A fluorescence turn-on probe for hydrogen sulfide and biothiols based on PET & TICT and its imaging in HeLa cells

Article abstract of DOI:10.1016/j.saa.2020.118839

In this paper, a photoinduced electron transfer (PET)& twisted intramolecular charge transfer (TICT)-based fluorescent probe (1) for detecting biothiols (GSH/Cys/Hcy) and hydrogen sulfide with fluorescence turn on was developed. The probe could recognize

Full text of DOI:10.1016/j.saa.2020.118839

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 244 (2021) 118839
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
journal homepage: www.elsevier.com/locate/saa
A fluorescence turn-on probe for hydrogen sulfide and biothiols based on
PET & TICT and its imaging in HeLa cells
a,
Xueqiong Zhang a, Xiaodong Jin a,b, Caiting Zhang a, Hui Zhong c, , Hongjun Zhu
⁎
⁎
a
Department of Applied Chemistry, College of Chemistry and Molecular Engineering, Nanjing Tech University, Nanjing 211816, China
b
Department of Criminal Science and Technology, Jiangsu Police Institute, Nanjing, Jiangsu 210031, China
c
Jiangsu Key Laboratory for Chemistry of Low-Dimensional Materials, School of Chemistry and Chemical Engineering, Huaiyin Normal University, Huaian 223300, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 30 June 2020
Received in revised form 1 August 2020
Accepted 10 August 2020
Available online 17 August 2020
In this paper, a photoinduced electron transfer (PET)& twisted intramolecular charge transfer (TICT)-based fluo-
rescent probe (1) for detecting biothiols (GSH/Cys/Hcy) and hydrogen sulfide with fluorescence turn on was de-
veloped. The probe could recognize hydrogen sulfide over primary ions and selectively detect GSH/Cys/Hcy over
other amino acids with fluorescence turn-on (an ESIPT process). H2S can be distinguished from GSH/Cys/Hcy
with wavelength shift by UV–Vis spectra. In addition, detection limits for H2S/GSH/Cys/Hcy of probe 1 were
1.42 μM (0–100 μM), 0.13 μM (0–40 μM), 0.27 (0–30 μM), 0.22 μM (0–40 μM), respectively. The proposed
thiolysis of the 2,4-dinitrochlorophenyl ether reaction in identification process was verified by the characteristic
peak in 1H NMR and HRMS spectra. Finally, the biological imaging experiments and low cytotoxicity investiga-
tions in HeLa cells demonstrated that probe 1 could provide a promising method for the determination of H2S
and biothiols in aqueous solution and living cells.
Keywords:
Chalcone
ESIPT
PET
Hydrogen sulfide
Biothiols
HeLa cells
© 2020 Published by Elsevier B.V.
1. Introduction
The current techniques for H2S and biothiols include
photoluminescence, electrochemical, colorimetry, chromatography,
As well known, hydrogen sulfide [1–3] plays many vital roles in
physiological and pathological processes. As an intracellular
gasotransmitter, H2S is useful in protecting heart and relaxing muscles
with 50–160 μM in the central nervous system and 10–100 μM in
blood [7,8]. The abnormal H2S level in vital organs may induce Down's
syndrome, Alzheimer's disease and diabetes [9,10]. Biothiols [4–6] also
occupy important positions in the series of vital processes. For example,
glutathione (GSH) is the most abundant biothiol with concentration at
2–20 μM in extracellular fluid and 2–10 mM in intracellular fluid, and
it acts an antioxitant to against toxins and free radicals. Exceptional
GSH level is related to Alzheimer's, diabetes, liver damage, cancers and
HIV infection [11,12]. Cysteine (Cys) can contribute in detoxification,
immunological competence, growth and protein synthesis at
3–200 μM. Deficiency of Cys has been proved to be linked to liver dam-
age, brain damage, hair loss, skin lesions and weakness [13–15]. Homo-
cysteine (Hcy) can promote cell proliferation and produce GSH with
concentration of 5–15 μM. Excessive Hcy level (>15 μM) has been re-
ported to be associated with myocardial infarction, stroke, venous
thromboembolism, vascular disease and Alzheimer's [16,17]. Therefore,
it is still a hot topic to detect and analyze hydrogen sulfide and biothiols
sensitively and selectively in biotics and physiology [18–21].
mass spectrometry and et al. [3,22–26]. When compared with other de-
tection techniques, fluorescent probes [27–30] have been reported for
photoluminescence materials which were performed excellently in
sensitivity, selectivity, simplicity, and operability. The special reaction
between fluorescent probes and mercapto compounds include nucleo-
philic substitution by thiol [31,32], cleavage reaction by mercapto group
[10,33], Michael addition [34], reduction reaction [35,36], thiolysis reac-
tion and et al. GSH/Cys/Hcy and H2S have the similar mercapto struc-
tural characteristics, so it is demanding to detect and discriminate
them [37,38]. On the other hand, it is still a challenge to provide a single
probe which could simultaneously distinct GSH/Cys/Hcy and H2S from
the new insight into multifaceted biological interaction.
The excited-state intramolecular proton transfer (ESIPT)-based dyes
[39–41] have been applied to design fluorescent probes for sensing
anions, cations, and small moleculars [41–44] in situ and vivo due to
its advantages like redder emission, larger Stock's shift and fluorescence
turn-on. It has been reported [41,45] that chalcone (compound 2) is an
ESIPT-active dye with red-emitting, this molecule has advantages like
easy synthesis, stable optical properties, hupotoxicity and et al. Inspired
by these virtues, chalcone was chosen as the fluorophore to construct
probe 1. Equipped with 2,4-dinitrochlorophenyl group as the receptor,
probe 1 presented fluorescence quenching by intramolecular PET&TICT
process. Probe 1 showed fluorescence turn on by an ESIPT process after
the thiolysis of the 2,4-dinitrochlorophenyl ether by H2S/thiols. And the
⁎
Corresponding authors.
E-mail addresses: huizhong@hytc.edu.cn (H. Zhong), zhuhj@njtech.edu.cn (H. Zhu).
https://doi.org/10.1016/j.saa.2020.118839
1386-1425/© 2020 Published by Elsevier B.V.

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X. Zhang et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 244 (2021) 118839
Scheme 1. Synthesis of probe 1
designed probe (1) showed high sensitivity and selectivity over other
primary ions and amino acids in the physiological environment. In addi-
tion, probe 1 could sense H2S in HeLa cells and had been proved low cy-
totoxicity in HeLa cells, MCF-10A and MCF-7 cells. All the results predict
probe (1) is a promising detection tool for H2S and biothiols (GSH/Cys/
Hcy).
2.2. Synthesis
Synthesis of 2′-hydroxy-4-(dimethylamino)chalcone (2). Com-
pound 2 was carried out according to the literature methods [44–47]
as a purplish solid.
Synthesis of 2′-(2,4-dinitro-phenoxy)-4-(dimethylamino) chalcone
(1). 2,4-Dinitrochlorobenzene (243 mg, 1.2 mmol), and compound 2
(267 mg, 1.0 mmol) was dissolved in 30 mL anhydrous dichlorometh-
ane under N2 atmosphere, then K2CO3 (138 mg, 1.0 mmol) was
added. The mixture was stirred at 30 °C for 12 h. The solution was con-
centrated under reduced pressure and the product was purified using
silica gel chromatography (petroleum ether: ethyl acetate = 10: 1 to
3: 1, v/v) to afford compound 1 as an orange solid (220 mg, yield:
51%). 1H NMR (400 MHz, CDCl3) δ 8.80 (d, J = 2.7 Hz, 1H), 8.26 (dd,
J = 9.3, 2.7 Hz, 1H), 7.84–7.77 (m, 1H), 7.61 (dd, J = 7.7, 1.5 Hz, 1H),
7.53–7.39 (m, 4H), 7.20 (d, J = 8.1 Hz, 1H), 7.06 (d, J = 15.6 Hz, 1H),
6.89 (d, J = 9.3 Hz, 1H), 6.65 (d, J = 8.9 Hz, 2H), 3.04 (s, 6H). 13C
NMR (100 MHz, CDCl3) δ 190.30, 156.51, 152.41, 150.67, 141.26,
138.56, 133.89, 132.97, 131.21, 130.95, 129.01,127.17, 122.24, 122.06,
121.78, 119.36, 118.06, 111.75, 77.37, 77.06, 76.74. HRMS (ESI, m/z)
calcd for C23H20N3O+6 [M + H]+: 434.13466, found: 434.13431
(Scheme 1).
2. Experimental
2.1. Materials and instrumentations
All chemicals used in this paper were obtained from Tansoole with-
out further purification. Silica gel (300–400 mesh, Qingdao Haiyang
Chemical Co.) was used for column chromatography. Dichloromethane
was processed with CaH2 and stored with 4 Å molecular sieves.
1H NMR, and 13C NMR spectra were measured on a Bruker AV-400
spectrometer with chemical shifts reported in ppm (in CDCl3 or d6-
DMSO, TMS as internal standard). UV–Vis spectra were acquired on a
Hewlett-Packard 8453 diode-array spectrometer. All fluorescence mea-
surements were recorded on a Hitachi-F-4600 fluorescence spectropho-
tometer. HI 2221 calibration check pH/ORP meter was used to measure
the pH of phosphate buffer.
Probe 1 GSH
Hcy
NaHS
458 nm
Cys
433 nm
1000
800
600
400
200
0
B
A
1.0
420 nm 426 nm
442 nm
Probe 1
Probe 1
Probe 1+GSH
Probe 1+Hcy
Probe 1+Cys
Probe 1+NaHS
Probe 1+GSH
Probe 1+Hcy
Probe 1+Cys
Probe 1+NaHS
0.8
0.6
0.4
0.2
0.0
500
550
600
650
700
400
450
500
550
Wavelength/nm
Wavelength/nm
Fig. 1. (A) Absorption spectra of probe 1 (50 μM) in the absence and presence of NaHS/GSH/Cys/Hcy (500 μM) in DMSO. (B) Fluorescence spectra of probe 1 (50 μM) toward NaHS/GSH/
Cys/Hcy (500 μM). λ ex = 480 nm, d ex = 5 nm, d em = 5 nm.

X. Zhang et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 244 (2021) 118839
3
Scheme 2. Luminous mechanism of probe 1 and compound 2.
3. Results and discussion
addition, the fluorescence intensity of probe 1 at 600 nm in DMSO-
PBS solution (5:5, v/v, pH = 7.40) remained stable for 0–72 h (as
shown in Fig. S2). Obviously, probe 1 performs high stability and
ought to be an applicable tool in physiological.
3.1. UV–Vis and fluorescence measurements for probe 1 toward NaHS and
thiols
Next, the time-dependent experiment was studied to monitor the
reaction dynamics between probe 1 (50 μM) and NaHS/GSH/Cys/Hcy
in DMSO-PBS (5:5, v/v, pH = 7.02, 12.5 mM) to evaluate the detection
time of reaction-based probes. Upon the addition of 10 equiv. of
NaHS/GSH/Cys/Hcy, respectively, the emission intensity of probe 1 at
600 nm increased gradually due to the ESIPT progress (Fig. 3). And the
Fig. 1(A) displayed the maximum absorption of probe 1 (50 μM) at
420 nm in DMSO at room temperature. Upon adding NaHS or biothiols,
new absorption bands appeared and centered at 458 nm (NaHS),
426 nm (GSH), 433 nm (Cys) and 442 nm (Hcy), respectively. Hence,
H2S and GSH/Cys/Hcy can be distinguished by the absorption spectra.
In the emission spectra, probe 1 behaved a weak emission peak at
575 nm (as shown in Fig. 1(B), λex = 480 nm), which might be due to
a
photoinduced electron transfer (PET) process with the 2,4-
Probe 1
dinitrophenyl moiety acting as the electron acceptor, and N, N-
dimethylaniline group as the electron donor (as seen in Scheme 2).
After adding NaHS and GSH/Cys/Hcy, the maximum emission of probe
1 red-shifted to 600 nm with obvious fluorescence enhancement,
which may be attributed to transformation between the normal form
and keto form of compound 2, respectively. The generation of com-
Probe 1+NaHS
800
600
400
200
pound
2 could be ascribed to the thiolysis of H2S and the
thiol-induced nucleophilic substitution and rearrangement reaction.
All these results demonstrate that probe 1 has the potential for being
an efficient spectroscopic probe for H2S and biothiols (GSH/Cys/Hcy).
For evaluating the application of probe 1 in physiological condition,
water content experiment and pH-response experiment were carried
out by fluorospectro photometer. The fluorescent intensity of probe 1
nearly unchanged with variation of water content and pH value.
While, water content reduction caused intensity increasing after
reacting with NaHS (see Fig. S1). This may be ascribed the excessive
water proportion would induce solid aggregated, therefore 50% water
content was selected in the spectra test. As seen in Fig. 2, the probe 1 be-
haved stably in DMSO-PBS solution with weak acid, however, neutral,
weak base, and the strong base would disturb the detection effect. In
4
5
6
7
8
9
10
pH
Fig. 2. Fluorescence spectra (λ = 600 nm) of probe 1 (50 μM) upon addition of 10 equiv. of
NaHS at different pH values in DMSO-PBS (5:5, v/v).

4
X. Zhang et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 244 (2021) 118839
2000
1500
1000
500
0
B
Probe 1
A
Probe 1+NaHS
Probe 1+GSH
Probe 1+Hcy
Probe 1+Cys
600
400
200
0
0
40
80
Time/min
120
160
0
20
40
Time/min
60
Fig. 3. Time-dependent fluorescence intensity (λ = 600 nm) changes of probe 1 (50 μM) upon addition of 10 equiv. of NaHS (A) and GSH/Cys/Hcy (B) in DMSO-PBS (5:5, v/v, pH = 7.40,
12.5 mM) at room temperature.
fluorescence intensity reached an optical stabilization platform after
100 min with NaHS, and 60 min with biothiols (GSH/Cys/Hcy),
respectively.
0.12 μM, 0.10 μM, respectively. The valid analyzing ranges of GSH/Cys/
Hcy are within the physiological ranges. The sensitivity tests indicated
that the chemodosimeter (probe 1) could detect biothiols (GSH/Cys/
Hcy) quantitatively at the physiological level by the fluorescence spec-
troscopy method.
3.2. Sensitive optical response of probe 1 toward H2S and biothiols
To assess the possibility of simultaneous detection for H2S and GSH/
Cys/Hcy, the UV–Vis and fluorescence of mixture of H2S and GSH with
probe 1 were investigated. Keeping the total concentrations of H2S
and GSH consistent, as the [H2S]/[GSH] ratio increased, the maxinum
absorption wavelength red-shifed from 426 nm to 458 nm with absorp-
tion increased (Fig. S3). In addition, as the [H2S]/GSH ratio increased, the
fluorescence intensity gradually decreased with a linear response
(Fig. S4, R2 = 0.9924). Therefore, the concentration of H2S can be mea-
sured by UV–Vis absorption at 458 nm, and the concentration of GSH
can be determined by emission intensity at 600 nm. Take advantages
of UV–Vis and emission spectra, probe 1 can detect GSH and H2S quan-
titatively when they coexist, probe 1 would be a potential tool in biolog-
ical samples.
Titration experiments were carried out by gradually adding NaHS to
evaluate the quantitative analysis of probe 1. The emission spectra were
collected after reacting completely. As shown in Fig. 4, the fluorescent
intensity at 600 nm was gradually enhanced with the addition of
NaHS, and exhibited an excellent linear relationship of emission inten-
sity with NaHS concentration (0–100 μM) in the DMSO/PBS (5:5, v/v)
solution (R2 = 0.9926). These concentrations are well within the
range that has been used to elicit physiological responses. The detection
limit was 1.42 μM based on 3σ/k (where σ is standard deviation of the
blank solution for 20 samples, and k is the slope of the calibration
curve).
Next, the thiolysis sensitivity of probe 1 toward different biothiols
(GSH, Cys, and Hcy) was also investigated to evaluate the quantitative
analysis of probe 1. It was found that there was an obvious fluorescence
turn-on after addition of amounts of GSH/Cys/Hcy at 600 nm. As shown
in Fig. 5, the fluorescent intensity showed a good linear relationship
with GSH/Cys/Hcy in the DMSO/PBS (5:5, v/v) solution. The linear
range are 0–40 μM for GSH (Y = 87.0105 + 21.8200X, R2 = 0.9965),
0–30 μM for Cys (Y = 71.4216 + 10.7624X, R2 = 0.9923), 0–40 μM
for Hcy (Y = 78.0580 + 13.3572X, R2 = 0.9939), respectively. The de-
tection limits of GSH, Cys, and Hcy were calculated to be 0.06 μM,
3.3. Selective optical response of probe 1 to various analytes and
amino acids
To evaluate the selectivity of probe 1 for NaHS, the fluorescence
spectra with sulfides (like S2O23−, SO32−, GSH, Hcy, Cys, S2O82−, HSO−3 ),
cations (Cr3+, Al3+, Zn2+, Ni+, Mn2+, etc) and anions (ClO−4 , I−, Br−
,
Cl−, H2PO−4 , PO34−, etc) were tested. As shown in Fig. 6(A), 1 displayed
negligible changes in emission intensity (at 600 nm) upon adding 10
200
150
100
50
B
200
150
100
50
A
NaHS
Probe 1+NaHS
0-100 μM
0
500
550
600
650
700
0
20
40
60
80
100
Wavelength/nm
Concentration/μM
Fig. 4. (A) Fluorescence spectra of probe 1 (50 μM) upon adding NaHS (0–100 μM). (B) The fluorescence intensity (λ = 600 nm) of probe 1 were linearly related to the concentration of
NaHS (0–100 μM) (R2 = 0.9926, CDL = 0.43*3/0.91 = 1.42 μM).

X. Zhang et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 244 (2021) 118839
5
1000
800
600
400
200
0
A
B1000
Probe 1+GSH
Probe 1+ GSH
0-40 μM
800
600
400
200
0
550
600
650
700
750
750
750
0
10
20
30
40
Wavelength/nm
Concentration/μM
400
350
300
250
200
150
100
50
C
500
400
300
200
100
0
D
Probe 1+Cys
Probe 1 + Cys
0-30 μM
0
550
600
650
700
0
5
10
15
20
25
30
Wavelength/nm
Concentration/μM
800
600
400
200
0
E
F
Probe 1+Hcy
Probe 1+ Hcy
0-40 μM
600
400
200
0
550
600
650
700
0
10
20
30
40
Wavelength/nm
Concentration/μM
Fig. 5. Titration fluorescence spectra of probe 1 (50 μM) upon addition of 10 equiv. of GSH (A, B), Cys (C, D), and Hcy (E, F) and intensity (λ = 600 nm) of probe 1 (50 μM) upon addition of
biothiols in DMSO-PBS (5:5, v/v, pH = 7.40, 12.5 mM) at room temperature.
equiv. of these interfering analytes except H2S, GSH, Hcy, and Cys. The
competitive experiments showed the fluorescent detection of H2S was
obstructed by metal ions (Zn2+, Cd2+, Hg2+) due to their complexation
toward S2−. Zn2+ is a kind of essential trace elements [48–51] in living
body, its interference in H2S detection needs to be taken seriously.
Meanwhile, according to the Ksp of ZnS, the concentrations of Zn2+ in
physiological condition is too low to interfere the H2S detection and
not enough to influence biological test when H2S and Zn2+ coexist.
Moreover, Cd2+ and Hg2+ are heavy metal ions, they are generally not
competitive experiments were also investigated by adding respective
biothiols (GSH/Hcy/Cys) to the mixture solution of probe 1 with
amino acids. The results indicated that the detection of GSH/Cys/Hcy
could survive possible interference from amino acids. All the results
demonstrated that probe 1 possesses a high selectivity toward hydro-
gen sulfide and biothiols (GSH/Hcy/Cys).
3.4. Mechanism verification of probe 1 for H2S
presented in living body. Therefore, the three metal ions (Zn2+, Cd2+
,
To validate the posed detection mechanism for H2S, the 1H NMR
spectra of probe 1 in d6-DMSO with or without NaHS at room tempera-
ture (Fig. 7) were tested. The hydroxyl hydrogen Ha (at ~13.20 ppm)
and aromatic proton Hb (at ~8.30 ppm), Hc (at ~6.95 ppm) and Hd (at
~6.75 ppm) appeared after adding NaHS in probe 1 solution, this phe-
nomenon indicated the cleavage of ether bond. The released
fluorophore compound 2 was also found by HRMS (calcd for
[M + H]+: 268.13321, found: 268.13348, Fig. S5). In addition, the
Hg2+) are negligible to disturb the H2S detection. These results could
suggest other species caused insignificant effects on H2S detection.
Next, probe 1 was subject to react with amino acids (Arg, Trp, Ile,
Pro, etc) that have a hydrosulfonyl and the structures were similar to
GSH/Hcy/Cys. The addition of 10 equiv. of amino acids exhibited almost
no changes with probe 1 in emission behaviour (Fig. 6(B), red column).
As seen in Fig. 6(B) (green, dark blue, and cerulean column), the

6
X. Zhang et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 244 (2021) 118839
800
600
400
200
0
A
Probe 1+analytes
Probe 1+analytes+NaHS
2
4
6
8 10 12 14 16 18 20 22 24 26
Analytes
Fig. 6. (A) Represents the addition of NaHS to the above analyte-containing solutions (bars show: 1. ClO−4 , 2. I−, 3. Br−, 4. Cl−, 5. H2PO−4 , 6. PO43−, 7. HSO4−, 8. Cr3+, 9. Al3+, 10. Zn2+, 11. Ni+
12. Mn2+, 13. Fe3+, 14. Cd2+, 15. Mg2+, 16. Hg2+,17. Thiourea, 18. Et3N, 19. Ethylenediamine, 20. S2O32−, 21. SO23−, 22.GSH, 23. Hcy, 24. H2O2, 25. t-BuOOH, 26. S2O28−, 27. NO2−, 28. HSO−3
,
,
29. Cys, 30. blank). (B) Represents the addition of GSH/Cys/Hcy to the amino acids.
wavelengths changed in UV–Vis spectra can be ascribed to the forma-
tion of 1-GSH, 1-Cys and 1-Hcy in recognition process (Fig. S6) when
responded to GSH/Cys/Hcy, and they had been verified by HRMS
(Fig. S7).
Fig. 8, the electron density localizes on the 2, 4-dinitrophenyl group in
the lowest unoccupied molecular orbital (LUMO) and the π electrons fo-
cused on the N, N-dimethylaniline group in the highest occupied molec-
ular orbital (HOMO), we suppose it could be corresponded with PET
[52,53] of probe 1. And the intramolecular rotation of 2, 4-
dinitrophenyl group in probe 1 (Fig. S8) is 67.6°, which is helpful to re-
sult in fluorescence quenching with TICT pattern [54,55]. While, the
electron intensity in the HOMO and LUMO of compound 2 spread over
the whole skeleton and the molecule structure is in the same plane
3.5. Theoretical calculation
To better understand the luminous mechanism, probe 1 and com-
pound 2 were examined by density function theory (DFT). As seen in
Fig. 7. The 1H NMR spectra of probe 1+ NaHS, probe 1+ compound 2, probe 1 and compound 2 only in d6-DMSO. [1] = [2] = 4.0 × 10−2 M, [NaHS] = 4.0 × 10−1 M.

X. Zhang et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 244 (2021) 118839
7
Fig. 8. Structural optimization and HOMO, LUMO orbitals of probe 1 and compound 2 by B3LYP/6-31G*.
which disturbed the TICT process. In addition, the alkenyl group and
keto is an excellent condition to form ESIPT form [38,56]. All these re-
sults make the recognition process showing a fluorescence turn-on.
The predicted mechanism of probe 1 toward RSH has been presented
in Fig. 9.
cytotoxicity. These results showed the developed probe 1 has the ability
as an excellent detecting tool to recognize H2S in living cells.
4. Conclusion
In this paper, a chalcone-probe (1) on the basis of 2, 4-dinitrophenyl
ether reaction has been synthesized and characterized. Probe 1 shows a
distinct fluorescence turn-on after adding H2S/GSH/Cys/Hcy by the
transformation from PET&TICT to ESIPT. Probe 1 possesses a high selec-
tivity and sensitivity toward both H2S and GSH/Cys/Hcy. Furthermore,
the application of probe 1 in HeLa cells indicates that probe 1 has
great potential for hydrogen sulfide and biothiols (GSH/Cys/Hcy) in bi-
ology and pathology.
3.6. Imaging of probe 1 in living cells
The above solution experiments revealed that probe 1 could detect
with high selectivity and sensitivity. In addition, the potential applica-
tion of probe 1 in HeLa cells was examined, and probe 1 (50 μM) showed
quite weak fluorescence (Fig. 9A) after incubating with HeLa cells for
30 min at 37 °C, the weak fluorescence revealed that probe 1 has suc-
cessfully infiltrated the living cells for intracellular H2S/biothiols
(Fig. S9). Next, the HeLa cells were continuously incubated with
200 μM and 500 μM NaHS, for another 2.5 h. Then, an obvious fluores-
cence intensity increase in HeLa cells (Fig. 9B and C) was observed,
which were attributed to the release of compound 2 via the thiolysis
of ether linkage. Moreover, the cytotoxicity of probe 1 was assessed
with various living cells (HeLa cells, MCF-7, MCF-10A, Fig. S10–12) by
MTT assays, the results presented that probe 1 has quite low cell
CRediT authorship contribution statement
Xueqiong Zhang: Methodology and Synthesis, Optical measure-
ment and data analyses, Writing- Original draft preparation;
Xiaodong Jin: Supervision, Writing- Reviewing and Editing;
Caiting Zhang: Optical measurement, Writing- Reviewing and
Editing;

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X. Zhang et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 244 (2021) 118839
Fig. 9. Images of probe 1 (50 μM) in HeLa cells with NaHS at 37 °C for 2.5 h at green channel (A-C, A, 0 μM; B, 200 μM; C, 500 μM, λ ex = 490 nm), (D-F), Bright-field.
[6] H. Zhang, L. Xu, W. Chen, et al., A lysosome-targetable fluorescent probe for simul-
Zhong Hui: Supervision and Funding acquisition.
Hongjun Zhu: Supervision and Funding acquisition.
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3 (2018) 2513–2517.
[7] B. Olas, Hydrogen sulfide in signaling pathways, Clin. Chim. Acta 439 (2015)
212–218.
Declaration of competing interest
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The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgements
This research was supported by the National Key Research and De-
velopment Program of China (2016YFB0301703), the National Natural
Science Foundation of China (21775051, 21375044), Natural Science
Foundation of Jiangsu Province (BK20191416), and the High-level In-
troduction of Talent Scientific Research Start-up Fund of Jiangsu Police
Institute (JSPI17GKZL402).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.saa.2020.118839.
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