Construction of ratiometric hydrogen sulfide probe with two reaction sites and its applications in solution and in live cells

Article abstract of DOI:10.1016/j.saa.2019.117391

Hydrogen sulfide (H₂S), as the third multifunctional signaling biomolecule, it acts as a neuromodulator in the human brain and is recognized as an important gas transmitter in human physiology. The abnormal concentrations of H₂S in human cells can result in several common diseases. Therefore, accurate, fast, and reliable methodologies are required for measuring the in vitro and in vivo concentrations of H₂S to further investigate its function. In this study, a novel DR–SO₂N₃ fluorescent probe containing the fluorophore Disperse Red 277 and a sulfonyl azide group was developed and exploited based on the structural characteristic of Disperse Red 277 that contains the active site easily can be attacked by HS^?. Therefore, this probe featured two reaction sites that involved the reduction and Michael addition of H₂S and exhibited rapid ratiometric fluorescence changes and high selectivity towards H₂S with a 619-fold enhancement factor. Further, the density functional theory (DFT)/time-dependent density functional theory (TDDFT) studies are conducted to understand the photophysical properties of DR–SO₂N₃ and the final product DRHS–SO₂NH₂, which makes the proposed mechanism more reasonable. Furthermore, the probe was successfully applied for the ratiometric fluorescence imaging of exogenous H₂S in living cells.

Full text of DOI:10.1016/j.saa.2019.117391

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 224 (2020) 117391
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Construction of ratiometric hydrogen sulfide probe with two reaction
sites and its applications in solution and in live cells
a
a
Deyang Fu a, Weiru Zhi a, Lihua Lv a, Yi Luo b, Xiaoqing Xiong a,b, , Xiaohui Kang
, Wei Hou , Jun Yan ,
⁎
b,c,⁎⁎
Hongjuan Zhao a, Laijiu Zheng a
a
Key Lab of Textile Cleaning, Dalian Polytechnic University, #1 Qinggongyuan, Dalian 116034, PR China
b
State Key Laboratory of Fine Chemicals, Dalian University of Technology, #2 Linggong Road, Dalian 116024, PR China
c
College of Pharmacy, Dalian Medical University, Western 9 Lvshun nan Road, Dalian 116044, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 2 April 2019
Received in revised form 28 June 2019
Accepted 14 July 2019
Available online 17 July 2019
Hydrogen sulfide (H2S), as the third multifunctional signaling biomolecule, it acts as a neuromodulator in the
human brain and is recognized as an important gas transmitter in human physiology. The abnormal concentra-
tions of H2S in human cells can result in several common diseases. Therefore, accurate, fast, and reliable method-
ologies are required for measuring the in vitro and in vivo concentrations of H2S to further investigate its function.
In this study, a novel DR–SO2N3 fluorescent probe containing the fluorophore Disperse Red 277 and a sulfonyl
azide group was developed and exploited based on the structural characteristic of Disperse Red 277 that contains
the active site easily can be attacked by HS−. Therefore, this probe featured two reaction sites that involved the
reduction and Michael addition of H2S and exhibited rapid ratiometric fluorescence changes and high selectivity
towards H2S with a 619-fold enhancement factor. Further, the density functional theory (DFT)/time-dependent
density functional theory (TDDFT) studies are conducted to understand the photophysical properties of DR–
SO2N3 and the final product DRHS–SO2NH2, which makes the proposed mechanism more reasonable. Further-
more, the probe was successfully applied for the ratiometric fluorescence imaging of exogenous H2S in living
cells.
Keywords:
Hydrogen sulfide probe
Ratiometric fluorescence changes
Two reaction sites
Fluorescence imaging
© 2019 Elsevier B.V. All rights reserved.
1. Introduction
amount of attention that is being devoted to H2S detection and determi-
nation in living systems. Currently, various methods, including both
H2S is a renowned, colorless, highly toxic, and acid gas with an un-
pleasant rotten egg odor [1–3]. H2S can affect the eyes as well as the re-
spiratory and central nervous systems even in low exogenous
concentrations. Endogenous H2S is produced using L–cysteine in the re-
actions that are catalyzed by cystathionine–synthase (CBS) or
cystathionine–lyase (CSE) [4] and transformed [5]. Therefore, H2S is
considered to be the third multifunctional signaling biomolecule along
with NO and CO [6] because it acts as a neuromodulator in the human
brain and is recognized as an important gas transmitter in human phys-
iology [7]. Currently, scientists have observed that the production of ab-
normal concentrations of H2S in human cells can result in several
common diseases [1,8], including Alzheimer's disease [9], Down's syn-
drome [10], and liver cirrhosis [11], which explains the increasing
chemical methods (iodometric, mercury, methylene blue colorimetric,
and lead acetate reaction rate) and physical methods (chromatography,
laser, and H2S sensor), are available for detecting H2S [12–14]; however,
all the aforementioned methods are complicated and time-consuming.
Therefore, accurate, fast, and reliable methodologies are still required
for measuring the in vitro and in vivo concentrations of H2S to further
investigate its function.
Recently, some fluorescent detection methods have been observed
to be particularly powerful because of their high sensitivities, specific-
ities, and minimal toxicities in cells and tissues [15–21]. Accordingly,
various fluorescent probes have been developed for the detection of
H2S based on different chemical reactions of H2S, including its reduction
property, high binding affinity towards Cu2+ ions [22], azide reduction
[12,23–33], specific nucleophilicity [34–36], and thiolysis of
dinitrophenyl ether [6,37]. Among these strategies, the introduction of
an azide or sulfonyl azide group into fluorescent dyes for its reduction
by H2S has been commonly applied. For example, Peng's group used
the “TURN-ON” fluorescence of the H2S probe 1–NH2 through the re-
duction of azide by H2S [38]. However, the “TURN-ON” fluorescence
⁎
Correspondence to: X. Xiong, Key Lab of Textile Cleaning, Dalian Polytechnic
University, #1 Qinggongyuan, Dalian 116034, PR China.
⁎⁎ Correspondence to: X. Kang, College of Pharmacy, Dalian Medical University, Western
9 Lvshun nan Road, Dalian 116044, PR China.
E-mail addresses: xiongxq@dlpu.edu.cn (X. Xiong), kangxh@dmu.edu.cn (X. Kang).
https://doi.org/10.1016/j.saa.2019.117391
1386-1425/© 2019 Elsevier B.V. All rights reserved.

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D. Fu et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 224 (2020) 117391
may be influenced because of environmental factors in real applications.
To avoid this negative interference, it is highly desirable to develop fast
response probes for H2S based on accurate ratiometric fluorescent
probes that can self-calibrate using two emission intensities
[30,39–41]. In addition, many reported H2S probes always simulta-
neously detected biothiols [1,42] such as cysteine (Cys), homocysteine
(Hcy), and glutathione (GSH), viscosity [43], HClO [23], NO [25] and so
on, thus, these probes showed low selectivity towards H2S. Even some
probes could discriminate H2S and the other species, the detection pro-
cess was very complicated through different emission channels. Thus,
H2S fluorescent probes with simple detection process and high selectiv-
ity should be proposed.
using the reduction and Michael addition of HS−. It is worth noting
that the new DR–SO2N3 probe exhibits physical properties that are sim-
ilar to those of Disperse Red 277. Furthermore, we have utilized the
DFT/TDDFT calculation for rationalizing the optical properties of DR–
SO2N3 and final product between DR–SO2N3 and H2S. Additionally, the
sensing properties of DR–SO2N3 were investigated to denote that this
probe exhibited rapid ratiometric fluorescence changes and high H2S
selectivity. Bioimaging studies have demonstrated that the DR–SO2N3
probe exhibited good permeability, low cytoxicity, and ratiometrically
detected endogenous H2S in living cells. These data provide us with a
new platform to study the function of H2S in biological systems.
Various characteristics, such as high quantum yield, long-
wavelength emission, and response to H2S by fluorescent changes,
should be considered while selecting a fluorophore. Coumarin and its
derivatives always own the aforementioned properties, thus, they are
commonly and extensively used in several applications, including cell
biology [44], lasers [45], sensors [46], and advanced photophysical sys-
tems. In particular, iminocoumarin (Disperse Red 277) is an important
coumarin dye whose derived fluorophores are renowned for their
bright fluorescent emissions and highly efficient quantum yields [47],
which are suitable for various biological applications. However, when
compared to coumarin, the usage of Disperse Red 277 in fluorescent
probes is still scarce and is limited to one report describing that the
iminocoumarin moiety can be incorporated as the fluorescent signaling
unit in the Zn2+ indicator [48]. To the best of our knowledge, Disperse
Red 277 and its derivatives have not been used so far for the develop-
ment of fluorescent probes that are capable of detecting and imaging
H2S, which would be highly valuable but even more challenging. As
matter of fact, the structure of Disperse Red 277 may provide the possi-
bility to detect H2S because the HS− group can take place nucleophilic
addition to an electron-poor α,β-unsaturated C_C double bond (site
II, Scheme 1).
2. Experimental section
2.1. Materials
The common reagents used in the experiments were all of analytical
grade. 4-N,N-diamino-2-hydroxybenzaldehyde, 2-cyanogen methyl
benzimidazole, 3-(2-benzimidazole)-7-diamine coumarin, malononitrile,
chlorosulfonic acid, and sodium azide were purchased from Aladdin. Rho-
damine B was purchased from Tianjin Fine Chemicals Development Cen-
ter. 1H NMR and 13C NMR spectra were recorded on a VARIAN INOVA-400
spectrometer with chemical shifts being reported as ppm (in DMSO, TMS
as internal standard). The following abbreviations are used to indicate the
multiplicities: s, singlet; d, doublet; t, triplet; q, quartet; m, multiple; and
br, broad. The mass spectrometric data were obtained on a Q-TOF Micro
mass spectrometer. Further, the UV–vis spectra were collected on a Perkin
Elmer Lambda 35 UV–vis spectrophotometer. The fluorescence measure-
ments were performed using a VAEIAN CARY Eclipse Fluorescence Spec-
trophotometer (Serial No. FL1109-M018), and the fluorescence images of
the MCF-7 cells were obtained using an Olympus FV1000 laser confocal
microscope.
By considering the aforementioned requirements and theoretical
facts, in this study, we describe the realization of an effective ratiometric
fluorescent probe (DR–SO2N3) for H2S based on the derivatization of
Disperse Red 277, which could discriminate H2S from other biothiols
2.2. Synthesis of DR–SO2N3
4-N,N-Diamino-2-hydroxybenzaldehyde (2.0 g, 0.01 mol) and 2-
cyanogen methyl benzimidazole (1,67 g, 0.01 mol) were dissolved in
Scheme 1. Design and synthetic routine of DR–SO2N3 and the rationale of the designing mechanism

D. Fu et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 224 (2020) 117391
3
ethanol (0.07 g, 0.001 mol), and pyridine was added as the catalyst
3. Results and discussion
into the reaction system at room temperature (25 °C). The mixture
was stirred for 24 h, and the product was filtered, washed with ethanol,
and dried. 3-(2-Benzimidazole)-7-diamine coumarin (2.0 g, 0.006 mol),
malononitrile (0.6 g, 0.009 mol), and ethylene glycol monomethyl ether
(20 mL) were further added. The mixture was refluxed for 5 h to obtain
Disperse Red 277 (1.9 g, 0.004 mol, yield 82%), which was used in the
subsequent step without any purification. In the ice water bath,
chlorosulfonic acid (30 mL) was added to Disperse Red 277 (2.0 g,
0.005 mol), and the mixture was refluxed at 130 °C for 3.5 h. After the
mixture was cooled to room temperature and poured into ice water,
an orange–red solid was precipitated. The precipitate was washed
using cold water until the pH was adjusted to approximately 5. The
DR–SO2Cl compound was obtained after being dried and grinded.
Next, sodium azide (0.124 g, 0.002 mol) dissolved in water/ethanol
(3 mL:3 mL) was added to a solution of DR–SO2Cl (0.5 g, 0.001 mol)
in ethanol (50 mL), and the mixture was stirred at 80 °C for 24 h. Fur-
ther, the solvents were evaporated under vacuum, and DR–SO2N3
(0.38 g, 0.0008 mol, yield 80%) was obtained as a dark red solid powder
(Rf = 0.35; 5% CH3OH in dichloromethane). 1H NMR (400 MHz,
DMSO d6) δ 9.32 (s, 1H), 8.85 (d, J = 8.3 Hz, 1H), 8.04 (d, J = 2.9 Hz,
2H), 7.82 (d, J = 9.0 Hz, 1H), 7.08–6.96 (m, 1H), 6.84 (s, 1H), 5.77 (s,
1H), 3.61 (q, J = 6.8 Hz, 4H), 1.21 (t, J = 6.9 Hz, 6H). 13C NMR
(100 MHz, DMSO d6) δ 161.53, 155.96, 154.42, 152.67, 149.76, 148.93,
137.61, 133.04, 131.50, 131.15, 123.84, 120.20, 117.06, 114.73, 112.67,
110.68, 105.45, 97.08, 49.06, 45.29, 12.90. MS (API ESI+): m/z: calcd
for C23H18N8O3S: 486.12; found 487.1301 M + 1.
3.1. Molecular design and synthesis of DR–SO2N3
The design of the DR–SO2N3 probe utilizing the coumarin dye Dis-
perse Red 277 is depicted in Scheme 1. The probe has two potential re-
action sites as fluorophores: a renowned fluorescence quencher
sulfonyl azide group that lies in the benzimidazole moiety as the reac-
tion site for H2S (site I) and an α,β-unsaturated bond in the coumarin
moiety as the Michael acceptor (site II). This structural feature was ex-
pected to contribute to the activity of the probe. We assumed that the
DR–SO2N3 probe could discriminate between H2S and other biothiols
and the biologically related species with high accuracy based on the re-
duction and nucleophilicity of HS−. Further, the reaction between DR–
SO2N3 and H2S would provide DR–SO2NH2 as an intermediate because
of the rapid reduction of HS− at site I. Subsequently, the Michael adduct
DRHS–SO2NH2 would be produced by intramolecular nucleophilic at-
tack of HS− at site II. These hypotheses are partially supported by the re-
cent publications that have investigated the intermediate and final
product of the reaction.25 The selective discrimination of H2S would be
feasible because of the weak reduction potential and nucleophilicity of
the remaining biothiols. Furthermore, the ratiometric monitoring of cel-
lular H2S could be accomplished, which will be useful for clarifying the
role of H2S in physiological processes. The structure of DR–SO2N3 was
elucidated using 1H and 13C nuclear magnetic resonance (NMR) spec-
troscopy and high-resolution mass spectrometry analyses (see
supporting information).
3.2. Photophysical properties and the DFT/TDDFT studies of DR–SO2N3
2.3. Spectroscopic and fluorescence quantum yield measurements
The photophysical properties of the DR–SO2N3 probe were initially
investigated using different solvents and were compared with those of
Disperse Red 277 (Tables S1 and S2). Further, the maximum absorption
and emission wavelengths of DR–SO2N3 were observed to be similar to
those of Disperse Red 277. In dimethyl sulfoxide (DMSO), the UV–vis
spectrum of DR–SO2N3 exhibited two main absorption peaks at 362
and 553 nm and two emission peaks at 460 and 583 nm. The fluores-
cence quantum yield of DR–SO2N3 (ΦDR–SO2N3) was established to
be 0.45 in DMSO (Table S1). The ΦDR–SO2N3 value was slightly lower
than that of Disperse Red 277 (Table S2), which indicated that the in-
troduction of the sulfonyl azide group did not completely quench the
fluorescence of DR–SO2N3.
To rationalize the optical properties of the new functional dye DR–
SO2N3, frontier molecular orbital analysis was conducted based on the
DFT/TDDFT calculation at the B3LYP/6–311 + G** level with Gaussian
09 in comparison with Disperse Red 277 (Fig. 1), considering the solva-
tion effect of DMSO using the solvent model density (SMD). The results
confirmed that the two strongest absorption bands that were experi-
mentally observed for Disperse Red 277 were obtained from the excited
states S1 and S5 and corresponded to two main electronic transitions
(highest occupied molecular orbital (HOMO) → lowest occupied molec-
ular orbital (LUMO), fab = 1.079 and λab = 492 nm; HOMO–3 → LUMO,
The relative fluorescence quantum yields were determined with
Rhodamine B as the standard and calculated using the following equa-
tion [49,50]:
2
Φx ¼ ΦsðFx=FsÞðAs=AxÞðλexs=λexxÞðnx=nsÞ ;
where Φx represents the quantum yield, F denotes the integrated area
under the corrected emission spectrum, A denotes the absorbance at
the excitation wavelength, λex denotes the excitation wavelength, n de-
notes the refractive index of the solution (because of the low concentra-
tions of the solutions (10−7–10−8 M), the refractive indices of the
solutions were replaced with those of the solvents); and the subscripts
x and s refer to the unknown and the standard, respectively.
2.4. Cell incubation and fluorescence imaging
The MCF-7 cells were obtained from the State Key Laboratory of Fine
Chemicals in the Dalian University of Technology. The cells were seeded
onto cover slips at a concentration of 2 × 104 cells mL−1 and cultured in
Dulbecco's modified eagle medium (DMEM) in an incubator (37 °C, 5%
CO2, and 20% O2). After 24 h, the cover slips were slightly rinsed thrice
using phosphate buffer saline (PBS) to remove the media and then cul-
tured in DMEM for later use. First, the DR–SO2N3 (10 μM) and Disperse
Red 277 (10 μM) dyes were added to the aforementioned cellular sam-
ples and incubated for 30 min; next, the samples were slightly rinsed
thrice using PBS and observed using an Olympus FV1000 confocal fluo-
rescence microscope to obtain pictures with white light and fluores-
cence with the help of a confocal fluorescence image captured using
100× objective lens. Then, the cells were treated using fresh medium
containing Na2S (150.0 μM) for an additional 15 min, and the corre-
sponding luminescence images were observed using the same imaging
method.
fab = 0.169, and λab = 332 nm), respectively (Table S3). The observa-
tion of the frontier molecular orbitals analysis reveals that HOMO and
LUMO are mainly located on the coumarin moiety, whereas HOMO–3
is mainly located on the benzimidazole moiety. Therefore, the two
remarkable absorption bands of Disperse Red 277 can be ascribed
to the π → π* transitions from the coumarin and benzimidazole
moieties to the coumarin moiety. Further, the calculated emission spec-
troscopic data denoted that the S1 → S0 (LUMO→HOMO, fem = 1.296,
and λem = 573 nm) and S5 → S0 (LUMO→HOMO–3, fem = 0.349, and
λem = 350 nm) transitions corresponded to two emission peaks at
584 and 463 nm, respectively (Table S4); this observation was in good
agreement with the experimental results. Further, when the benzene
ring of the benzimidazole moiety in Disperse Red 277 was modified
using the sulfonyl azide group for generating DR–SO2N3, two main

4
D. Fu et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 224 (2020) 117391
Fig. 1. The frontier molecular orbital plots of Disperse Red 277 and DR–SO2N3 in DMSO; they are involved in vertical excitation (UV–vis absorption) and emission.
absorption bands were observed for DR–SO2N3, which were attributed
to the HOMO→LUMO (fab = 1.167 and λab = 489 nm) and
HOMO→LUMO+2 (fab = 0.225 and λab = 358 nm) transitions
(Table S5). The electron transfers from the coumarin moiety resulted
in a short wavelength absorption band at 358 nm because LUMO+2
was mainly located on the benzimidazole and sulfonyl azide moieties.
Meanwhile, the calculated emission data of DR–SO2N3 were similar to
that of Disperse Red 277. Thus, the emission transitions were attributed
to S1 → S0 (LUMO→HOMO, fem = 1.358, and λem = 563 nm) and S4 → S0
(LUMO+2 → HOMO, fem = 0.448, and λem = 371 nm) (Table S6). Even
though the optical results for both Disperse Red 277 and DR–SO2N3 that
are obtained from the frontier molecular orbital analysis did not
completely agree with the experimental results, these results shed
some light on the luminescence mechanism of DR–SO2N3. Regardless,
it can be concluded that the high fluorescence quantum yield of this sys-
tem may be useful for detecting H2S.
experiments. Further, the absorbance and fluorescence intensity
changes of the DR–SO2N3 probe were recorded upon the addition of
Na2S because Na2S is commonly used as the source of H2S. Upon the ad-
dition of 1 equiv. Na2S, a slight increase could be observed in the fluores-
cence intensity of the bands at 435 and 584 nm. This is most likely
because of the reaction of the fluorescence quencher sulfonyl azide
with H2S producing the intermediate DR–SO2NH2 by the reduction of
azide to amine. Further, the mixture was treated using the gradient con-
centrations of Na2S (from 3 to 150 equiv.), and a remarkable decrease in
the fluorescence intensity of the peak could be observed at 584 nm,
whereas the emission peak gradually increased at 435 nm (Fig. 2). The
enhanced emission at 435 nm may be associated with the formation
of the final product DRHS–SO2NH2. These changes in fluorescence in-
tensity after the occurrence of the reaction imply that the DR–SO2N3
probe can ratiometrically respond to the target H2S. The ratiometric re-
sponse of the fluorescence intensities at 435 and 584 nm (F435 nm/F584
nm) increased from 0.16 to 98.98 before and after the reaction, with a
final 619-fold enhancement factor. As depicted in Fig. S1, the fluores-
cence signal ratio F584 nm/F435 nm was linearly related with the concen-
tration of Na2S ranging from 0 to 28 μM. Under well-established
sensing conditions, the limit of detection was calculated to be 2.46 μM
3.3. Sensing properties of DR–SO2N3 to H2S
To understand the sensing behavior of the DR–SO2N3 probe towards
H2S, it was subjected to the absorbance and fluorescence titration
Fig. 2. (A) Fluorescence spectral changes of DR–SO2N3 (5.0 μM in the DMSO/HEPES buffer = 6/4) after incubation with different Na2S concentrations (0, 0.5, and 1.0 equiv.).
(B) Fluorescence spectral changes of DR–SO2N3 (5.0 μM in DMSO/HEPES buffer = 6/4) after incubation with different concentrations of Na2S (3.0 → 150 equiv.). Each spectrum was
recorded after 60 s. The inset denotes the color change of DR–SO2N3 (5.0 μM) in the absence of Na2S (left) and the presence of 150 equiv. Na2S (right).

D. Fu et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 224 (2020) 117391
5
Fig. 3. Absorption spectral changes of DR–SO2N3 (5.0 μM in DMSO/HEPES buffer = 6/4)
after incubation with different concentrations of Na2S (0 → 150 equiv.). Each spectrum
was recorded after 60 s. The inset denotes the color change of DR–SO2N3 (5.0 μM) in the
absence of Na2S (left) and the presence of 150 equiv. Na2S (right).
Fig. 5. Absorption spectral changes of Disperse Red 277 (5.0 μM in DMSO/HEPES buffer =
6/4) after incubation with different concentrations of Na2S (0 → 150 equiv). Each
spectrum was recorded after 60 s. The inset denotes the color change of Disperse Red
277 (5.0 μM) in the absence of Na2S (left) and the presence of 150 equiv. Na2S (right).
according to the definition by 3σ/k (where σ is the standard deviation of
the blank sample and k is the slope of the linear regression equation).
Further, the results of the absorption experiments are presented in
Fig. 3. As can be observed, the initial absorption peak of DR–SO2N3 at
366 nm decreased and exhibited a 36-nm blue shift to 330 nm after
the addition of Na2S. Furthermore, a new peak could be observed at
386 nm with a distinct isosbestic point at 355 nm, whereas the absorp-
tion peak at 555 nm exhibited a drastic decrease, which was accompa-
nied by an apparent change in color from pink to almost colorless
(inset of Fig. 3). The absorption peak at 330 nm may be assigned to
the DR–SO2NH2 intermediate, whereas the absorption peak at 355 nm
may be attributed to the final product DRHS–SO2NH2. This indicates
that the conjugation between coumarin and benzimidazole sulfonyl
azide was broken because of the Michael addition of HS− to site II in
the probe, which allowed for the colorimetric detection of H2S by the
naked eye. Based on these results, it can be concluded that changes in
absorption and emission are likely to be associated with the formation
of the final product DRHS–SO2NH2.
slight enhancement when the concentration of Na2S became 1 equiv.,
whereas the peak at 435 nm remained virtually unaltered. Conversely,
the latter fluorescence intensity at 435 nm increased after the addition
of more than 3 equiv. Na2S, whereas that at 584 nm decreased, which
was consistent with the results of the Na2S titration experiments. All
the changes with respect to the fluorescence intensities were observed
to occur within 60 s, indicating the rapid response ability of the DR–
SO2N3 probe to H2S.
To investigate the selectivity of DR–SO2N3, which is another key fac-
tor for a probe, it was treated with various relevant interference species
(such as representative amino acids, metal ions, and anions) and mon-
itored by emission spectroscopy. The results denoted that no noticeable
changes were observed in the fluorescence histogram upon the addition
of biothiols (Cys, Hcy, and GSH), metal ions (K+, Cu2+, Al3+, Ca2+, and
NH4+), and anions (Br−, NO3−, I−, PO43−, HSO3−, SCN−, and SO23−), aminos
(glycine, alanine, and valine), reactive oxygen species (H2O2, ClO−), and
reactive nitrogen species (NO−2 ), which confirmed that the DR–SO2N3
probe could distinguish H2S from other biothiols and relevant interfer-
ence (Fig. S2). The excellent performance of the DR–SO2N3 probe ren-
ders it applicable for sensing intracellular H2S.
By considering the relevance of the response time for fluorescence
probes, the time-dependent fluorescence intensities at 584 and
435 nm at varied concentrations of HS− were monitored. As can be ob-
served from Fig. 4, the fluorescence intensity at 584 nm exhibited a
Fig. 6. Fluorescence spectral changes of Disperse Red 277 (5.0 μM in DMSO/HEPES
buffer = 6/4) after incubation with different concentrations of Na2S (0 → 150
equiv). Each spectrum was recorded after 60 s. The inset denotes the fluorescent
change of Disperse Red 277 (5.0 μM) in the absence of Na2S (left) and the presence
of 150 equiv. Na2S (right).
Fig. 4. Time course of the fluorescence response (λem = 435 and 584 nm) of the DR–SO2N3
probe (5.0 μM) in an aqueous solution (5.0 μM in DMSO/HEPES buffer = 6/4) with respect
to the presence of 1 and 150 equiv. Na2S and an excitation at 340 nm.

6
D. Fu et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 224 (2020) 117391
Scheme 2. Reaction mechanism for Disperse Red 277 and HS−
.
3.4. Reaction mechanism
concentration of Na2S became 5.0 μM (1 equiv.). Furthermore, the
ratiometric response of the fluorescence intensities at 418 and 586 nm
(F418 nm/F586 nm) increased from 0.054 to 0.27, with a final 5-fold en-
hancement factor. This indicates that the sulfonyl azide group of DR–
SO2N3 and the basic fluorophore Disperse Red 277 play important
roles in the highly selective and sensitive detection of H2S. The spectral
studies indicated that the sensing response of Disperse Red 277 to Na2S
was most probably because of the formation of a new DRHS compound.
Further, the possible response mechanism for the formation of DRHS is
depicted in Scheme 2.
Because the isolation of the proposed final products DRHS–
SO2NH2 and DRHS failed, further evidence is required to corroborate
their formation. Thus, mass spectrometry analysis of the products
was performed after the reaction between DR–SO2N3, Disperse
Red 277, and Na2S. As depicted in Figs. S3 and S4, the peaks of the
key products DRHS–SO2NH2 (m/z = 493.1) and DRHS (m/z =
414.1) were observed in the reaction mixture, which is in accordance
To further confirm our hypothesis and investigate the real detection
mechanism, the following experiments are performed. First, the absorp-
tion and emission changes of Disperse Red 277 upon the addition of dif-
ferent concentrations of Na2S were measured to verify whether the
fluorophore Disperse Red 277 played any role in the detection of H2S
by the DR–SO2N3 probe. It was observed that the absorptions at 329
and 555 nm apparently decreased while a new absorption peak ap-
peared at approximately 374 nm (Fig. 5). This was accompanied by a
color change from pink to almost colorless (inset of Fig. 5). Accordingly,
the emission wavelength completely disappeared at 586 nm, whereas
the intensity of the wavelength slightly increased at 418 nm upon exci-
tation at 380 nm (Fig. 6). All the aforementioned results indicate that a
new compound DRHS can be produced after the addition of HS− to the
Disperse Red 277 solution. To our surprise, no fluorescence enhance-
ment was observed in the fluorescent spectra at 586 nm when the
Fig. 7. Frontier molecular orbitals of DRHS and DRHS-SO2NH2.

D. Fu et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 224 (2020) 117391
7
on the coumarin and represented the π* orbital. Thus, both the n → π*
and π → π* transitions generated the absorption peak observed in the
experiment. Further, the LUMO→HOMO−2 (fem = 0.164 and λem
=
407 nm) transition of DRHS was responsible for the emission peak of
418 nm in the experiment (Table S6). The calculated spectroscopic
data for DRHS–SO2NH2 revealed almost similar electronic transitions
to those of DRHS (Table S7), except for the HOMO−2 of DRHS–
SO2NH2, which was mainly located on the S atom, and the absorption
peak was mainly ascribed to the n → π* transition. The LUMO→HOMO
−2 transition (fem = 0.187 and λem = 407 nm) contributed to the emis-
sion at 435 nm for DRHS–SO2NH2 (Table S8). Thus, the theoretical cal-
culation is also in good agreement with the experimental mass spectra
and our proposed mechanism.
3.5. The applications of probe DR–SO2N3
To evaluate the biological applicability of DR–SO2N3 and verify its
permeability and cytotoxicity, the effect of DR–SO2N3 on cell prolifera-
tion was determined using the MTT assay in the living MCF-7 cell lines
according to the reported literature. The accumulation behavior of
DR–SO2N3 was assessed, and the viability of the cells was observed
(Fig. 8). Meanwhile, the cytotoxicity for Disperse Red 277 was also in-
vestigated using the living MCF-7 cells. The DR–SO2N3 probe and Dis-
perse Red 277 showed good permeability and no cytotoxicity to living
cells in 0–30 μM (Fig. 8), which confirmed the applicability of the DR–
SO2N3 probe and Disperse Red 277 for endogenous H2S imaging.
Next, the ability of DR–SO2N3 for detecting the changes in exoge-
nous H2S in living cells was examined using confocal microscopy. The
MCF-7 cells generated fluorescence in both the blue and red channels
upon incubation using DR–SO2N3 (10 μM) for 0.5 h at 37 °C, (Fig. 9).
The cells were further treated with fresh medium containing Na2S
(150 μM) for an additional 15 min. We observed that in the presence
of Na2S, the luminescence intensity (blue channel) of the MCF-7 cells in-
creased, whereas the fluorescence intensity (red channel) of the MCF-7
cells decreased (Fig. 9). As can be observed from Fig. 9, the Iblue/Ired ratio
increased upon the exogenous addition of H2S, which indicated that the
DR–SO2N3 probe could quantitatively and ratiometrically detect the
exogeneous H2S in living cells. Meanwhile, we also investigated
whether Disperse Red 277 can realize the ratiometric detection of H2S
in living cells. As depicted in Fig. S4, the fluorescence intensity of the
Fig. 8. MTT assay using the formazan absorbance at 570 nm for the biological toxicity of
the MCF-7 cells in the presence of DR–SO2N3 and Disperse Red 277 after different
incubation times. The mean values and standard deviations were obtained from three
independent experimental determinations.
with our hypothesis and the results of the absorption and emission
spectroscopy.
According to the aforementioned experiment and the proposed
mechanism, when different concentrations of Na2S were added to
DR–SO2N3 and Disperse Red 277, the DRHS–SO2NH2 and DRHS com-
pounds were obtained, and the relative long-wavelength absorption
and emission bands disappeared. To clarify this phenomenon, we also
performed DFT/TDDFT studies on DRHS-SO2NH2 and DRHS with the
B3LYP/6–311 + G** method. The computational results denoted that
no strong absorption peak was observed to be higher than 400 nm,
which was consistent with the experimental observations. As depicted
in Fig. 7, the relatively long absorption peak at 375 nm can be attributed
to the HOMO−2 → LUMO transition (DRHS: fab = 0.136 and λab
=
313 nm; DRHS–SO2NH2: fab = 0.236 and λab = 316 nm) (Table S5).
In case of the frontier molecular orbitals, the HOMO−2 of DRHS was
mainly occupied by the lone-pair electrons of the S atom and the π elec-
trons of the benzimidazole moiety, while the LUMO was mainly located
Fig. 9. Confocal fluorescence images of MCF-7 stained with DR–SO2N3 (10 μM) for 0.5 h before and after incubation with Na2S (150 μM) for 15 min. (a) Bright images of the cells;
(b) fluorescence images of the blue channels collected at 415–460 nm; (c) fluorescence images of the red channels collected at 575–620 nm; (d) overlap of the fluorescence and
bright images; (e) ratiometric images of Iblue/Ired; excitation wavelength at 405 nm. (For interpretation of the references to color in this figure legend, the reader is referred to the
web version of this article.)

8
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Acknowledgements
This work was supported by the NSF of China (21606032, 21704011
and 21706021, scientific research general project No. L2015053 from
Liaoning Provincial Education Department, the doctoral scientific re-
search foundation No. 201501192 of Liaoning province, the State Key
Laboratory of Fine Chemicals (KF 1608 and KF1713), and Dalian science
and technology innovation fund (2018J12GX056), Dalian youth science
and technology star fund (2018RQ46).
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