A near-infrared fluorescent probe based on SNAr reaction for H2S/GSH detection in living cells and zebrafish

Article abstract of DOI:10.1016/j.dyepig.2018.11.050

Hydrogen sulfide (H₂S) and biothiols such as cysteine (Cys), homocysteine (Hcy) and glutathione (GSH), play critical roles in physiological and pathological processes. However, due to the challenge in sensing H₂S and biothiols simultaneously, it is of great importance to solve this problem in biological processes. Herein, a near-infrared fluorescent probe (HZ-NBD) based on S_NAr reactions was exploited to achieve the sensitive and selective detection of H₂S and glutathione (GSH) simultaneously in the same conditions. The design strategy was employing dicyanoisophorone based fluorescent dye as the fluorophore, the NBD moiety as a response unit and a quencher of fluorophore. The probe exhibited a low limit of detection to H₂S (13.2 nM) and GSH (112 nM) as well as a favourable large stokes shift (112 nm). The reaction mechanism was investigated via mass spectra. What's more, owing to the advantages of low cytotoxicity, fast response and strong anti-interference ability, the probe HZ-NBD was successfully applied to bioimage H₂S and GSH in HepG2 cells and zebrafish.

Full text of DOI:10.1016/j.dyepig.2018.11.050

Accepted Manuscript
A near-infrared fluorescent probe based on S Ar reaction for H S/GSH detection in
N
2
living cells and zebrafish
Han Zhang, Xiang Xia, Hong Zhao, Guo-Ning Zhang, Dao-Yong Jiang, Xing-Ying
Xue, Jiao Zhang
PII:
S0143-7208(18)32256-3
https://doi.org/10.1016/j.dyepig.2018.11.050
DYPI 7194
DOI:
Reference:
To appear in: Dyes and Pigments
Received Date: 10 October 2018
Revised Date: 14 November 2018
Accepted Date: 24 November 2018
Please cite this article as: Zhang H, Xia X, Zhao H, Zhang G-N, Jiang D-Y, Xue X-Y, Zhang J, A near-
infrared fluorescent probe based on S Ar reaction for H S/GSH detection in living cells and zebrafish,
N
2
Dyes and Pigments (2018), doi: https://doi.org/10.1016/j.dyepig.2018.11.050.
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ACCEPTED MANUSCRIPT
A near-infrared fluorescent probe based on SNAr reaction for
H2S/GSH detection in living cells and zebrafish
Han Zhang,a, † Xiang Xia,a, † Hong Zhao,a,* Guo-Ning Zhang,b Dao-Yong Jiang,a Xing-Ying Xue,a Jiao
Zhang,a
a School of Chemistry and Chemical Engineering, Southeast University, Nanjing, 211189, China.
†: Han Zhang and Xiang Xia contributed equally to this work.
Corresponding author E-mail adress: zhaohong@seu.edu.cn
Abstract
Hydrogen sulfide (H2S) and biothiols such as cysteine (Cys), homocysteine (Hcy) and glutathione
(GSH), play critical roles in physiological and pathological processes. However, due to the challenge in
sensing H2S and biothiols simultaneously, it is of great importance to solve this problem in biological
processes. Herein, a near-infrared fluorescent probe (HZ-NBD) based on SNAr reactions was exploited
to achieve the sensitive and selective detection of H2S and glutathione (GSH) simultaneously in the
same conditions. The design strategy was employing dicyanoisophorone based fluorescent dye as the
fluorophore, the NBD moiety as a response unit and a quencher of fluorophore. The probe exhibited a
low limit of detection to H2S (13.2 nM) and GSH (112 nM) as well as a favorable large stokes shift (112
nm). The reaction mechanism was investigated via mass spectra. What’s more, owing to the
advantages of low cytotoxicity, fast response and strong anti-interference ability, the probe HZ-NBD
was successfully applied to bioimage H2S and GSH in HepG2 cells and zebrafish.
Key words: dicyanoisophorone, near-infrared emission, SNAr, fluorescent probe

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1.Introduction
Hydrogen sulfide (H2S) and biothiols such as cysteine (Cys), homocysteine (Hcy) and glutathione
(GSH), play crucial roles in many physiological processes[1-4]. Generally speaking, In living organism,
the endogenous H2S can be produced from cysteine (Cys) and homocysteine (Hcy) by
cystathionine-γ-lyase
(CES),
cystathionine-β-synthase
(CBS),
and
3-mercaptopyruvate
sulfurtransferase (3-MST), which exists in the cardiovascular system, liver, kidneys and brain[5-7].
What’s more, a series of studies have demonstated that it is not the H2S that performs straightly as an
antixidant to protect neutons from oxidative stress, it achieves neuromodulation via adding the level
of GSH. H2S itself can’t rescue cells from oxidative stress but it could induce the generation of a major
and potent antioxidant GSH[8, 9]. Notably, there is a close correlation between the physiological level
of biothiols and H2S (Fig.S1)[10]. However, the abnormal amounts of H2S and GSH are related to
some human diseases. For example, abnormal production of H2S may cause human diseases including
diabetes, Alzheimer's disease and Down syndrome and the concentration of GSH is directly linked to
cancer, cardiovascular disease and Alzheimer's disease[11-14]. Hence, it is urgently required to
develop sensitive and selective methods to monitor H2S and GSH for the early diagnosis and therapy
of some related diseases.
Fluorescent probes have gained more and more attention due to their sensitivity, selectivity,
simplicity and real-time detection[15-19]. To date, various fluorescent probes have been developed
for the detection of H2S or GSH[20-28]. For instance, Xiong et al.[29] have reportd a NIR fluorescent
probe based on cyanine for H2S and Xia et al.[30] have synthesized a BODIPY-based turn on
fluorescent probe with red emission to discriminate GSH over Cys and Hcy. Encouragingly, further
efforts have been contributed to discover a single probe for the synchronous detection of H2S and
GSH. On the basis of coumarin, Li et al.[31] and Chen et al. [32]designed two fluorescent probe, which
achieved the simultaneous detection of H2S and GSH. Nevertheless, limitations such as short emission
wavelength, still exist in previously reported probe, which greatly restricts their biological
applications. To avoid that, the design and implementation of near-infrared (NIR, 650～900 nm)
fluorescent probes are of crucial importance, which have admirable merits (deep tissue penetration,
low background interference and minimum photo damage) for monitoring the target molecule in vivo
and in vitro[33-38]. Therefore, it is of great value to develop a NIR fluorescent probe for simultaneous

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detection of H2S and GSH.
(a)
(b) Previous work
(c) Present work
Scheme. 1(a) The relation diagram among H2S, biothiols generation and metabolic pathways. (b) Previous work: recent
reports for the detection of H2S and GSH simultaneously. (c) Present work: a fluorescent probe for detection of H2S/GSH in
zebrafish imaging photograph.
Keeping the above in perspective, a near-infrared fluorescent probe HZ-NBD for the
simultaneous detection of H2S/GSH was exploited. It is well known that the dicyanoisophorone based
dyes is used as a colour reporting group owing to its eminent optical properties and
biocompatibility[39-42], and 4-aromatic hydroxyl NBD derivatives can be used to distinguish Cys/Hcy
from GSH and H2S[43-46]. Utilizing the isophorone-based fluorescent dye as the fluorophore, the NBD
moiety as a response unit and a quencher of fluorophore, a NIR emission fluorescent probe HZ-NBD

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was exploited. As expected, the probe HZ-NBD itself showed negligible fluorescence, while a
remarkable fluorescence enhancement at 672 nm appeared via the sulfhydryl-promoted SNAr
reaction in the presence of H2S/GSH. Compared to the previously reported probe, the probe HZ-NBD
exhibited many merits as follows: the synchronous discrimination of H2S/GSH; emission in the NIR
light region; simple synthetic route; high sensitivity with a detection limit of 13.2 nM (H2S) and 112
nM (GSH). Apart from that, the probe HZ-NBD achieved the “naked-eye” detection. Finally, the probe
HZ-NBD realized the bioimage of H2S and GSH in HepG2 cells and zebrafish with low cytotoxicity.
2. Experimental section
2.1 Materials and methods
All reagents and solvents were commercially purchased. Solvents were purchased from
Sinopharm Chemical Reagent Co. Ltd (Shanghai, China) and used without further refinement.
The 4-chloro-7-nitro-1,2,3-benzoxadiazole (NBD-Cl) was purchased from Adamas and 3- (N,
N-diethyl amino) phenol, isophorone and malononitrile were purchased from Sinopharm
Chemical Reagent Co. Ltd (Shanghai, China). Glutathione (GSH), cysteine (Cys), and
homocysteine (Hcy) were purchased from Macklin Biochemical Co. Ltd (AR, Shanghai, China),
Shanghai HuiXing Biochemical Reagent Co., Ltd, and Tokyo Chemical Industry Co., Ltd (AR,
Shanghai, China), respectively. Histidine (His), aspartic acid (Asp), threonine (Thr), tyrosine
(Tyr), lysine (Lys), glutamic acid (Glu), glycine (Gly), Valine (Val), phenylalanine (Phe), Leucine
(Leu), proline (Pro), Sodium Chloride (NaCl), Sodium fluoride (NaF), Sodium iodide (NaI),
Sodium hypochlorite (NaClO), Sodium sulfate (Na2SO4), Sodium nitrate (NaNO3), Sodium
Carbonate (Na2CO3) and Sodium Acetate (AcONa) were purchased from Sinopharm Chemical
Reagent Co. Ltd (AR, Shanghai, China). Sodium Hydrogen Sulfite (NaHSO3) and Sodium
hydrosulfide (NaSH) were purchased from Aladdin (AR, Shanghai, China). A stock solution
(1×10-3M) of HZ-NBD was prepared in THF and then diluted to the desired concentration prior
to the spectroscopic mesurements. The various analytes were dissolved in double-distilled
water and its stock solution concentration was 1×10-2 M.
1H NMR and 13C NMR spectra were recorded on a Bruker DMX600 NMR spectrometer
using CDCl3 as solvents for dissolving the samples and tetramethylsilane (TMS) as the internal
standard. The mass spectra were obtained using an Ultraflex II (MALDI-TOF) spectrometer.

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UV-visible absorption spectra were determined on a Shimadzu UV-3600 spectrophotometer.
Fluorescence spectra were measured on a HORIBA FL-4 Max spectrometer; 3 × 1 × 1 cm quartz
cuvettes were used for recording absorption and emission spectra. Fluorescence imaging of
cells was performed using confocal microscopy (OLMPUS, FV3000). All pH measurements
were made on a pHS-3C pH meter and the value of pH of the solution was adjusted by NaOH
or HCl. All the measurement experiments were performed at about 298.0 K.
2.2 Synthesis of Intermediate compounds and target compound
The synthesis of compound 1 and 2 were prepared according to the references[26, 47].
2.2.1 Synthesis of HZ-1
An equal equivalent of 4-(N, N-diethylamino)-2-hydroxybenzaldehyde (2) (0.48 g, 2.5
mmol) and active 2-(3,5,5-trimethylcyclohex-2-enylidene) malononitrile (1) (0.46 g, 2.5 mmol)
were heated in a small volume of ethanol under reflux in the presence of a few drops of
piperidine catalyst. The reaction progress was monitored by TLC. After the reaction
completed, the reaction mixture was cooled and extracted with CH2Cl2. Finally, the collected
organic layers were dried over anhydrous MgSO4, concentrated under vacuum and purified by
silica chromatography (eluted with petroleum/ethyl acetate = 5/1, v/v) to give dark purple
1
powders HZ-1 0.18 g, yield: 20%. H NMR (600 MHz, CDCl3, TMS): δ 7.38 (d, J = 8.8 Hz, 1H),
7.36 (d, J = 15.9 Hz, 1H), 6.99 (d, J = 15.9 Hz, 1H), 6.72 (s, 1H), 3.40-3.36 (m, 4H), 2.55 (s, 2H),
2.47 (s, 2H), 1.20 (t, J = 7.0 Hz, 6H). 13C NMR (150 MHz, CDCl3, TMS): δ 169.43, 156.72, 156.53,
129.63, 120.75, 114.58, 113.83, 43.08, 39.16, 29.66, 28.02, 12.66. HRMS calcd for C23H27N3O
[HZ-1 + H]+: 362.21541. Found 362.22121.
2.2.2 Synthesis of HZ-NBD
A solution of HZ-1 (0.36 g, 1 mmol), NBD-Cl (0.24 g, 1.2 mmol) and a few drops of Et3N
catalyst in CH2Cl2 were stirred at room temperature overnight. Then, the mixture solution was
evaporated under reduced pressure and purified by silica chromatography (eluted with
1
petroleum/ethyl acetate = 2/1, v/v) to give dark red powders HZ-NBD 0.13 g, yield: 25%. H
NMR (600 MHz, CDCl3, TMS): δ 8.44 (d, J = 8.2 Hz, 1H), 7.67 (d, J = 9.0 Hz, 1H), 6.98 (d, J = 16.0
Hz, 1H), 6.84 (d, J = 15.9 Hz, 1H), 6.71-6.70 (m, 2H), 6.54 (d, J = 8.3 Hz, 1H), 6.36 (d, J = 2.5 Hz,

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1H), 3.42-3.38 (m, 4H), 2.50 (s, 2H), 2.18 (s, 1H), 1.57 (s, 3H), 1.21 (t, J = 7.0 Hz, 6H). 13C NMR
(150 MHz, CDCl3, TMS): δ 168.94, 154.19, 154.17, 152.98, 150.46, 144.22, 133.37,. 130.87,
128.87, 126.67, 122.64, 44.74, 42.93, 38.89, 31.89, 29.66, 27.88, 12.54. HRMS calcd for
C29H28N6O4 [HZ-NBD + Na]+: 547.21720. Found 547.20988.
3. Results and discussion
3.1 The design and synthesis of NIR emission fluorescent probe HZ-NBD
A near infrared emission fluorescent probe HZ-NBD has been reported. Herein, utilizing
the intermediate 1 as start material, the π-conjugate system was enlarged to NIR region
through Knoevenagel condensation reaction by reacting with intermediate 2. Additionally, the
HZ-1 presented large stokes shift (112 nm), meaning that it could be regarded as a favourable
platform for the design of NIR fluorescent probe. On the basis, the NBD as a quenching group
was introduced. Due to the strong electron-withdrawing ability of NBD, the intramolecular
charge transfer (ICT) process was inhibited that caused the quenching of fluorescence. When
adding H2S/GSH, the free fluorophore HZ-1 was produced by a nucleophilic substitution (SNAr)
reaction, generating a red emission. Besides, the structures of HZ-NBD and intermediates
were confirmed by 1H NMR, 13C NMR and HRMS (Fig. S7-S16).
Scheme. 2 The synthetic route of HZ-NBD
3.2 Specific detection for H2S and GSH
First of all, the selectivity of probe HZ-NBD were examined upon addition of H2S/GSH and other
-
−
analytes (F-, Cl−, ClO-, I-, AcO−, NO3 , CO32-, SO42-, HSO3 , Hcy, Cys, His, Asp, Tyr, Lys, Thr, Gly, Phe, Pro,
Leu, Ser, Val). As shown in Fig. 1, the probe HZ-NBD itself showed pink colours under sunlight and
non-fluorescence with excited at 365 nm by hand UV-lamp, when adding H2S/GSH, the colour of the
solution changed from pink to purple red along with a strong red emission. Besides, according to the
Fig. 2, the probe with H2S/GSH presented a remarkable “off-on” fluorescence signal with a red

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emission at 672 nm, while no significant changes were observed with other analytes, especially for
Cys and Hcy, which may disturb the detection of H2S/GSH. This can be explained by the nucleophilic
aromatic substitution (SNAr) reaction promoted by the sulfhdryl group of H2S/GSH. Since the NBD
group was removed via forming NBD-SH/NBD-S-GSH, the fluorophore HZ-1 was released with NIR
emission. As a result, these results proved that HZ-NBD displayed a high selectivity toward H2S and
GSH. Subsequently, the competition experiments were carried out to further demonstrate the
selective recognition of HZ-NBD toward H2S and GSH. Upon adding 100 μM H2S or GSH into the
solution of probe in the presence of 100 μM other analytes (seeing Fig. S1 and S2), the co-existence of
other analytes did not interfere with the capability of probe to detect H2S and GSH. As a result, these
results proved that HZ-NBD displayed a high selectivity toward H2S and GSH in the presence of other
analytes.
Sunlight
Ultraviolet
Fig. 1 Photographs of probe HZ-NBD (1×10-5M) with various analytes (100 μM of NaSH, GSH, Cys, Hcy, His, Tyr, Thr, Lys, Phe,
-
-
Gly, Trp, Ser, Val, Leu, Pro, HSO3 , Cl-, AcO-, NO3 , CO32-, SO42-, F-, I-, ClO- ) were recorded at room temperature. a: sunlight. b:
fluorescence observed irradiation with 365 nm.
Fig. 2 Fluorescence response of probe HZ-NBD (1×10-5M) in THF/PBS (v/v 1:1, pH=7.4) mixture solution upon addition
-
-
of 10 equiv. of NaSH, GSH, Cys, Hcy, His, Tyr, Thr, Lys, Phe, Gly, Trp, Ser, Val, Leu, Pro, HSO3 , Cl-, AcO-, NO3 , CO32-, F-, I-,
2-
ClO-, SO4
(b) The bar graph of fluorescence response at 672 nm of probe HZ-NBD with analytes (λex = 480 nm, slit: 3
/3 nm) at r.t.

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3.3 UV-vis and fluorescence response of HZ-NBD to different concentrations of
H2S/GSH
Fig. 3(a)/(b) Fluorescence spectra of HZ-NBD (10 μM) to various concentrations of H2S (0-14 equiv) and GSH (0-70 equiv) in
THF/PBS (v/v 1:1, pH=7.4) mixture solution. Inset: the linear plot of fluorescence intensity changes at 672 nm with the range of
H2S concentrations (0–5 equiv) and GSH concentrations (16-50 equiv). (c)/(d) The corresponding fluorescence intensity changes
of probe HZ-NBD (10 μM) at 672 nm toward different concentrations of H2S (0-14 equiv)/GSH (0-70 equiv) (λex = 480 nm, slit: 3
/3 nm).
Then, the titration experiments were conducted on the solution of probe HZ-NBD (10
μM) with an incremental addition of H2S/GSH. Initially, the free probe HZ-NBD exhibited an
absorption band centered at 520 nm in THF/PBS (v/v 1:1, pH = 7.4) aqueous medium. Upon
gradual addition of H2S/GSH, the solution of HZ-NBD displayed a red-shift from 520 nm to 560
nm in the UV spectrums accompanied by a drastic increase (seeing Fig. S3(a) and 3(b)). This
can be ascribed to the removal of the NBD group from probe, which restored the parent
fluorescent dye HZ-1. Meanwhile, the fluorescence spectra of HZ-NBD (10 μM) with an
increasing addition of H2S/GSH were performed. The probe itself exhibited insignificant
fluorescence (Ф = 0.0045), upon the addition of different amounts of the H2S (Ф = 0.080), it

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presented a distinct fluorescence intensity at 672 nm and reached the saturation state (
～
36-fold) after adding 10 equiv. H2S. For another, after adding incremental GSH (Ф = 0.067), the
fluorescence emission at 672 nm was observed and achieved the maximum fluorescence
intensity (
～29-fold) with 60 equiv. GSH. Furthermore, the fluorescence intensity changes of
probe HZ-NBD at 672 nm versus the amounts of H2S (0–14 equiv.) and GSH (0-70 equiv.) were
revealed as shown in Fig. 3(c) and 3(d). The fluorescence intensity was linear to the
concentration of H2S (0-5 equiv.) and GSH (16-50 equiv.), and the detection limit (3σ/K[48])
was calculated to be 13.2 nM (H2S) and 112 nM (GSH), respectively. These observations
implied that the probe HZ-NBD showed high sensitivities toward H2S/GSH.
3.4 Sensing mechanism of HZ-NBD towards H2S/GSH
Subsequently, to get better insight into the proposed mechanism (Scheme 3), the MS
analysis of H2S was performed to confirm the sensing process of probe HZ-NBD with H2S. It
was most likely happened as follows: Upon adding H2S/GSH, the sulfhydryl group of H2S and
GSH initially attacked the 4-position of NBD, the ether bond was broken at the same time,
which illustrated the occurrence of a nucleophilic aromatic substitution (SNAr) reaction, the
fluorescence was turn on. Additionally, the distinction of nucleophilicity of GSH and H2S
resulted in the diverse reaction rate. As shown in Fig. S5 and Fig. S6, the m/z peaks of
362.21871 and 219.08689 should be attributed to the decomposition products HZ-1 and
NBD-SH, proving the fluorophore HZ-1 was indeed formed, which was forcefully consistent
with our vision and could reasonably illustrate the sensing process of HZ-NBD to H2S.
Scheme. 3 Proposed sensing mechanism of probe HZ-NBD towards H2S and GSH

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3.5 Time/ pH-dependence of HZ-NBD to H2S/GSH
The time and pH dependent experiments of the probe HZ-NBD towards H2S/GSH were
carried out. Firstly, the kinetic studies were carried out (Fig. 4a). when adding NaSH, the
fluorescence intensity at 672 nm showed the fastest growth rate and reached a plateau within
400 s, secondly was probe with GSH saturated in 500 s. As for Cys and Hcy, the fluorescence
enhancement was insignificant. Therefore, the order of reaction rate was
NaHS>GSH>Hcy>Cys, which was consistent with selective experimental results and literature
reports[49]. In addition, to explore the biological application of the probe, the effect of pH (3.0
-12.0) on the fluorescence intensity was conducted. In the presence and absence of H2S and GSH, the
fluorescence signal remained stable over a wide pH range from 7.0 to 10.0. It meant that the
probe HZ-NBD exhibited a wide range of pH application particularly in physiological
conditions.
Fig. 4 (a) Time-dependent fluorescence intensity changes of HZ-NBD (10 μM) at 672 nm upon addition of 10 equiv. of H2S, GSH,
Cys and Hcy in in THF/PBS(v/v 1:1, pH=7.4) mixture solution. (b) The pH effect on the fluorescence intensity of HZ-NBD (10 μM)
at 672 nm with H2S/GSH (100 μM) and without H2S/GSH.
3.6 Fluorescence imaging in living cells and cytotoxicity assay
Inspired by the above-mentioned excellent performance of the probe HZ-NBD, the
applicability of the probe HZ-NBD in cellular imaging was further investigated. Before confocal
fluorescence imaging, MTT assays were carried out on HZ-NBD, which indicated that probe
HZ-NBD exhibited low cytotoxicity to living cells (Fig. S4). Then, cell imaging experiments were
conducted in HepG2 cells. As depicted in Fig. 5, HepG2 cells were pretreated with
N-ethylmaleimide (500 μM) (NEM, a scavenger of biothiols) for 30 min followed by treated
with the probe HZ-NBD (10 μM) for another 30 min as control, no fluorescence exhibited.

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There was red fluorescence shown after HepG2 cells loaded with HZ-NBD (10 μM) solely, this
can be attributed to the existence of the endogenous H2S and GSH. Finally, HepG2 cells were
cultivated with probe (10 μM) for 30 min, and subsequently incubated with NEM (500 μM) for
30 min, GSH (200 μM)/NaSH (200 μM) for another 30 min, a remarkable red fluorescence was
observed. Above all, these results clearly demonstated that the probe HZ-NBD has good
biocompatibility and can serve as a fluorescent probe for sensing endogenous and exogenous
H2S/GSH in living cells.
Probe + NEM + GSH
Probe + NEM + NaSH
Probe + NEM
Probe only
a1
a2
a3
b1
b2
b3
c1
d1
c2
d2
c3
d3
Fig. 5 Fluorescence imaging of probe HZ-NBD responding to H2S/GSH in HepG2 cells. (a) the cells incubated with NEM (500 μM)
for 30 min then incubated with probe (10 μM) for 30 min (b) the cells incubated with only probe (10 μM) for 30 min (c)/(d) the
cells incubated with NEM (500 μM) for 30 min then incubated with probe (10 μM) for 30 min, thereafter incubated with H2S
(200 μM)/GSH (200 μM) for another 30 min, respectively. (λex = 480 nm, collected at the red channel (640-720 nm). Scale bar
represents 20 μm).
3.7 Fluorescence imaging in living zebrafish
To certify that the fluorescence imaging of living bodies can be realized by HZ-NBD, the
transparent zebrafish was employed. As shown in Fig.6, no fluorescence was observed when the
zebrafish pretreated with NEM (500 μM) for 30 min were further incubated with probe HZ-NBD (10
μM) for another 30 min. Then, zebrafish only treated with probe HZ-NBD (10 μM) for 30 min, red
fluorescence was displayed inside the zebrafish. Finally, when zebrafish were cultivated with probe

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(10 μM) for 30 min, then added NEM (500 μM) for 30 min, GSH (200 μM)/NaSH (200 μM) for another
30 min, red fluorescence emerged inside the zebrafish. It implied that probe HZ-NBD could
successfully enter zebrafish and detect H2S/GSH via a turn-on fluorescence signal response.
Probe + NEM
Probe only
Probe + NEM + NaSH
Probe + NEM + GSH
a1
a2
a3
b1
b2
b3
c1
d1
c2
d2
c3
d3
Fig. 6 Fluorescence imaging of HZ-NBD responding to H2S/GSH in zebrafish. (a) the cells incubated with NEM (500 μM) for 30
min then incubated with probe (10 μM) for 30 min (b) the cells incubated with only probe (10 μM) for 30 min (c)/(d) the cells
incubated with NEM (500 μM) for 30 min then incubated with probe (10 μM) for 30 min, thereafter incubated with H2S (200
μM)/GSH (200 μM) for another 30 min, respectively. (λex = 480 nm, collected at the red channel (640-720 nm)).
4.Conclusion
In summary, a NIR emission fluorescent probe HZ-NBD to detect H2S and GSH that
contain isophorone-based fluorophore linked to a quenching group NBD was constructed. The
probe HZ-NBD showed high selectivity toward H2S/GSH via the sulfhydryl-promoted SNAr
reaction to produce the fluorophore HZ-1 and could react with H2S in a short time (within 7
min). Besides, the probe achieved the naked detection of H2S/GSH, corresponding to a colour
change from pink to purple red. Apart from that, the probe has many other merits such as a
relatively large stokes shift (λem – λabs = 112 nm), a wide pH range (7
～10), low cytotoxicity,
biocompatibility and low detection limit for H2S (13.2 nM) and GSH (112 nM). As a result, it
can be utilized to detect H2S/GSH in living cells and zebrafish. Taken together, the numerous
advantages of HZ-NBD make it a promising prospect in biological and clinical investigations.

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Acknowledgements
We thank the financial support from the Jiangsu Ainaji Neoenergy Science & Technology Co., Ltd.
(8507040091) and the Fundamental Research Funds for the Central Universities (3207045420).
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Highlight
1. Near infrared emission at 672 nm.
2. Distinguish H2S and GSH simultaneously over Hcy and Cys.
3. Fluorescence imaging in zebrafish/cells in vivo and vitro.
4. High sensitivity (13.2 nM for H2S, 112.0 nM for GSH) and selectivity.