A colorimetric and ratiometric fluorescent probe with a large stokes shift for detection of hydrogen sulfide

Article abstract of DOI:10.1016/j.dyepig.2015.06.037

Developing probes for selective and sensitive detection of hydrogen sulfide (H₂S) has received much research attention, because H₂S is an environmental toxin as well as an important signaling molecule to regulate physiological and pathological processes. In this work, a new colorimetric and ratiometric fluorescent probe (Probe 1) for H₂S detection was synthesized by employing dicyanoisophorone based fluorescence dye as a fluorophore and azide group as the response unit. The synthesized Probe 1 showed a long emission wavelength (λ_em = 643 nm) and large stokes shift (λ_em - λ_abs = 163 nm). Based on the H₂S-induced reduction of azide group to amino group, Probe 1 showed high response speed, sensitivity, and selectivity toward HS^- under room temperature. Moreover, Probe 1 can ratiometrically respond to HS^- and the detection limit is as low as 0.13 μM. It was proved that Probe 1 is suitable for quantitatively detecting HS^- ions in river water samples. The numerous advantages of Probe 1 make it be potentially used for quantitative detection of H₂S in environment and living organisms.

Full text of DOI:10.1016/j.dyepig.2015.06.037

Accepted Manuscript
A colorimetric and ratiometric fluorescent probe with a large stokes shift for detection
of hydrogen sulfide
Kaiqiang Xiang, Yunchang Liu, Changjiang Li, Baozhu Tian, Tianzhong Tong, Jinlong
Zhang
PII:
S0143-7208(15)00256-9
10.1016/j.dyepig.2015.06.037
DYPI 4840
DOI:
Reference:
To appear in: Dyes and Pigments
Received Date: 3 May 2015
Revised Date: 19 June 2015
Accepted Date: 22 June 2015
Please cite this article as: Xiang K, Liu Y, Li C, Tian B, Tong T, Zhang J, A colorimetric and ratiometric
fluorescent probe with a large stokes shift for detection of hydrogen sulfide, Dyes and Pigments (2015),
doi: 10.1016/j.dyepig.2015.06.037.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
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ACCEPTED MANUSCRIPT
Graphical abstract

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A colorimetric and ratiometric fluorescent probe with a large
stokes shift for detection of hydrogen sulfide
Kaiqiang Xiang, Yunchang Liu, Changjiang Li, Baozhu Tian*, Tianzhong Tong, Jinlong Zhang*
a
Key Lab for Advanced Materials and Institute of Fine Chemicals, East China University of Science
and Technology, 130 Meilong Road, Shanghai 200237, PR China
b
Department of Chemistry, Huangshan University, Huangshan 245041, China
Corresponding author. Tel.: +86 21 64252062; fax: +86 21 64252062.
E-mail address: baozhutian@ecust.e du.cn;
Jlzhang@ ecust.edu.cn

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Abstract: Developing probes for selective and sensitive detection of hydrogen sulfide (H S) has received
2
much research attention, because H S is an environmental toxins as well as an important signaling
2
molecule to regulate physiological and pathological processes. In this work, a new colorimetric and
ratiometric fluorescent probe (Probe 1) for H S detection was synthesized by employing
2
dicyanoisophorone based fluorescence dye as a fluorophore and azide group as the response unit. The
synthesized Probe 1 showed a long emission wavelength (λem = 643 nm) and large stokes shift (λem – λabs
=
163 nm). Based on the H S-induced reduction of azide group to amino group, Probe 1 showed high
2
−
response speed, sensitivity, and selectivity toward HS under room temperature. Moreover, Probe 1 can
−
ratiometrically respond to HS and the detection limit is as low as 0.13 ꢀM. It was proved that Probe 1 is
−
suitable for quantitatively detecting HS ions in river water samples. The numerous advantages of Probe 1
make it can be potentially used for quantitative detection of H S in environment and living organisms.
2
Keywords: Fluorescence probe; hydrogen sulfide; stokes shift; isophorone; ratiometric detection

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1
. Introduction
As a member of the reactive sulfur species family, hydrogen sulfide (H S) has drawn much attention
2
due to its effects on environmental toxins and poisons for centuries [1]. Usually, it is largely generated in
coal and natural gas processing, petroleum industries, biogas production, automobile tail gas, and sewage
treatment plants [2, 3]. It can damage the human nerve and respiratory systems, causing people to lose
consciousness or even die at very low concentrations (ppm levels) [4]. However, in biological systems,
H S is usually produced from L-cysteine in reactions catalyzed by enzymes such as cystathionine
2
β-synthase (CBS ), cystathionine γ-lyase (CSE), and 3-mercaptopyruvate sulfur transferase (3MST), as
well as from nonenzymatic processes [5]. Following nitric oxide (NO) and carbon monoxide (CO), H2S
has been considered to be the third gasotransmitter to regulate cardiovascular, neurona, immune,
endocrine, and gastro-intestinal systems [6-8]. Many studies have shown that H S takes part in many
2
physiological processes, such as angiogenesis [9], vasodilation [10], apoptosis [11], regulation of
inflammation [12], and neuromodulation [13]. In addition, it also has been proved that abnormal H2S
production is linked to human diseases such as Alzheimer's disease [14], Down's syndrome [15],
hypertension [10], and liver cirrhosis [16]. Therefore, the quantitative detection of H S is of great
2
significance for both environmental and biological systems [17].
Considerable efforts have been devoted to exploring the effective strategies to measure H S [18].
2
Among these available methods, fluorescent imaging is considered to be one of the most promising
approaches because of its sensitivity and simplicity [19, 20]. So far, fluorescence probes for detection of
H S are mainly based on three types of reactions [17]: (1) H S reductive reactions: reducing azides to
2
2
amines, reducing nitro/azanol to amines, and reducing selenoxide to selenide [21-28]; (2) H2S
nucleophilic reactions: Michael addition reactions, dual nucleophilic reactions, double bond addition
reactions, and thiolysis reactions [29-33]; (3) Copper sulfide precipitation reaction [34-36].
Recently, colorimetric and ratiometric fluorescent probes have received extensive research attention,
owing to their unique advantages such as low cost, simply pretreatment and naked-eye mode in a
high-throughput fashion [37-42]. However, most H S probes still have the drawbacks of the single
2
detection window and short excitation wavelengths [43]. So it is desirable to design some H S probes
2
with double detection window and red/or NIR emission [44-48].
Dicyanoisophorone based fluorescent dyes are famous for their excellent properties in
Dye-sensitized solar cells [49,50] and organic nonlinear optical crystals [51,52] due to their typical

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donor-π-acceptor (D-π-A) structure, longer emission wavelength in the NIR region, and large Stokes shift
from the ultrafast intramolecular charge transfer (ICT) [53]. However, to the best of our knowledge,
dicyanoisophorone based fluorescent dyes have rarely been employed as fluorescence probes. Here, we
designed a novel H S fluorescence probe (Probe 1) with double detection window and red emission by
2
employing dicyanoisophorone based fluorescent dye as the fluorophore and azide group as the response
unit (Scheme 1). As expected, H S can react with the azide group of Probe 1 and release the amino group.
2
Probe 1 displayed high sensitivity and selectivity towards H S in red region.
2
2
. Experimental
2
.1. Synthesis
2
.1.1. Synthesis of compound 4
-Nitrobenzyl alcohol (3.80 g, 24.8 mmol) was dissolved in EtOH (30.0 mL), followed by addition of
4
FeCl (0.1g) and activated carbon (0.5g). After refluxed for 2 h, hydrazine hydrate (5.0 g, 100 mmol) was
3
added dropwise to the above mixture at the same temperature. After stirred for another 3 h, the mixture
was filtrated and the filter liquor was concentrated under vacuum. The obtained precipitation was purified
by silica column chromatography using Petroleum/Ethyl acetate (2:1, v/v) to give a white solid (2.75g,
1
9
0%). H-NMR (400 MHz, DMSO): δ = 6.95 (d, J = 8 Hz, 2H), 6.50 (d, J = 8 Hz, 2H), 4.92 (s, 2H), 4.79
13
(
t, J = 8 Hz, 1H), 4.28 (d, J = 8 Hz, 2H) (Fig. S1). C-NMR (100 MHz, DMSO): δ=145.97, 131.13,
1
28.78, 115.20, 65.16 (Fig. S2).
2
.1.2. Synthesis of compound 3
2
.15 g (17.5 mmol) of 4-aminobenzyl alcohol was added in 20 mL of 10% HCl aqueous solution at
0
°C, followed by addition of 1.45 g (21.0 mmol, 1.2 equiv) sodium nitrite dissolved in dry 10 mL CH Cl .
2
2
After the mixture was stirred at 0 °C for 1 h, 4 mL of sodium azide (1.88 g, 28.9 mmol) aqueous solution
was added drop-wise and stirred overnight. Then, the mixture was quenched with brine and extracted with
ethyl acetate (100 mL × 3). The combined organic layer was dried over anhydrous sodium sulfate and
concentrated in vacuum. Finally, the crude product was purified by flash chromatography
1
(
Petroleum/Ethyl acetate = 4:1) to give a yellow oil (2.21 g, 85 %). H-NMR (400 MHz, DMSO): δ =
6
.95 (d, J = 8 Hz, 2H), 6.50 (d, J = 8 Hz, 2H), 4.92 (s, 2H), 4.79 (t, J = 8 Hz, 1H), 4.28 (d, J = 8 Hz, 2H)
13
(
Fig. S3). C-NMR (100 MHz, DMSO): δ = 145.97, 131.13, 128.78, 115.20, 65.16 (Fig. S4).
2
.1.3. Synthesis of Compound 2
Compound 3 (2.0 g, 13.4 mmol) was dissolved in CH Cl (100 mL), followed by addition of
2
2

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pyridinium dichromate (6.06 g, 16.1 mmol). After the mixture solution was stirred at room temperature
for 4 h, the reaction solution was filtered through 130 g of silica and the silica was washed with 1.5 L of
CH Cl . Then, the filter solution was concentrated under vacuum and the crude product was purified by
2
2
silica column chromatography using Petroleum/Ethyl acetate (5:1, v/v) to obtain a pale yellow oil (1.63 g,
1
8
3 %). H-NMR (400 MHz, CDCl ): δ = 9.94 (s, 1H), 7.88 (d, J = 8 Hz, 2H), 7.15 (d, J = 12 Hz, 2H) (Fig.
3
1
3
S5). C-NMR (100 MHz, CDCl ): δ = 190.58, 146.23, 133.21, 131.52, 119.46 (Fig. S6).
3
2
.1.4. Synthesis of Probe 1
Compound 2 (100 mg, 0.68 mmol), 2-(3,5,5-trimethyl-2-cyclohexen-1-ylidene)-Propanedinitrile (126
mg, 0.68 mmol), and piperidine (5 drops) were in sequence dissolved in acetonitrile (20 mL) and refluxed
under N for 3 h. Then, the mixture solution was concentrated under vacuum and the crude product was
2
purified by silica column chromatography using Petroleum/Ethyl acetate (4:1, v/v) to give a yellow solid
1
(
102 mg, 48%). H-NMR (400 MHz, CDCl ): δ = 7.50 (d, J = 12 Hz, 2H), 7.05 (d, J = 8 Hz, 2H), 6.95 (t,
3
13
J = 8 Hz, 2H), 6.84 (s, 1H), 2.60 (s, 2H), 2.46 (s, 2H), 1.08 (s, 6H) (Fig. S7). C-NMR (100 MHz,
CDCl ): δ=169.20, 153.68, 141.33, 132.53, 128.80, 123.61, 119.67, 113.51, 112.75, 78.69, 42.98, 39.19,
3
+
3
2.04, 28.03 (Fig. S8). HRMS (ESI): calcd. For [C H N + H] 314.1406; found 314.1404 (Fig. S9).
19
17
5
2
.2. Characterization
1
13
H NMR and C NMR spectra were recorded on a Bruker AM400 NMR spectrometer with TMS as
internal standard. HRMS was conducted on a Waters LCT Premier XE spectrometer. Absorption spectra
were measured with a SHIMADZU UV-2450 spectrophotometer at room temperature. The Fluorescence
spectra were obtained on a SHIMADZU RF-5301PC fluorescence spectrophotometer by using 480 nm
line of Xe lamp as excitation source at room temperature. HPLC analyses were performed on an
InertSustain C18 column (5 ꢀm, 4.6 mm × 250 mm) using a Shimadzu HPLC system that consists of two
LC-20AD pumps and a SPD-M20A UV-vis detector at 450 nm with methanol (flow rate, 0.8 mL/min)
and water (flow rate, 0.2 mL/min) as eluents.
−
2
.3. Measurements of HS in river water samples
Two water samples were collected from Qing-Chun River of East China University of Science
&
Technology and Huang-Pu River of Shanghai, respectively. Before use, the water samples were filtered
and the pH was adjusted with a PBS buffer (20 mM, pH 7.4). Then, the two samples were spiked with
−
different concentrations of HS (5, 10, 20 μM). Prior to detection, they were further treated with probe 1

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−
to give the final mixtures (3.0 mL) containing probe 1 (10 μM) and HS (5, 10, 20 μM). The fluorescence
emission signals at 643 nm and 562 nm were measured with a SHIMADZU UV-2450 spectrophotometer.
3
. Result and discussion
3
.1. Synthesis route
The synthetic route of Probe 1 was illustrated in scheme 1. Firstly, compound 4 was synthesized by
reducing 4-nitrobenzyl alcohol in EtOH. Then, compound 3 and Compound 2 were obtained by
diazotization and Sarrett oxidation, respectively. Finally, Probe 1 was prepared by the Kenevenagel
condensation reaction in acetonitrile. The structures of Probe 1, compound 4, compound 3, and
Compound 2 were confirmed by NMR and HRMS spectroscopy.
(
Scheme 1)
3
.2. Absorption and emission properties
Fig. 1 shows the time dependent UV-vis and fluorescence emission spectra of Probe 1 after adding
NaHS in DMSO/PBS buffer solution (50/50, v/v, pH = 7.4, 20 mM). In the absence of NaHS, Probe 1 (10
4
-1
-1
ꢀ
M) showed a strong absorption peak at 420 nm (ε = 5.6 × 10 M cm ) and weak fluorescence emission
peak at 562 nm (Fig. 1A and 1C). After addition of NaHS (1 mM), the absorption band at 420 nm
4
-1
-1
decreased gradually, together with the growth of absorbance band at 480 nm (ε = 4.6 × 10 M cm , ꢁλ
abs
=
60 nm), which can be easily observed by the naked eyes. Meanwhile, the emission peak at 562 nm
decreased and a new strong emission peak at 643 nm appeared (ꢁλem = 81 nm) (Fig. 1C). Apparently,
Probe 1 exhibited a large stokes shift of 163 nm (λabs = 480 nm, λem = 643 nm) in PBS buffer (Fig. 2),
making the excitation and emission bands can be clearly separated, which is desired in fluorescence
microscopy studies [44]. Additionally, both absorbance and emission spectra reached a plateau within 20
−
min (Fig. 1B and 1D), indicating Probe 1 has a faster response speed to HS ions. As shown in Fig. 1D,
the fluorescence intensity ratio at 643 nm and 562 nm (I643/I562) before adding NaHS (t = 0) was 0.15, and
it increased to 24.15 when the concentration of NaHS was 1 mM (t = 20 min). The corresponding
signal-to-background ratio was calculated to be 161, implying that Probe 1 is a competent ratiometric
sensor for H S detection.
2
(
(
Fig. 1)
Fig. 2)
3
.3. Response kinetics

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The reaction kinetics of Probe 1 upon addition of NaHS was investigated by analyzing the change of
fluorescence intensity ratio I643/I562 as a function of reaction time. As shown in Fig. 3, under various
NaHS concentrations, the emission intensity ratios I643/I562 increased with increasing reaction time and
reached a plateau within 20 min, revealing that the reaction between Probe 1 and NaHS follows a
pseudo-zero-order kinetics, that is, the reaction rate almost has no change with the variation of NaHS
concentration. Additionally, the Probe 1 displayed a good stability in aqueous solution because no
observable fluorescence was detected in the absence of NaHS during an identical measuring time.
(
Fig. 3)
3
.4. Effect of pH
Since the fluorescence signal of a probe may be influenced by the pH of detection system, we
−
further investigated the fluorescence response of Probe 1 towards HS in the pH range of 5.7-8.0. As
shown in Fig. 4, in the absence of NaHS, the fluorescence intensity ratio I643/I562 of Probe 1 kept near to 0
in the wide pH range. In the presence of NaHS, the fluorescence intensity ratio I643/I562 showed a slight
increase in the pH range of 5.7-6.2 and kept nearly unchanged in the pH range of 6.2-8.0. The decrease of
fluorescence intensity ratio I643/I562 at lower pH might be ascribed to two probable reasons: one is that the
+
−
higher H ion concentration will decrease the reaction speed between Probe 1 and HS ; the other is that
+
the higher H ion concentration will directly decrease the fluorescence intensity of Compound 2 which is
−
the reaction product of Probe 1 and HS (Scheme 2). To find out the actual reason, we further compared
the effect of pH on the fluorescence intensity ratio I643/I562 of Compound 2. As shown in Fig. S10, I643/I562
of Compound 2 also exhibited a downward trend with the decrease of pH in the range of 5.7-6.2. At lower
pH, the amino group of Compound 2 will be protonated, leading to the decrease of fluorescence intensity
at 643 nm. The above results confirmed that the decrease of fluorescence intensity ratio I643/I562 of Probe
1
at lower pH is relative to the protonation of amino group of its reaction production—Compound 2.
(
Fig. 4)
3
.5. Sensitivity
−
The response sensitivity of Probe 1 towards HS was investigated by the fluorescence titration
experiments in DMSO/PBS buffer solution (50/50, v/v, pH = 7.4, 20 mM). As shown in Fig. 4, in the
absence of NaHS, Probe 1 exhibited weak fluorescence centered at 562 nm. As the increase of NaHS
concentration from 0 to 100 ꢀM, the emission band at 562 nm decreased gradually, along with the
enhancement of emission band at 643 nm. Fig. 5 shows the plot of fluorescence intensity ratio

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I643/I562 vs. NaHS concentration in the range of 0-100 μM. Notably, the plot showed good linearity with a
correlation coefficient of R = 0.9927, indicating that Probe 1 is a competent probe for quantitative
−
detection of HS . The detection limit for H S was determined to be as low as 0.13 ꢀM at 3σ/κ, where σ is
2
the standard deviation of blank measurements and κ is the slope obtained from the ratiometric calibration
curve. It is worth mentioning that this detection limit is lower than that of previously reported H S probe
2
based on azide group (Table S1) [54−59].
(
Fig. 5)
3
.6. Response mechanism
Similar to the previously reported azide-based H S probes, the fluorescence shift and response
2
enhancement of Probe 1 towards H S is most likely ascribed to the H S-induced reduction of the azide
2
2
group to an amino group [57,58] (Scheme 2). To verify the above proposed reaction mechanism, the
HPLC and mass spectra of the reaction product of Probe 1 and NaHS were measured and compared with
those of Probe 1 and Compound 2. As shown in Fig. 6 and Fig. S11, the reaction product exhibited the
same HPLC and mass spectra as Compound 2, confirming the correct of the proposed reaction
mechanism.
(
Scheme 2)
Fig. 6)
(
3
.7. Selectivity
Selectivity is another very important parameter to evaluate the performance of a new fluorescent
probe. Especially, for the probes with potential application in environmental and biological systems, a
highly selective response to the target species is necessary [60]. Herein, to evaluate the selectivity of
Probe 1 to H S, we examined the UV−vis absorption and fluorescence spectra of Probe 1 toward 20 kinds
2
−
−
−
−
−
−
2−
2−
+
2+
3+
of environment and biology-related species (F , Cl , Br , I , ClO , AcO , SO , S O3 , K , Zn , Fe ,
4
3
2
3
+
2+
2+
Al , Cu , Ca , Hcy, Cys, GSH, Glucose, H O , Citric Acid). As shown in Fig. 7A and 7B, the UV−vis
2
2
−
absorption and fluorescence spectra only respond to HS , and have no/or little response to other species,
−
indicating that probe 1 has a high selectivity to HS . Fig. 8 presents the fluorescence ratio I643/I562 of
Probe 1 toward the different species. In the presence of NaHS (1 mM), the fluorescence intensity ratio
I643/I562 increased 161-fold. In contrary, I643/I562 almost had no change toward other species. The above
results demonstrated that Probe 1 is highly selective to H S over other environment and biology-related
2
species and can meet the selective requirement of the environmental and biological applications.

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(
Fig. 7)
(
Fig. 8)
−
3
.8. Detection of HS in environment water samples
−
We further investigated the detection ability of Probe 1 toward HS in environmental river water by
collecting Qing-Chun River of East China University of Science & Technology and Huang-Pu River of
Shanghai as the two representative water samples. First, the water samples were filtered and different
−
concentrations of HS solution (5, 10, 20 μM) were separately added. Then, the fluorescence emission
−
signals at 643 nm and 562 nm were collected, respectively. As shown in Fig. 9, the recovered HS
concentrations of all measuring points matched well with the corresponding spiked concentrations. This
−
result indicated that Probe 1 can be potentially used for the quantitative detection of HS ions in
environmental river water.
(
Fig. 9)
4
. Conclusion
In summary, we successfully synthesized a new colorimetric and ratiometric fluorescent probe
Probe 1) for H S detection by employing dicyanoisophorone based fluorescence dye as the fluorophore
(
2
and azide group as the response unit. Based on the H S-induced reduction of azide group to amino group,
2
−
Probe 1 showed high response speed, sensitivity, and selectivity toward HS under room temperature.
Meanwhile, Probe 1 exhibited a relatively large stokes shift (163 nm), corresponding to a color change
from yellow to pink, which can be differentiated by naked eyes. Moreover, Probe 1 can ratiometrically
−
respond to HS and the detection limit is as low as 0.13 ꢀM. It has been proved that Probe 1 can be
−
competent to quantitatively detect HS ions in river water samples. The numerous advantages of Probe 1
make it can be potentially used for quantitative detection of H S in environment and living organisms.
2
Acknowledgment
This work has been supported by the National Natural Science Foundation of China (21277046,
2
1173077, 21377038), the Shanghai Committee of Science and Technology (13NM1401000), the
Research Fund of Education of the People’s Republic of China (JPPT-125-4-076), and the National Basic
Research Program of China (973 Program, 2013CB632403).

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Figure and Scheme captions
Scheme 1. Synthetic route of Probe 1.
Scheme 2. Proposed reaction mechanism for selective detecting H S.
2
Fig. 1. Time dependent UV−vis (A, B) and fluorescence spectra (C, D) of Probe 1(10 μM) upon addition
of NaHS (1 mM). All spectra were recorded in DMSO/PBS buffer solution (50/50, v/v, pH = 7.4, 20 mM).
Inset (A): Color changes observed from solution of Probe 1 after addition of NaHS (1 mM). λ =480 nm.
ex
Slit width = 5 nm/5 nm.
Fig. 2. Absorbance and emission spectra of Probe 1 and Compound 2 in DMSO/PBS buffer solution
(
50/50, v/v, pH = 7.4, 20 mM). Dash line: Probe 1; solid line: Compound 2. λ = 480 nm. Slit width = 5
ex
nm/5 nm.
Fig. 3. Changes of fluorescence intensity ratios (I643/I562) of Probe 1 as a function of reaction time under
different NaHS concentrations (0, 0.1, 0.4, 0.7, 1 mM) in DMSO/PBS buffer solution (50/50, v/v, pH =
7
.4, 20 mM). λ = 480 nm. Slit width = 5 nm/5 nm.
ex
Fig. 4. Effect of pH on the fluorescence intensity ratio I643/I562 of Probe 1 (10 μM) in
DMSO/NaH PO -Na HPO solution (50/50, v/v, 20 mM) with (●) or without (▪) NaHS (1 mM). λ =480
2
4
2
4
ex
nm. Slit width = 5 nm/5 nm.
Fig. 5. Fluorescence spectra (A) of Probe 1 (10 ꢀM) towards different concentrations of NaHS (0−100
M) in DMSO/PBS buffer solution (50/50, v/v, pH = 7.4, 10 mM). B) Plot of fluorescence intensity
ratio 643/I562 vs. NaHS concentration (0−100 ꢀM). λ = 480 nm. Slit width = 5 nm/5 nm.
ꢀ
I
ex
Fig. 6. HPLC chromatograms of Probe 1, Compound 2, and the reaction product of Probe 1 and NaHS.
Fig. 7. UV−Vis (A) and fluorescence (B) spectra of Probe 1 toward various species (1 mM). λ =480 nm.
ex
Slit width = 5 nm/5 nm.
−
Fig. 8. Fluorescence intensity ratio I643/I562 in the presence of different species (1 mM): (1) blank; (2) HS ;
−
−
−
−
2−
2−
−
−
−
2+
3+
(
3) F ; (4) Cl ; (5) Br ; (6) I ; (7) SO ; (8) S O3 ; (9) ClO ; (10) OAc ; (11) Cl ; (12) Zn ; (13) Al ;
3 2 4
2
+
3+
2+
(
14) Ca ; (15) Fe ; (16) Cu ; (17) Hcy; (18) Cys; (19) GSH; (20) Glucose; (21) H O (22) Citric acid.
2 2;
λex = 480 nm. Slit width = 5 nm/5 nm.
−
Fig. 9. Measured concentrations of HS ions in “Qing-Chun River water” and “Huang-Pu River water” by
probe 1 (10 ꢀM). The measurements were conducted in DMSO/PBS buffer solution (50/50, v/v, pH = 7.4,
2
0 mM) at room temperature.

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Scheme 1. Synthetic route of Probe 1
Scheme 2. Proposed reaction mechanism for detecting H2S

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Fig. 1. Time dependent UV−Vis (A, B) and fluorescence spectra (C, D) of Probe 1(10 μM) upon addition
of NaHS (1 mM). All spectra were recorded in DMSO/PBS buffer solution (50/50, v/v, pH = 7.4, 20 mM).
Inset (A): Color changes observed from solution of Probe 1 after addition of NaHS (1 mM). λ =480 nm.
ex
Slit width = 5 nm/5 nm.
Fig. 2. Absorbance and emission spectra of Probe 1 and Compound 2 in DMSO/PBS buffer solution
(
50/50, v/v, pH = 7.4, 20 mM). Dash line: Probe 1; solid line: Compound 2. λ = 480 nm. Slit width = 5
ex
nm/5 nm.

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Fig. 3. Changes of fluorescence intensity ratios (I643/I562) of Probe 1 as a function of reaction time under
different NaHS concentrations (0, 0.1, 0.4, 0.7, 1 mM) in DMSO/PBS buffer solution (50/50, v/v, pH =
7
.4, 20 mM). λ = 480 nm. Slit width = 5 nm/5 nm.
ex
Fig. 4. Effect of pH on the fluorescence intensity ratio I643/I562 of Probe 1 (10 μM) in
DMSO/NaH PO -Na HPO solution (50/50, v/v, 20 mM) with (●) or without (▪) NaHS (1 mM). λ =480
2
4
2
4
ex
nm. Slit width = 5 nm/5 nm.

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Fig. 5. Fluorescence spectra (A) of Probe 1 (10 ꢀM) towards different concentrations of NaHS (0−100
ꢀ
M) in DMSO/PBS buffer solution (50/50, v/v, pH = 7.4, 10 mM). B) Plot of fluorescence intensity
ratio 643/I562 vs. NaHS concentration (0−100 ꢀM). λ = 480 nm. Slit width = 5 nm/5 nm.
I
ex
Fig. 6. HPLC chromatograms of Probe 1, Compound 2, and the reaction product of Probe 1 and NaHS.

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Fig. 7. UV−vis (A) and fluorescence (B) spectra of Probe 1 toward various species (1 mM). λ =480 nm.
ex
Slit width = 5 nm/5 nm.
−
Fig. 8. Fluorescence intensity ratio I643/I562 in the presence of different species (1 mM): (1) blank; (2) HS ;
−
−
−
−
2−
2−
−
−
−
2+
3+
(
3) F ; (4) Cl ; (5) Br ; (6) I ; (7) SO ; (8) S O3 ; (9) ClO ; (10) OAc ; (11) Cl ; (12) Zn ; (13) Al ;
3 2 4
2+
3+
2+
(
14) Ca ; (15) Fe ; (16) Cu ; (17) Hcy; (18) Cys; (19) GSH; (20) Glucose; (21) H O (22) Citric acid.
2 2;
λex = 480 nm. Slit width = 5 nm/5 nm.

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−
Fig. 9. Measured concentrations of HS ions in “Qing-Chun River water” and “Huang-Pu River water” by
probe 1 (10 ꢀM). The measurements were conducted in DMSO/PBS buffer solution (50/50, v/v, pH = 7.4,
2
0 mM) at room temperature.

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Highlights
ꢀ
ꢀ
ꢀ
ꢀ
A ratiometric fluorescence probe for H S was synthesized using dicyanoisophorone as fluorophore.
2
The probe showed long emission wavelength (643 nm) and large stokes shift (163 nm).
The Probe exhibited high response speed and selectivity toward H S.
2
−
The Probe showed high sensitivity toward HS and the detection limit is as low as 0.13 μM.

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Supplementary Information
A colorimetric and ratiometric fluorescent sensor with
a large stokes shift for detection of hydrogen sulfide
Kaiqiang Xiang, Yunchang Liu, Changjiang Li, Baozhu Tian*, Tianzhong Tong, Jinlong Zhang*
Key Lab for Advanced Materials and Institute of Fine Chemicals, East China University of Science
and Technology, 130 Meilong Road, Shanghai 200237, PR China.
Corresponding author: baozhutian@ecust.edu.cn;
jlzhang@ecust.edu.cn.

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Table S1. Comparison of determined detection limit with those of some literatures.
Linear range
Probe
λ /λ (nm)
ex em
Detection limit
Reference
(
ꢀM)
3
30/370
30/465
Talanta. 2014
0-100
2.4 μM
3
121, 122−126.
Anal.
Chem.2013, 85,
875 7881.
Chem.
Commun. 2013,
9, 3890 3892.
7
60/480
20/670
0−6
0.1μM
7
−
5
0−250
3.05 μM
4
−
Anal. Chim.
Acta. 2015,
4
10/478
10/702
2
−100
0.22 μM
0.1μM
4
859, 59−65
Chem.
Commun. 2013,
49, 502 504.
5
35/566
35/620
0
−40
5
−
New J. Chem.
2014, 38,
6
40/750
10−80
0.22 μM
0.13 μM
2770
−2773.
480/562
80/643
0−100
This Work
4
1
Fig. S1. H NMR spectrum of compound 4 in CDCl
3

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1
3
Fig. S2. C NMR spectrum of compound 4 in CDCl
3
1
Fig. S3. H NMR spectrum of compound 3 in DMSO
1
3
Fig. S4. C NMR spectrum of compound 3 in DMSO.

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1
Fig. S5. H NMR spectrum of Compound 2 in CDCl .
3
13
Fig. S6. C NMR spectrum of Compound 2 in CDCl .
3
1
Fig. S7. H NMR spectrum of Probe 1 in CDCl .
3

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1
3
Fig. S8. C NMR spectrum of Probe 1 in CDCl .
3
Fig. S9. HRMS spectrum of Probe 1.
Fig. S10. Effect of pH on the fluorescence intensity ratio (I /I ) of Compound 2 (10 μM) in
6
43 562
DMSO/NaH PO -Na HPO solution (50/50, v/v, 20 mM).
2
4
2
4

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Fig. S11. HRMS spectrum of the reaction production of Probe 1 and NaHS.