Q. Sun, H. Liu, Y. Qiu et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 254 (2021) 119620
cystathionine b-synthase (CBS) and 3-mercaptopyruvate sulfur-
transferase (3-MST)/cysteine aminotransferase (CAT) in various
tissues and organs [6–9]. In addition, H2S is closely related to var-
ious physiological pathways, such as inflammation, apoptosis,
vasodilation, angiogenesis, neuromodulation, insulin signaling,
oxygen sensing and ischemia reperfusion injury [10–14]. Further-
more, accumulating evidence confirms that the imbalance level
of H2S will lead to a series of diseased such as liver cirrhosis, Down
syndrome, Alzheimer’s disease, diabetes, cirrhosis and heart dis-
ease [15–18]. Nevertheless, these possible molecular events of
H2S and its specific mechanisms are still unclear. Therefore, in
order to investigate H2S biology and H2S-related diseases, efficient
technology and method for visualization of H2S in cells and animal
model is an imperative solving problem.
diluted from the stock solution. The excitation wavelength of all
fluorescence spectra was 450 nm.
Limit of detection. The limit of detection (LOD) was calculated
depending on the following formula (1), which was reported in
the previous literatures [31–33]. The fluorescence spectrums of
probe NT-SH were measured for 10 times to obtain the standard
deviation
r. The linear relationship between the fluorescence
intensity (550 nm) and the dose of NaHS was measured to get
the slop k.
LOD ¼ 3
r
=k
ð1Þ
Quantum yield. The fluorescence quantum yields of NT-SH and
NT were detected according to following Eq. (2) based on the
reported literatures [34–36]. The subscripts c and s represented
Fluorescence probe detection technology are the most powerful
method for the detection of H2S in complicated biological systems
owing to its simple, rapid response, good sensitivity, high sensitiv-
ity and non-invasive [19–21]. To date, a great quantity of fluores-
cent probes have been constructed and applied to the detection
of intracellular H2S [22,23]. These fluorescent probes for H2S are
designed based on three mechanisms including reduction of azides
and nitro/azanol to amines, nucleophilic reaction of Michael addi-
tion and formation of copper sulfide precipitation [24–30].
Although many great successes of fluorescent probes for H2S have
been reported, only few of them are used for real-time imaging and
sensing of H2S in vivo (Table S1).
Herein, we rationally constructed a fluorescent turn-on probe
NT-SH for sensitive and selective sensing of H2S in vitro and
in vivo. The construction of NT-SH contained two parts: the naph-
thalimide part was acted as the fluorophore and the 2,4-
dinitrobenzenesulfonyl (DNBS) part was acted as the quenching
group via photoinduced electron transfer (PET) effect and recogni-
tion group for reaction with H2S, as illustrated in Scheme 1. NT-SH
exhibited excellent water solubility, low fluorescence background
and good biocompatibility due to the obvious advantages of naph-
thalimide fluorophore. More importantly, NT-SH was applied for
sensing and visualizing of H2S in A549 cells and zebrafish with
low fluorescence background and high sensitivity, which revealed
NT-SH had the potential ability for imaging and detection of H2S
in vitro and in vivo.
fluorescein (
U = 0.98) and sample respectively. U was the quan-
tum yield of the reference compound and samples. The subscripts
A represented the absorbance and the subscripts F mean the fluo-
rescence integral area.
Fs ꢁ A
Us
¼
c Uc
ð2Þ
Fc ꢁ As
Cell Imaging. A549 cells were cultured in RPMI-1640 complete
medium including penicillin and streptomycin on a cell culture
flask in the cell incubator. A549 cells were initial digested by tryp-
sin, and then cultured overnight on a 12-well plate. After incuba-
tion with NT-SH (10
three times to remove the remaining NT-SH. Then, various doses
of NaHS (0, 25, 50 and 100 M) were adding into the A549 cells
for next 30 min. Finally, the A549 cells was imaged at green chan-
nel (510–550 nm) by the Olympus IX71 microscope.
Zebrafish Imaging. zebrafish was cultured in 100 mL medium
containing 1-phenyl-2-thiourea (PTU) at 28 °C for 48 h. For the
imaging experiments, 10 lM NT-SH was adding into the cultured
medium of zebrafishes at 28 °C for 1 h. Then, the medium contain-
ing NT-SH was carefully removed to reduce the fluorescence back-
ground. And the zebrafish was imaged directly as the control
group. In the experimental groups, these zebrafishes were further
co-cultured with various doses of NaSH (0, 25, 50, 100 lM) for
2 h. Finally, all the zebrafishes were imaged at green channel
lM) for 30 min, the A549 cells were washed
l
(510–550 nm) by Olympus SZX16 microscope.
2. Materials and methods
3. Results and discussion
2.1. Materials and instruments
3.1. Design and synthesis of probe NT-SH
All the reagents used in the experiments were analytical grade
reagents obtained from Sigma-Aldrich. These reagents could be
directly used without further purification. The A549 living cells
and zebrafish larvae were purchased from China Zebrafish
Resource Center (Wuhan city of China). The PBS buffers with differ-
As we know, naphthalimide was a widely used fluorophore due
to its practical advantages, such as large Stokes’ shift, high quan-
tum yield, favorable water solubility and biocompatibility
[37,38]. Base on this, a water-soluble fluorescence probe NT-SH
for sensing of H2S was constructed by connecting the recognition
group DNBS and the fluorophore NT via a simple nucleophilic reac-
tion, as shown in Scheme 1. NT-SH probe showed a low fluores-
cence background due to the PET effect of DNBS group. while the
breaking sulfonate bond by H2S produced fluorescence turn-on
phenomenon through the released of fluorophore. To evaluate
the response performance of NT-SH for H2S, NT-SH and NT were
synthesized according to the synthetic route, as displayed in
Scheme S1. These synthesized compounds were identified by 1H
NMR, 13C NMR and HR-MS in the Support Information, as shown
in Figs. S1-S9.
ent pH were prepared by STARTER 3100C pH meter. The 1H and 13
C
NMR spectrums were tested using a Varian Inova 500 NMR Spec-
trometer. The UV–vis and fluorescence spectra were recorded by
multifunctional enzyme labeling instrument (SpectraMax M5,
USA). The fluorescence images of A549 cells and zebrafishes were
performed by inverted fluorescence microscopy (Olympus IX71,
Japan).
General Optical Measurements. 10 mM NT-SH was dissolved in
DMOS as the stock solution. The PBS buffers with different pH (4.0,
5.0, 6.0, 7.0, 7.4, 8.0 and 9.0) were prepared for testing the pH
effect. Various analytes (100
l
M) including inorganic metal salt
Spectra response of NT-SH toward H2S. As shown in Fig. S10, the
pKa1 and pKa2 of hydrogen sulfide are calculated to be 6.88 and
14.15, respectively. Therefore, under the test condition (pH = 7.4),
the main forms of H2S are HS- and S2- in vitro and in vivo. Hence,
NaHS can be used as a source of H2S. To verify the response capa-
(FeSO4, HgCl2, CuCl, BaCl2, MnSO4, NaNO3, NaF, NaI, NaCl, CH3-
COONa), and amino acids (Leu, Cys, Asp, Arg) were prepared to
evaluate selectivity. All the test concentrations of NT-SH used in
the spectra experiment and bioimaging were 10 lM, which were
2