLa cells to investigate
whether the fluorescent probe was able to detect biological thiols
We then examined the influence of pH on fluorescence before and the redox changes mediated by thiols and ROS. After being
and after adding 2 equiv. of GSH (Fig. S4, ESI†). The result incubated with FSeSeF for 5 min and washed with physiological
demonstrated that there was no significant variation in fluores- saline three times, living HeLa cells were observed by confocal
cence intensity near the physiological pH, indicating that FSeSeF laser scanning fluorescent microscopy. As shown in Fig. 3a, green
would work well under the physiological pH conditions. Selectivity fluorescence was observed inside the cells, indicating that FSeSeF
of the probe toward thiols was then examined by screening its could penetrate cell membranes and image cellular thiols. How-
response to other biologically relevant substances. As shown in ever, when the HeLa cells were pretreated with N-ethylmaleimide,
Fig. 2a, no remarkable enhancements of fluorescence intensity a well-known thiol scavenger that selectively reacts with thiols
for amino acids, ROS, or reducing agents were observed in the through Michael addition,25 and then incubated with FSeSeF
emission spectra except for the thiol-containing compounds, under the same conditions, cellular fluorescence turned to be
demonstrating that FSeSeF was highly selective for thiols over extremely faint (Fig. 3b). In contrast, as shown in Fig. 3c, an
other competing analytes.
increased green fluorescence was observed when the cells were
To study the response velocity of FSeSeF to thiols, we preincubated for 1 day with a-lipoic acid which could increase the
performed a time-dependent analysis. Fig. 2b illustrates that production of GSH.12d,26 Relevant bright field transmission images
the probe exhibited a prompt response to GSH (within several confirmed that the cells were still viable (ESI†). In addition, the
milliseconds). The best fitting of the kinetic response of FSeSeF MTT assay in HeLa cells with probe concentrations from 0.01 mM
c
392 Chem. Commun., 2013, 49, 391--393
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designing fluorescent probes, and provided a tool for further
investigation of relevant biological processes in thiols and ROS.
This work was supported by NSFC (No. 21273234 and
21203192) and the National Basic Research Program of China
(2013CB834604).
Notes and references
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Fig. 3 Confocal fluorescence images of the changes in thiol concentration and
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To investigate whether FSeSeF was responsive to the redox
changes mediated by thiols and ROS in vivo, we performed reversi-
bility measurement of the probe in HeLa cells. Treating the same
FSeSeF-incubated cells with H2O2 resulted in decreased cellular
fluorescence intensity (Fig. 3e). Moreover, to rule out that the weak
intracellular fluorescence compared with that shown in Fig. 3d was
not due to photobleaching or loss of fluorescent probes, we added a-
lipoic acid into the culture solution of HeLa cells and found
increased intracellular fluorescence (Fig. 3f). Taken together, these
results suggest that FSeSeF can be developed as a fluorescence probe
for the redox changes mediated by thiols and ROS in living cells. We
next sought to investigate the intracellular localization of FSeSeF in
HeLa cells using Hoechst 33342 to label the nucleus,27 which
demonstrated that the fluorophore was capable of penetrating the
nuclear membranes and dispersing in the whole cell (Fig. 3g–i).
In summary, we had successfully designed and synthesized a
diselenide-containing fluorescent probe (FSeSeF) for detecting
thiols and the redox changes mediated by thiols and ROS both
in aqueous medium and living cells. The fluorescent probe for
thiols exhibited several advantages, including high selectivity and
speedy response. Furthermore, FSeSeF could respond to the redox
changes mediated by thiols and ROS both in aqueous medium
and living cells. This work highlighted a promising strategy for
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c
This journal is The Royal Society of Chemistry 2013
Chem. Commun., 2013, 49, 391--393 393