A highly selective fluorescent probe for fast detection of hydrogen sulfide in aqueous solution and living cells

Article abstract of DOI:10.1039/c2cc36141h

A new ratiometric fluorescence probe E1 based on an excited-state intramolecular proton transfer (ESIPT) mechanism for detection of hydrogen sulfide (H₂S) is reported. E1 responds to H₂S quickly and showed a 30-fold fluorescence enhancement in 2 minutes. Moreover, E1 can detect H₂S quantitatively with a detection limit as low as 0.12 μM in aqueous solution. Its potential for biological applications was confirmed by employing it for fluorescence imaging of H₂S in living cells.

Full text of DOI:10.1039/c2cc36141h

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A highly selective fluorescent probe for fast detection of hydrogen sulfide
in aqueous solution and living cellsw
Zheng Xu,z Lin Xu,z Ji Zhou, Yufang Xu, Weiping Zhu and Xuhong Qian*
Received 16th July 2012, Accepted 14th September 2012
DOI: 10.1039/c2cc36141h
A new ratiometric fluorescence probe E1 based on an excited-state
intramolecular proton transfer (ESIPT) mechanism for detection of
hydrogen sulfide (H2S) is reported. E1 responds to H2S quickly and
showed a 30-fold fluorescence enhancement in 2 minutes. Moreover,
E1 can detect H2S quantitatively with a detection limit as low as
0.12 lM in aqueous solution. Its potential for biological applications
was confirmed by employing it for fluorescence imaging of H2S in
living cells.
Hydrogen sulfide (H2S) has emerged as an important endogenous
gasotransmitter recently. It plays important roles in many
biological processes, such as neurotransmission, vasorelaxation,
cardioprotection, and anti-inflammation.1 It has been shown that
Scheme 1 Synthetic route for probe E1.
the endogenous H2S effects the relaxation of vascular smooth
In this communication, we report a new excited-state intra-
muscle, accommodates cardiovascular tissue and the inhibition
of vascular smooth muscle cell proliferation.2 It also regulates the
molecular proton transfer (ESIPT) based fluorescent probe E1. It
myocardial contractile force and other physiological processes.1b
exhibits high selectivity, detection kinetics and a sub-micromolar
detection limit of H2S and meets the requirements of practical
applications. 2-(20-Hydroxyphenyl)benzothiazole (HMBT) was
Therefore, selective detection of H2S has received significant
attention.
chosen as the fluorophore by virtue of large Stokes shift, good
photostability and ratiometric detection capability.7
Many approaches, such as colorimetric, electrochemical analysis
and gas chromatography, have been employed to measure and
trace H2S.3 However, relatively high cost, time-consuming
The probe E1 was synthesized concisely as shown in Scheme 1.
Its structure was confirmed by 1H NMR, 13C NMR, and HRMS
processes and lack of temporal and spatial resolution prohibit
spectra (ESI,w Sections S2 and S8).
At first, the selectivity of E1 was measured in aqueous solution
them from having applications in many biological studies.
Therefore, fluorescent techniques are extremely attractive due
(20 mM Tris-HCl, pH 7.4, 1 mM cetyltrimethylammonium
to their simplicity, high sensitivity and real-time detection.
bromide CTAB). As shown in Fig. 1 and Fig. S3 (ESIw),
To date, several fluorescent probes for H2S have been
reported.4 However, the reported kinetics of H2S detection is
not yet optimal and this greatly limits their potential in real
applications since catabolism of H2S is very fast. Besides, the
detection limits of those probes are not low enough to
effectively assay biological sulfides, typically from 10 to 300 mm,5
or even lower.6 Therefore, the design of fluorescent probes
for H2S with improved kinetics and detection limit is of
pressing need.
Shanghai Key Laboratory of Chemical Biology, State Key Laboratory
of Bioreactor Engineering, School of Pharmacy, East China
University of Science and Technology, Shanghai, 200237, China.
E-mail: xhqian@ecust.edu.cn; Fax: +86 21 6425 2603;
Tel: +86 21 6425 3589
w Electronic supplementary information (ESI) available: Synthetic
details and spectroscopic data. See DOI: 10.1039/c2cc36141h
Fig. 1 Ratiometric fluorescence responses (F483nm/F356nm) of E1 to
NaHS and other biological thiols in 20 mM Tris-HCl buffer with
1 mM CTAB (pH 7.4). Bar 1, 3 and 5: E1 (10 mM) + amino acid
(100 mM); bar 2, 4 and 6: E1 (10 mM) + amino acid (100 mM) + NaHS
z Z. Xu and L. Xu contributed equally to this work. Dr L. Xu now
works at East China Nomal University.
(100 mM) (lex = 295 nm).
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Chem. Commun., 2012, 48, 10871–10873 10871

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free E1 displayed a weak fluorescence band at 356 nm. Upon
addition of NaHS (a commonly employed H2S donor), the
fluorescence intensity at 356 nm decreased and the fluores-
cence intensity at 483 nm dramatically increased. The 127 nm
red-shift of emission indicated that H2S reacted with E1 and
led to the cleavage of ester groups. Addition of other bio-
logical thiols, such as cysteine (Cys), homocysteine (Hcy) and
glutathione (GSH), did not lead to any fluorescence intensity
fluctuation (Fig. 1).
Fig. 2 Schematic illustration of PET from sulphur donors to fluorophore
in E1.
High selectivity toward H2S in the presence of other competitive
species is a very important feature to evaluate the performance of
the fluorescent probe E1. Therefore, the competition experiments
were also conducted. Fig. 1 indicates that these biological
thiols showed no obvious interference in H2S detection. The
above results suggested that E1 is a highly selective fluorescent
probe for H2S in aqueous solution.
(20 mM, pH 7.4, 2 : 8 v/v or 4 : 6 v/v) with 50 mM NaHS.
Within 1 hour of reaction, E1 produced obvious turn-on
response (Fig. 3a and Fig. S4, ESIw). Next, we treated E1
(10 mM) in Tris-HCl buffer (20 mM, pH 7.4) with 1 mM
CTAB, there was no change in the fluorescence intensity without
NaHS (Fig. S2a, ESIw). Upon treatment with 1 mM NaHS, a slight
fluorescence intensity decrease at wavelength 356 nm and a new
emerging fluorescence at wavelength 487 nm were observed.
With increasing amount of NaHS, the fluorescence intensity at
356 nm diminished, and the fluorescence band at 487 nm
increased drastically. Fluorescence spectra display minimal
changes when the added H2S went beyond 30 mM (Fig. 3).
A linear relationship was found between the fluorescence
enhancement at 487 nm and the hydrogen sulfide concentration
from 0 to 10 mM (Fig. 4). The detection limit of the probe was
estimated to be 0.12 mM. These results demonstrated that E1
could detect H2S quantitatively.
As shown in Scheme 2, such a good selectivity can be
attributed to the H2S receptor 2-(pyridin-2-yl-disulfanyl)benzoic
acid (PBA). By means of PBA, E1 can react with H2S to form
the intermediate E2. However, E2 is a SH containing com-
pound, which can spontaneously cyclize with ester groups to
release the fluorophore HMBT and the other by-product E3.
This mechanism has been proved by reaction between E1 and
1
NaHS, and reaction product E3 was confirmed by H NMR,
13C NMR (ESI,w Section S8). Mono-thiols such as cysteine,
homocysteine and glutathione can react with E1 in an analogous
fashion to yield E4, which does not contain sulfhydryl group.
Therefore, E4 cannot undergo an intramolecular nucleophilic
attack to release HMBT.
To our surprise, the fluorescent intensity of free E1 is very
weak. We propose that the fluorescence was quenched via the
following two pathways. On the one hand, the lone electron
pairs of the sulphur atoms could potentially quench the excited
state via the photo-induced electron transfer (PET) mechanism
(Fig. 2).8 Further, the high order of molecular flexibility of E1
could have also contributed.9
Then we tested the fluorescence properties of the
probe E1. Initially we investigated the fluorescence sensing
behavior of E1 (10 mM) in different EtOH/Tris-HCl buffer
Fig. 3 (a) Time-dependent fluorescence spectral changes of E1 with
H2S (E1 10 mM, NaHS 50 mM) in EtOH/Tris-HCl buffer (20 mM, pH
7.4, 4 : 6 v/v). Time points represent 5, 10, 20, 30, 40, 50, 60, 70 and
80 min. (b) Fluorescence enhancement of E1 at 487 nm upon reaction
with H2S. Concentrations of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20 and 30 mM of
NaHS were reacted with 10 mM E1, in 20 mM Tris-HCl buffer with
1 mM CTAB (pH 7.4). Slite 10, 5 (lex = 295 nm).
Scheme 2 Proposed detection mechanism of E1 against H2S/thiols.
10872 Chem. Commun., 2012, 48, 10871–10873
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short as 2 minutes with reaching the maximum fluorescent
intensity.
We also explored the potential applications of E1 in
biological systems. Incubation of Hela cells with E1 for
30 min at 37 1C was followed by the addition of NaHS (with
or without CTAB) and then incubation for another 30 min. As
shown in Fig. 6, Hela cells with only E1 show low fluorescence,
while in the presence of NaHS, Hela cells show strong
fluorescence and stronger with CTAB. This result demon-
strates that E1 has potential in visualizing H2S levels change
of living cells.
In summary, a new ESIPT-based ratiometric fluorescence
probe E1 for H2S was reported. E1 responds to H2S very
quickly and showed a 30-fold fluorescence enhancement in
2 minutes. Moreover, E1 can detect H2S quantitatively with a
detection limit as low as 0.12 mM. The potential for biological
applications of E1 was confirmed by employing it for fluores-
cence imaging of H2S in living cells.
Fig. 4 Hydrogen sulfide concentration-dependent fluorescence intensity
changes of E1 in 20 mM Tris-HCl buffer with 1 mM CTAB (pH 7.4).
Concentrations of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 mM of NaHS were reacted
with 10 mM E1. Slite 10, 5 (lex = 295 nm).
We are grateful for the financial support from National Basic
Research Program of China (973 Program, 2010CB126100),
the National High Technology Research and Development
Program of China (863 Program 2011AA10A207), the China
111 Project (Grant B07023), and the Shanghai Leading Academic
Discipline Project (B507).
Notes and references
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Fig. 5 Reaction time profile of E1 and H2S (E1 10 mM, H2S 50 mM)
in 20 mM Tris-HCl buffer with 1 mM CTAB, pH 7.4. Slite 10, 5 (lex
295 nm).
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Fig. 6 Images of H2S detection in Hela cells using E1 (100 mM) at 37 1C.
(a) Bright-field image of Hela cells incubated with E1 for 60 min.
(b) Bright-field image of Hela cells incubated with E1 for 60 min with
NaHS (100 mM) added for the final 30 min. (c) Bright-field image of Hela
cells incubated with E1 for 60 min with NaHS (100 mM) and CTAB
(1 mM) added for the final 30 min. (d) Fluorescence image of Hela cells
incubated with E1 for 60 min. (e) Fluorescence image of Hela cells
incubated with E1 for 60 min with NaHS (100 mM) added for the final
30 min. (f) Fluorescence image of Hela cells incubated with E1 for 60 min
with NaHS (100 mM) and CTAB (1 mM) added for the final 30 min.
Since the catabolism of H2S is very fast in vivo, we also
performed time-based experiments to study the kinetics of E1
reacting with H2S. As shown in Fig. 5, the reaction time was as
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c
This journal is The Royal Society of Chemistry 2012
Chem. Commun., 2012, 48, 10871–10873 10873