A ratiometric fluorescent probe for hydrogen sulfide based on an excited-state intramolecular proton transfer mechanism

Article abstract of DOI:10.1016/j.dyepig.2013.07.033

A ratiometric fluorescent probe for hydrogen sulfide (H₂S) has been developed based on modulation of the excited-state intramolecular proton transfer (ESIPT) process of 2-(2′-hydroxy-3′-methoxyphenyl) benzothiazole (HMBT). The probe was prepared by coupling HMBT with 3,3′-dithiodipropionic acid, and it only displays enol-like fluorescence emission at 374 nm. However, upon introducing H₂S in neutral solution, the protecting groups of the probe was removed via the tandem nucleophilic substitution/cyclization reaction, thereby retrieving the ESIPT process of HMBT, which resulted in a decrease of the emission band at 374 nm along with a concomitant increase of a new fluorescence peak at 478 nm. The fluorescent intensity ratio at 478 and 374 nm (I₄₇₈/I₃₇₄) increases linearly with H₂S concentration in the range 0.5-10 μM. The proposed probe shows excellent selectivity toward H₂S over other common anions and biothiols.

Full text of DOI:10.1016/j.dyepig.2013.07.033

Dyes and Pigments 99 (2013) 871e877
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Dyes and Pigments
journal homepage: www.elsevier.com/locate/dyepig
A ratiometric fluorescent probe for hydrogen sulfide based on an
excited-state intramolecular proton transfer mechanism
*
Qian Huang, Xiao-Feng Yang , Hua Li
Key Laboratory of Synthetic and Natural Functional Molecule Chemistry of Ministry of Education, College of Chemistry & Materials Science,
Northwest University, Xi’an 710069, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A ratiometric fluorescent probe for hydrogen sulfide (H2S) has been developed based on modulation of
the excited-state intramolecular proton transfer (ESIPT) process of 2-(20-hydroxy-30-methoxyphenyl)
benzothiazole (HMBT). The probe was prepared by coupling HMBT with 3,30-dithiodipropionic acid, and
it only displays enol-like fluorescence emission at 374 nm. However, upon introducing H2S in neutral
solution, the protecting groups of the probe was removed via the tandem nucleophilic substitution/
cyclization reaction, thereby retrieving the ESIPT process of HMBT, which resulted in a decrease of the
emission band at 374 nm along with a concomitant increase of a new fluorescence peak at 478 nm. The
fluorescent intensity ratio at 478 and 374 nm (I478/I374) increases linearly with H2S concentration in the
Received 1 June 2013
Received in revised form
25 July 2013
Accepted 26 July 2013
Available online 6 August 2013
Keywords:
Hydrogen sulfide
Fluorescent probe
Ratiometric
range 0.5e10
m
M. The proposed probe shows excellent selectivity toward H2S over other common anions
and biothiols.
Ó 2013 Elsevier Ltd. All rights reserved.
Excited-state intramolecular proton transfer
Benzothiazole
Nucleophilicity
1. Introduction
three types: i) taking advantage of a reduction between the azide
group (or nitro group) and H2S [13e23]; ii) exploiting the unique
Hydrogen sulfide (H2S) is being increasingly recognized as the
third member of endogenous gaseous transmitter family along
with nitric oxide (NO) and carbon monoxide [1,2]. H2S is involved in
a wide range of physiological functions, such as modulation of
blood pressure, reduction of ischemia reperfusion injury [3],
regulation of inflammation [4], and suppression of oxidative stress
[5]. Abnormal levels of H2S are associated with a series of diseases,
including diabetes, hypertension, stroke and Alzheimer’s disease
[6,7]. Therefore, selective detection of H2S has received significant
attention in recent years.
Several methods such as colorimetric [8,9] and electrochemical
[10,11] assays have been developed for H2S detection. Among them,
the methylene blue method [12] is the most commonly used,
however, tedious and complicated sample pretreatment prohibit it
from having applications in many biological studies. Fluorescence
techniques are superior in H2S detection for its high sensitivity,
selectivity, convenience and real time detection. Since 2011, several
fluorescent probes for H2S have been exploited based on different
mechanisms [13e31]. In general, these probes are divided into
nucleophilic character of H2S [24e28]; iii) binding affinity with
copper(II) ion complexes [29e31]. Unfortunately, most of these
probes exhibit a response to H2S with changes only in fluorescence
intensity, which may be influenced by factors independent from
the target species concentration such as environmental or instru-
mental effects. By contrast, ratiometric fluorescent probes allow the
measurement of emission intensities at two different wavelengths.
This should provide a built-in correction for the above mentioned
environmental effects and gives greater precision to the data
analysis relative to single-channel detection [32]. However, to our
best knowledge, only a few examples of ratiometric fluorescent
probes for the selective detection of H2S in biological systems have
been reported thus far [15,33]. Therefore, there remains a need to
develop ratiometric fluorescent probes for H2S with new sensing
mechanisms.
The excited-state intramolecular proton transfer (ESIPT) chro-
mophores are particularly useful for developing ratiometric probes
as they exhibit unique and beneficial photophysical properties
including a large Stokes shift which is a desired optical response
for any fluorescent probe as it minimizes artifacts such as self-
absorption and an inner-filter effect [34,35]. In recent years,
O-functionalized 2-(20-hydroxyphenyl)benzothiazoles (HBT) have
been developed as ratiometric fluorescent probes for a variety of
* Corresponding author. Tel.: þ86 29 88302604; fax: þ86 29 88303798.
E-mail address: xfyang@nwu.edu.cn (X.-F. Yang).
0143-7208/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.dyepig.2013.07.033

872
Q. Huang et al. / Dyes and Pigments 99 (2013) 871e877
analytes [36e41]. In these samples, the phenolic OH-caged HBT
derivatives are converted to the starting HBT by the analyte-
triggered reactions, with the conversions offering ratiometric re-
sponses because HBT and their O-functionalized derivatives give
rise to the keto and enol-like emissions, respectively.
spectrometer (Bruker Daltonics Corp.,USA) in electrospray ioniza-
tion (ESI) mode. 1H and 13C NMR spectra were recorded on an
INOVA-400 spectrometer (Varian Unity), using tetramethylsilane
(TMS) as the internal standard. The pH was measured with a
Sartorius PB-10 pH meter.
Inspired by the strategy developed by Xian’s group [24], we
report a new ratiometric fluorescent probe for H2S based on its
unique dual nucleophilicity. Probe 2 was constructed by coupling 2-
(20-hydroxy-30-methoxyphenyl)benzothiazole (HMBT) with 3,30-
dithiodipropionic acid. HMBT, a typical ESIPT compound, was
selected as the fluorophore because its ester and free form would
give the enol and keto emission bands upon photoexcitation, which
facilitates the ratiometric probe design. As expected, probe 2 results
in enol-like emission due to the blockage of ESIPT. However, upon
introducing H2S in neutral buffer solution, the protecting group of
probe 2 was readily removed via the tandem nucleophilic substi-
tution/cyclization reaction, thereby retrieving the ESIPT process of
HMBT. Accordingly, it was observed a gradual decrease in the
fluorescence emission at 374 nm and a progressive increase of a
new emission band around 478 nm. The fluorescent intensity ratio
at 478 and 374 nm (I478/I374) is linearly related to H2S concentration
2.2. Synthesis of probe 1 and 2
HMBT was synthesized according to the method we reported
previously [40]. To a suspension of 2,20-dithiodipropionbenzoic
acid (0.153 g, 0.5 mmol) in anhydrous dichloromethane (10 mL)
was added 1-(3-dimethylaminopropyl)-3 -ethylcarbodiimide hy-
drochloride (EDC, 0.21 g, 1.1 mmol), 4-dimethylaminopyridine
(DMAP, 5 mg) and HMBT (0.257 g, 1.0 mmol). The mixture was
stirred at room temperature overnight and then the solvent was
removed under reduced pressure. The resulting residue was puri-
fied by silica gel column chromatography (petroleum ether/ethyl
acetate, 1:1, v/v) to give the target compound 1 as off-white solids
(0.27 g, 68%) (Scheme 1). IR (ATR, cmꢂ1), 2937, 1729, 1583, 1470,
1268, 1184, 1088, 1028, 897, 740. 1H NMR (400 MHz, CDCl3):8.52 (d,
J ¼ 7.6 Hz, 2H), 7.98 (q, J ¼ 7.7 Hz, 4H), 7.91 (d, J ¼ 8.4 Hz, 2H), 7.84
(d, J ¼ 8.0 Hz, 2H), 7.507e7.409 (m, 6H), 7.36 (q, J ¼ 6.9 Hz, 4H), 7.16
(d, J ¼ 8.0 Hz, 2H), 3.91 (s, 6H). 13C NMR (100 MHz, CDCl3): 164.00,
162.26, 152.90, 152.15, 141.92, 138.09, 135.56, 133.84, 132.84, 127.67,
127.10, 126.68, 126.36, 126.26, 125.86, 125.40, 123.46, 121.51, 121.48,
114.31, 56.55. HRMS (ESI) [M þ H]þ m/z 785.0975, [M þ Na]þ m/z
807.0802, calcd for C42H29N2O6S4 785.0908; C42H28N2O6S4Naþ
807.0728.
in the range 0.5e10
mM. The proposed probe exhibits excellent
selectivity toward H2S over other common anions and biothiols and
has been used to measure H2S levels in biological fluids.
2. Experimental
2.1. Materials and instrumentations
Probe 2 was prepared by condensation of HMBT with 3,30-
dithiodipropionic acid using the same procedure described above.
IR (ATR, cmꢂ1), 2926, 1763, 1578, 1481, 1270,1103, 996, 770. 1H NMR
(400 MHz, CDCl3): 8.08 (d, J ¼ 8.0 Hz, 2H), 7.89 (d, J ¼ 7.6 Hz, 4H),
7.48 (t, J ¼ 7.6 Hz, 2H), 7.35 (q, J ¼ 7.2 Hz, 4H), 7.08 (d, J ¼ 8.0 Hz, 2H),
3.87 (s, 6H), 3.26 (t, J ¼ 6.8 Hz, 4H), 3.17 (t, J ¼ 6.8 Hz, 4H). 13C NMR
(100 MHz, CDCl3):169.44, 162.27, 152.91, 151.94, 137.96, 135.44,
127.38, 126.93, 126.41, 125.49, 123.44, 121.51, 121.43, 114.13, 56.37,
34.48, 32.93. HRMS (ESI) [M þ Na]þ m/z 711.0761, calcd for
Unless otherwise stated, all reagents were purchased from
commercial suppliers and used without further purification. Sol-
vents were purified and dried by standard methods prior to use.
Twice-distilled water was used throughout the experiments. Flash
chromatography was performed using Qingdao Haiyang silica gel
(200e300 mesh). The fluorescence spectra and relative fluores-
cence intensity were measured with a Shimadzu RF-5301 spec-
trofluorimeter with a 10 mm quartz cuvette. UV/Vis spectra were
determined with a Shimadzu UV-2550 spectrophotometer. IR
spectra were recorded on a Bruker Vertex 70 FT-IR spectrometer
using a diamond ATR attachment. GCeMS analyses employed an
Agilent GCeMS system (GC model 6890/MS model 5973) equipped
C
34H28N2O6S4Naþ 711.0728.
2.3. General procedure for the spectra measurement
with a DB-5 MS column (30 m ꢀ 0.25 mm ꢀ 0.5
m
m) programmed
In a set of 10 mL volumetric flasks containing 1.0 mL of TriseHCl
(0.1 M, pH 7.4), 1.0 mL of CTAB (0.01 M) and 100 mL of probe 2
from 140 ꢁC (2 min) to 280 ꢁC at 20 ꢁC minꢂ1. High-resolution mass
spectra were collected using
a
Bruker micrOTOF-Q II mass
(1.0 mM), different concentrations of analytes were added and the
COOH
S
S
COOH
S
O
N
O
EDC, DMAP
DCM, rt
OCH3
S
HO
OCH3
N
S
2
HOOC
HOOC
1
S
S
O
HMBT
N
O
S
EDC, DMAP
OCH3
DCM, rt
S
2
2
Scheme 1. Synthesis of compound 1 and 2.

Q. Huang et al. / Dyes and Pigments 99 (2013) 871e877
873
reaction mixture was diluted to 10.0 mL with water. The resulting
solution was kept at 37 ꢁC for 25 min, and then the absorption or
unclear. Obviously, to successfully design a ratiometric H2S probe,
the 2,20-dithiodibenzoyl group has to be replaced by another di-
sulfide compound, which should not quench the fluorescence of
HMBT.
fluorescence spectra were recorded. The fluorescence ratio of I478
/
I374 was calculated with the excitation and emission wavelengths at
310 and 478/374 nm, respectively.
It is known that 3-(disulfanyl)-propionic acid can cyclize spon-
taneously to afford 1,2-dithiolan-3-one (4) as a stable derivative of
the linear persulfide [45]. Based on this fact, we envisioned that
3,30-dithiodipropionic acid may be exploited to develop new
ratiometric fluorescent H2S probes. With this ideal in mind, we
constructed compound 2 as a candidate for a ratiometric fluores-
cent H2S probe.
3. Result and discussion
3.1. Development of the probe and its sensing mechanism toward H2S
To monitor H2S in biological systems, the key is to discriminate
H2S from other biological nucleophiles, especially biothiols such as
cysteine (Cys), homocysteine (Hcy) and glutathione (GSH). The
double nucleophilicity of H2S has been employed in constructing
fluorescent probes by introducing a disulfide and an ester group as
the two electrophilic sites for reaction with H2S [24,27]. Unfortu-
nately, these probes are unsymmetrical disulfides, theoretically,
there are two possible entries for H2S-mediated nucleophilic sub-
stitution (Scheme S1, Supplementary data) [42]. Hence, parts of H2S
will consumed on the undesirable pathways (route b in Scheme S1),
which will decrease the sensitivity of the method. On the other
hand, if the probe is symmetrical to disulfide bond, only one per-
sulfide intermediate will be formed via the nucleophilic substitu-
tion and no H2S is wasted in the formation of persulfide that do not
contribute to the fluorescence signal measured, and thus facilitates
the H2S measurement. Based on the above considerations, we set
out to develop symmetrical disulfide-containing probes for H2S.
In our first attempt to develop ratiometric fluorescent H2S
probe, we constructed compound 1 by coupling HMBT with 2,20-
dithiodipropionbenzoic acid and expected it shows selective
response to H2S because it has similar recognition moiety to that of
the probe reported by Xian et al. [24]. The fluorescence sensing
behavior of 1 toward H2S was then examined in pH 7.4 TriseHCl
buffer (10 mM) with 1 mM CTAB. Surprisingly, probe 1 gives almost
no enol-like emission of HMBT, even though the hydroxyl groups of
HMBTs in probe 1 are masked by the ester groups. Thus only a
fluorescence turn-on instead of ratiometric response was observed
upon introducing NaHS (a commonly employed H2S donor) to the
solution of 1 (Fig. 1). We reasoned that the fluorescence quenching
may occur attributed to the high order of molecular flexibility of
compound 1 [43,44], however, the detailed mechanism remains
The absorption and fluorescence spectra of compound 1 and 2
were measured and compared with that of HMBT. As can be seen
from Fig. 2a, probe 1, 2 and HMBT exhibit a broad absorption band
centered at 310 nm, while the absorbance intensity of 1 and 2 are
much higher (ca. 2-fold) than that of HMBT because they contain
two HMBT units. Furthermore, the fluorescence spectra of 1, 2 and
HMBT were recorded under identical conditions (Fig. 2b). It can be
observed that HMBT displays a major emission bands centered at
478 nm (keto emission), whereas for 2, it gives rise to a major
emission peak around 374 nm (enol-like emission) due to the
prevention of the ESIPT process. In the case of compound 1, how-
ever, almost no fluorescence emissions are observed. The above
results are due to the high order of molecular flexibility. The above
results indicate that compound 2 may provide a ratiometric fluo-
rescence response to H2S.
0.5
HMBT
a
probe
probe
1
0.4
0.3
0.2
0.1
0.0
2
300
350
400
450
500
Wavelength (nm)
800
900
600
700
600
500
400
300
200
100
0
400
750
HMBT
probe
probe
200
b
1
0
600
2
0
2
4
6
8
NaHS
μ
C ( M)
450
300
150
0
350
400
450
500
550
600
Wavelength (nm)
350
400
450
500
550
600
Fig. 1. Fluorescence spectra of 1 (10
mM) upon addition of increasing concentrations of
Wavelength (nm)
NaHS (0e8 M) in 1.0 mM CTAB media buffered at pH 7.4 (TriseHCl buffer, 10 mM) for
m
25 min. The inset figure shows the plot of the fluorescence intensity at 478 nm as a
function of NaHS concentration. Data were acquired at 37 ꢁC with excitation at
lex ¼ 310 nm.
Fig. 2. Absorption (a) and fluorescence spectra (b) of 1, 2 and HMBT (both 10
1.0 mM CTAB media buffered at pH 7.4 (TriseHCl buffer, 10 mM).
mM) in

874
Q. Huang et al. / Dyes and Pigments 99 (2013) 871e877
Next, the fluorescence sensing behavior of probe 2 toward H2S
4
3
2
1
0
was examined in pH 7.4 TriseHCl buffer (10 mM) with 1 mM CTAB
and the results are shown in Fig. 3, it was observed that the probe
itself gives only fluorescence emission at 374 nm. However, upon
addition of an increasing amount of NaHS to the solution of 2, the
emission band of probe 2 at 374 nm gradually decreased with the
concomitant growth of a new emission band at 478 nm due to the
restoration of ESIPT process. The large shift in the emission spectra
(up to 104 nm) allows the significant separation of the emission
peaks before and after treatment with NaHS, and thus a ratiometric
measurement can be realized. The emission ratios of fluorescence
intensity at 478 and 374 nm (I478/I374) exhibit a change from 0.21 in
Y = 0.2937X + 0.0652
r = 0.9947
the absence of NaHS to 29.4 in the presence of NaHS (200 mM), and
the final enhancement factor is about 140-fold. Furthermore, the
emission ratios (I478/I374) were plotted as a function of NaHS con-
centration and a typical calibration graph was obtained as shown in
Fig. 4. The I478/I374 value was linearly related to the concentration of
0
2
4
6
8
10
NaHS concentration ( M)
μ
sulfite in the range 0.5e10
(3 ). These results demonstrate that probe 2 can detect H2S
quantitatively.
mM with a detection limit of 0.068 mM
d
Fig. 4. Plot of emission ratios at 478 and 374 nm (I478/I374) as a function of NaHS
concentration when using probe 2 (10 mM). Other conditions are the same as those
Based on previous studies [24,45], we propose the reaction
mechanism as follows (shown in Scheme 2). HSꢂ, the major form of
H2S in physiological pH conditions (ca. 72.4%) [46], can undergo
nucleophilic reaction with probe 2 to form the intermediate 3. As 3
is a thiol-containing compound, it can attack the ester, cyclizing to
form dithiolanone 4 and simultaneously releasing the free HMBT. In
the case of other biothiols (such as Cys), a similar nucleophilic
substitution can occur to generate a disulfide (5). However, product
5 does not contain a sulfhydryl group and cannot undergo the
subsequent intramolecular nucleophilic attack to release HMBT.
Therefore, probe 2 shows a selective response toward H2S over
biothiols. The above sensing mechanism has been proved by mass
spectrometry analysis of the product generated from the incuba-
tion of 2 with NaHS in acetone-H2O (50:10, v/v) solution. A
prominent peak at m/z 258.0590 corresponding [HMBT þ H]þ
(calcd 258.0589 for C14H12NO2S) is clearly observed in the HRMS
data (Fig. S2, Supplementary data). Unfortunately, the peak corre-
sponding to 4 cannot be observed in the above HRMS spectrum
because 4 will decompose upon direct MS analysis [45]. Further-
more, the above solution was analyzed by GCeMS, and the mo-
lecular ion m/z 120 (Mþ) and fragments m/z 64 [SeS]þ and m/z 55
[O]C]CHeCH2]þ for the GC fraction at 3.67 min on the gas
described in Section 2.3.
chromatogram is observed (Fig. S3, Supplementary data), which
provides strong evidence that compound 4 is indeed generated in
the above sensing process.
3.2. Effect of surfactant media and reaction time
Initially, the fluorescence sensing behavior of probe 2 toward
H2S was investigated in EtOH/TriseHCl buffer (20/80, v/v, pH 7.4,
10 mM). Unfortunately, only a slight increase in emission ratio (I478
I374) was observed upon introducing NaHS to the solution of 2
(Fig. S4, Supplementary data). It is precedented that the nucleo-
philic substitution reaction between thiols and electrophiles are
catalyzed by cetyltrimethylammonium bromide (CTAB) [47,48],
thus we tested the detection of H2S with probe 2 in CTAB micelles.
The time course of the emission ratio (I478/I374) of probe 2
/
(10
mM) in the presence of NaHS (10 equiv) was studied in CTAB
micelles and the results are shown in Fig.
5
and Fig. S5
(Supplementary data). Upon addition of NaHS, the emission ratio
(I478/I374) of probe 2 showed a dramatic increase in I478/I374 values,
which essentially reached a maximum in 25 min. Meanwhile, the
I478/I374 of free probe 2 displayed no noticeable changes under
identical conditions. Under pseudo-first-order kinetic conditions,
the observed rate constant (kobs) for NaHS was determined to be
0.122 ꢃ 0.003 minꢂ1 (Fig. S6, Supplementary data) [49].
1400
1200
1000
800
600
400
200
0
The rate enhancement of the proposed reaction in CTAB micelles
is explained as follows: the cationic surfactant CTAB can adsorb
counter ions HSꢂ, so HSꢂ concentration should be much higher on
CTAB micelles surface than in the bulk solution. On the other hand,
as probe 2 is hydrophobic, it can be incorporated into CTAB micelles
effectively. Therefore, higher concentrations of HSꢂ and the probe
around CTAB micelles will lead to a higher reaction rate. In addition,
it was observed that the keto emission of HMBT at 478 nm was
significantly increased in CTAB media (Fig. S7, Supplementary data),
which is favorable for ratiometric sensing of H2S with probe 2. On
the basis of above considerations, CTAB micellar medium was
selected in the evaluation of the selectivity and sensitivity of probe
2 toward H2S.
350
400
450
500
550
600
Wavelength (nm)
Fig. 3. Fluorescence spectra of probe 2 upon addition of increasing concentrations of
NaHS (0e200 M) in 1.0 mM CTAB media buffered at pH 7.4 (TriseHCl buffer, 10 mM)
for 25 min. Inset: images of 2 (10 M) in the absence and presence of NaHS (10 equiv)
under
UV lamp at 365 nm. Data were acquired at 37 ꢁC with excitation at
lex ¼ 310 nm.
3.3. Selectivity studies
m
m
To evaluate selectivity of probe 2 for H2S, changes of the fluo-
rescence spectra of 2 caused by other anions and biothiols were
a

Q. Huang et al. / Dyes and Pigments 99 (2013) 871e877
875
HS S
O
HO
OCH3
O
N
O
N
O
S
H2S
+
S
O
OCH3
OCH3
S
S
S
N
S
4
HMBT
2
2
3
ESTPT ON λem=478 nm
ESTPT OFF λem=374 nm
COOH
NH2
S S
COOH
H2N
HS
O
O
OCH3
N
S
5
ESTPT OFF
=374 nm
λem
Scheme 2. Proposed mechanism of discrimination of H2S from Cys when using 2.
therefore investigated under physiological pH conditions. As dis-
played in Fig. 6a, reaction of NaHS with probe 2 gave dramatic
changes in fluorescence spectra. By contrast, no observable changes
in emission spectra were observed upon addition of other anions
(such as Fꢂ, Brꢂ, Iꢂ, NO3ꢂ, SCNꢂ, HSOꢂ3 , S2O23ꢂ, SO24ꢂ, HSOꢂ3 , Acꢂ and
citrate) and biothiols (e.g., Cys, Hcy, GSH) indicating that probe 2
Biotechnology. Co., Ltd, Shanghai, China) samples, which were then
diluted appropriately with water and analyzed according the pro-
cedure described in Section 2.3. As shown in Fig. 7, we can measure
the H2S concentration in FBS in the range of 50e600
mM. This
concentration range can meet the requirement for H2S assay in
human blood [29]. Therefore, it can be concluded that probe 2 is
can specifically react with H2S. In addition, the emission ratios (I478
/
I374) were measured for the above analytes (Fig. 6b), and it was
observed that only HSꢂ gave a pronounced enhancement, and no
significant variations were observed for other analytes at the same
reaction conditions, which implies that the selectivity of 2 to H2S
over other anions and biothiols is remarkably high.
Furthermore, to examine whether probe 2 can still retain its
sensing response to H2S in the presence of a large amount of bio-
thiols, detection of H2S with probe 2 was carried out in the presence
of GSH (0.1 mM). As displayed in Fig. S8, probe 2 can still be used for
H2S assay even with the involvement of a high concentration of
GSH, which confirms that probe 2 offers a good selectivity for H2S.
1000
a
-
2 only, F-, Br-, I-, NO3 , SCN-,
Ac-, Citrate, S2O32-, SO4
HSO3-, Hcy, Cys, GSH
800
2-
,
HS-
600
400
200
0
3.4. Sensing of H2S in fetal bovine serum
350
400
450
500
550
600
We next examined the usefulness of probe 2 for fluorescence
sensing of H2S in biological fluids. Different amounts of NaHS were
spiked to fetal bovine serum (FBS, which was obtained from Sangon
Wavelength (nm)
4
3
2
1
0
b
18
15
12
9
Probe
Probe
2
2
+ NaHS
6
3
1
2
3
4 5 6 7 8 9 10 11 12 13 14 15
0
Fig. 6. (a) Fluorescence spectral changes of probe 2 (10
anions and biothiols in 1.0 mmol Lꢂ1 CTAB media buffered at pH 7.4 (TriseHCl buffer,
10 mmol Lꢂ1) for 25 min. NaHS, 10
M; other anions and biothiols, 100 M. (b)
mM) upon addition of various
0
5
10
15
20
25
30
35
40
m
m
Reaction time (min)
Emission intensity ratio ((I478/I374) of probe 2 in the presence of various species in
1.0 mmol Lꢂ1 CTAB media buffered at pH 7.4 (TriseHCl buffer, 10 mmol Lꢂ1). 1) 2 only;
Fig. 5. Time-dependent changes of the emission ratios (I478/I374) of probe 2 (10
presence of NaHS (10 equiv) in 1.0 mM CTAB media buffered at pH 7.4 (TriseHCl buffer,
10 mM). Data were acquired at 37 ꢁC with excitation at lex ¼ 310 nm.
mM) in
2) Fꢂ; 3) Brꢂ; 4) Iꢂ; 5) NO3ꢂ; 6) SCNꢂ; 7) Ac-; 8) Citrate; 9) S2O23ꢂ; 10) SO42ꢂ; 11) HSOꢂ3
;
12) Hcy; 13) Cys; 14) GSH; 15) HSꢂ. Data were acquired at 37 ꢁC with excitation at
lex ¼ 310 nm.

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Q. Huang et al. / Dyes and Pigments 99 (2013) 871e877
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0
1
2
3
4
5
6
NaHS concentration(10-4 M)
Fig. 7. Plot of emission ratios (lex ¼ 320 nm) at 478 and 374 nm (I478/I374) as a function
of NaHS concentration in fetal bovine serum samples when using probe 2 (20 mM).
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capable of sensing H2S in biological fluids such as serum. It was also
observed that emission ratio in FBS was lower compared to that
obtained in TriseHCl buffer solutions. This is presumably due to the
scavenging of the added HSꢂ by serum electrophiles [50].
4. Conclusion
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In summary, we have developed compound 2 as a new ratio-
metric fluorescent probe for H2S. It is operated by modulation of
the ESIPT process of the probe via an H2S-induced nucleophilic
substitution/cyclization tandem reaction. Notably, probe 2 features
a ratiometric fluorescent response to H2S with a remarkable red
shift in emission wavelength (w104 nm) and displays high selec-
tivity for H2S over other anions and biothiols. The developed probe
can be broadly applicable to measure H2S levels in biological
samples.
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Acknowledgments
This research was supported by the National Natural Science
Foundation of China (No. 21275117, 21175106)), the Science &
Technology Department (No. 2012JM2004) and the Education
Department (No. 12JK0518) of Shaanxi Province of China.
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probe for fast detection of hydrogen sulfide in aqueous solution and living
cells. Chem Commun 2012;48:10871e3.
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probes for hydrogen sulfide. Org Lett 2012;14:2184e7.
Appendix A. Supplementary data
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Development of a highly selective fluorescence probe for hydrogen sulfide.
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cells. Inorg Chem 2012;51:2454e60.
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.dyepig.2013.07.033.
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