Dual-functional multi-application probe: Rapid detection of H2S and colorimetric recognition of HSO3? in food and cell

Article abstract of DOI:10.1016/j.dyepig.2020.108656

Since hydrogen sulfide (H₂S) and bisulfite (HSO₃^?) are widely used in the biological environment and industrial production, many fluorescent probes have been developed to detect them individually. However, designing and sy

Full text of DOI:10.1016/j.dyepig.2020.108656

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Dual-functional Multi-application Probe: Rapid Detection of H S and Colorimetric
2
-
Recognition of HSO in Food and Cell
3
Keli Zhong, Lin Chen, Xiaomei Yan, Yiwei Tang, Shuhua Hou, Xuepeng Li, Lijun Tang
PII:
S0143-7208(20)31353-X
DOI:
https://doi.org/10.1016/j.dyepig.2020.108656
DYPI 108656
Reference:
To appear in: Dyes and Pigments
Received Date: 14 February 2020
Revised Date: 24 April 2020
Accepted Date: 17 June 2020
Please cite this article as: Zhong K, Chen L, Yan X, Tang Y, Hou S, Li X, Tang L, Dual-functional Multi-
-
application Probe: Rapid Detection of H S and Colorimetric Recognition of HSO in Food and Cell,
2
3
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-
H2S and Colorimetric Recognition of HSO3 in Food and Cell”. (ID:
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Date signed:
2020.4.22

TOC
Dual-functional Multi-application Probe: Rapid Detection of
-
H S and Colorimetric Recognition of HSO in Food and Cell
2
3
a
a
b
c
a
a
Keli Zhong, Lin Chen, Xiaomei Yan, Yiwei Tang, Shuhua Hou, Xuepeng Li,* and
a
Lijun Tang*

Dual-functional Multi-application Probe: Rapid Detection of
-
H S and Colorimetric Recognition of HSO in Food and Cell
2
3
a
a
b
c
a
a
a
Keli Zhong, Lin Chen, Xiaomei Yan, Yiwei Tang, Shuhua Hou, Xuepeng Li,* and Lijun Tang*
a. College of Chemistry and Chemical Engineering, College of Food Science and Technology, Bohai
University; Food Safety Key Lab of Liaoning Province; National & Local Joint Engineering
Research Center of Storage, Processing and Safety Control Technology for Fresh Agricultural and
Aquatic Products; Bohai University, Jinzhou, 121013, China.
E-mail: lixuepeng@bhu.edu.cn (X. Li), ljtang@bhu.edu.cn (L. Tang)
b. College of Laboratory Medicine, Dalian Medical University, Dalian, 116044, China
c. College of Food Science and Technology, Agricultural University of Hebei, Baoding 071001,
China

-
Abstract: Since hydrogen sulfide (H S) and bisulfite (HSO ) are widely used in the
2
3
biological environment and industrial production, many fluorescent probes have been
developed to detect them individually. However, designing and synthesizing a sensor
-
that recognizes both H S and HSO remains a challenge. In this study, we developed a
2
3
-
new dual-function probe TCF-DNs for the dual channel detection of H S and HSO in
2
3
EtOH/H O (2/8, v/v, HEPES 10 mM, pH=7.4). The color of TCF-DNs changed from
2
lilac to light-blue on treatment with H S, and a visible color change from lilac to
2
-
colorless occurred on treatment with HSO as well. The easy-to-synthesize TCF-DNs
3
can recognize H S through rapid near-infrared fluorescence enhancement with a
2
detection limit of 13.5 nM, however, the probe exhibits fluorescence quenching toward
-
HSO . The sensing mechanism of TCF-DNs is based on the removal of
3
-
2
,4-dinitrobenzenesulfonate unit by HS and the occurrence of a nucleophilic addition
-
for HSO . In addition, the application study indicates that probe TCF-DNs can detect
3
-
HSO in granulated sugar, and sensitively detect H S in red wine, real water, living
3
2
cells. Meanwhile, TCF-DNs can be used to prepare a cheap test paper strip for the rapid
and qualitative detection of H S by the naked eye and to serve as an indicator to detect
2
H S gas.
2
Keywords: Hydrogen sulfide; Bisulfite; Fluorescent probe; Dual-function; Imaging;
Multi-application

Introduction
Hydrogen sulfide (H S), an irritating gas, can lead to difficulty breathing, rapid entry
2
into a coma and death due to respiratory paralysis when there appears acute
poisoning.[1, 2] As one of the endogenous signal molecules, H S plays a vital role in
2
neuromodulation, apoptosis processes.[3-5] Furthermore, H S, deemed to be a common
2
ingredient in the fermentation of wine, will cause a decline in the quality of wine and
give rise to huge economic losses as well as food safety issues.[6, 7] It’s worth noting
that H S is difficult to analyze accurately because the fast decomposition of H S will
2
2
cause fluctuations in concentration.[8-10] Hence, there is an urgent need for fast
response and highly selective detection of H S.
2
-
Bisulfite (HSO ), with the similar nucleophilic and reducing reactivity of H S, is
3
2
widely used as a food bleaching agent, an inhibitor of enzymatic and non-enzymatic
browning, and a very effective antibacterial agent in the sugar industry.[11-16] The
-
HSO levels in food are strictly limited in many countries. For instance, according to
3
the National Standards of the People’s Republic of China (GB 2760), the residual
-
amount of HSO in sugar (calculated as SO ) shall not exceed 0.1 g/kg. Therefore, new
3
2
-
analytical methods available for the detection of HSO levels in foods are important for
3
the safety of the consumers.[17]
To date, although there are many traditional and classic methods to detect H S and
2
-
HSO , the fluorescence method is more popular because of the advantages of high
3
sensitivity, time-saving and real-time imaging etc.[18-24] At present, abundant
-
small-molecule fluorescent probes for the separate sensing H S or HSO has been
2
3

widely reported.[25-32] Due to the dual-functional probe can reduce the synthetic
workload, save the cost, and avoid spending a lot of time exploring the test conditions,
it is more and more favored by people.[33] But the similar nucleophilicity and
-
reducibility of H S and HSO , it is difficult to detect two analytes using one
2
3
probe.[34-37] Only a few instances have been found, for example, Zhang et al.
developed a new coumarin-based fluorescent probe detecting sulfite and sulfide in
CTAB micelle and differential imaging in living cells in 2014.[38] Wang group
constructed a bifunctional probe with sensitive fluorescence response to H S and
2
colorimetric recognition sulfite in PBS containing 10% DMSO (v/v), and the probe can
image endogenous and exogenous H S in live cells in 2016.[39] Yang et al. reported a
2
dual-site fluorescent probe for separate detection of H S in real water samples and
2
bisulfite in sugar and water samples in 2019.[40] Although these probes exhibited good
selectivity, satisfactory sensitivity and potential applications, they generally show the
slow response rate to detect H S and complex synthesis process as well as poor
2
application in the food sample. Therefore, construction of an easy-to-prepare
dual-functional probe that can rapidly recognize H S and respectively detect H S and
2
2
-
HSO in food has attracted our attention.
3
In our previous work, we used TCF-1 as a fluorophore and synthesized a
near-infrared (NIR) emission fluorescent probe for exclusive recognition of H S through
2
-
the thiolysis of 2,4-dinitrophenyl (DNP) ether triggered by HS to release TCF-1,
involving eliminating the d-PET and recovering the ICT process.[41] The obtained
fluorescent probe displays high selectivity and sensitivity to H S with a simple synthetic
2

process, NIR emission and multi-application (Scheme1). In this work, utilizing the same
strategy, we adopted 2,4-dinitrobenzenesulfonyl (DNs) to replace DNP and synthesize
probe TCF-DNs (Scheme 2). It’s worth noting that the strongly electron-withdrawing
DNs group, as a functional trigger moiety by nucleophilic thiolate anion, constantly
been used to design thiol probes. Unexpectedly, probe TCF-DNs cannot recognize thiol,
but can quickly sense H S (2.5 min) through colorimetry and fluorescence, and has an
2
-
excellent colorimetric response to HSO . What is more, the thiols don’t interfere with
3
-
the recognition of H S/HSO by probe TCF-DNs. In addition, application research
2
3
shows that TCF-DNs can detect H S in real water, red wine and living cells and sense
2
-
HSO in sugar. TCF-DNs can be used to prepare a cheap test paper strip for the rapid
3
and qualitative detection of H S by the naked eye and to serve as an indicator to detect
2
H S gas. As far as we know, the dual-functional multi-application probe with rapid
2
-
detection of H S and colorimetric recognition of HSO in food and cell was rarely
2
3
reported.
Scheme 1. Dual functional fluorescent probe in this work.

Experimental section
Synthesis of TCF-DNs
Compound TCF-1 (374.4 mg, 1.0 mmol), triethylamine (151.5 mg, 1.5 mmol),
2
,4-dinitrobenzenesulfonyl chloride (319.9 mg, 1.2 mmol) were dissolved in dry
CH Cl (15 mL) under ice bath, and stirred for 12h at ambient temperature. Then the
2
2
solvent was removed by vacuum distillation, the crude product was purified by column
chromatography using ethyl acetate/petroleum ether (1/3, v/v) as eluent to give
o
1
TCF-DNs as purple solid (326.4 mg, 54.0%). m.p. 243.6-244.0 C; H NMR (400 MHz,
DMSO-d ) δ 9.15 (d, J = 2.2 Hz, 1H), 8.70 (dd, J = 8.8, 2.2 Hz, 1H), 8.42 (d, J = 8.8 Hz,
6
1
H), 8.05 (d, J = 9.3 Hz, 1H), 7.93 (d, J = 15.9 Hz, 1H), 6.95 (d, J = 15.9 Hz, 1H), 6.87
(dd, J = 9.3, 2.5 Hz, 1H), 6.31 (d, J = 2.5 Hz, 1H), 3.39 (q, J = 7.2 Hz, 4H), 1.69 (s, 6H),
1
3
1
1
.03 (t, J = 7.2 Hz, 6H). C NMR (101 MHz, DMSO-d ) 177.9, 175.4, 152.4, 152.0,
6
50.7, 148.5, 140.5, 134.1, 132.0, 131.5, 129.1, 128.1, 121.6, 114.3, 113.6, 112.7, 112.4,
+
1
11.1, 104.8, 98.9, 94.6, 52.7, 45.0, 25.6, 12.7. HRMS (ESI ) calcd for
+
+
C H N NaO S [M+Na] : 627.1269, found:627.0507.
28
24
6
8
General procedure for spectra measurement
The 50 mM salt stock solutions of various anions were prepared using redistilled
water. The 10 μM TCF-DNs solution in EtOH/H O (2/8, v/v, HEPES 10 mM, pH= 7.4)
2
was prepared using the 1 mM stock solution of TCF-DNs in DMSO. UV-Vis absorption
spectra were evaluated using 20 μM solution of TCF-DNs. For the detection of H S and
2
-
HSO , the resulting solution of TCF-DNs was incubated separately with appropriate
3
-
-
HS (NaHS used for H S production)/HSO for 5 min and 70 min at ambient
2
3

temperature, and then was used for UV-Vis absorption and fluorescence experiment.
The excitation wavelength was 560 nm and slit width was all 10 nm.
Preparation of HEPES buffer solution
4
-hydroxypiperazineethanesulfonic acid (HEPES, 1.192 g) was dissolved with a
small amount of distilled water in the 500 mL beaker. Then the pH of HEPES solution
was adjusted to 7.4 with 10% NaOH, and finally the concentration of HEPES solution
was prepared to 10 mM with distilled water in a volumetric flask of 500 mL.
Application procedures of TCF-DNs in red wine and sugar samples
The red wine and granulated sugar were purchased from different manufacturers in a
supermarket of Jinzhou (China). The red wine was diluted 100 times, then was used to
prepare a EtOH/wine (2/8, v/v) mixed solution. Different concentrations of NaHS were
added into the 10 μM TCF-DNs solutions prepared with the aforesaid mixed solution,
and then the fluorescence signals (643 nm) of TCF-DNs solutions were recorded.
The 0.5 g granulated sugar and stock solution of TCF-DNs (1 mM, 250 μL) were
added to a volumetric flask of 25 mL. The EtOH/H O (2/8, v/v, HEPES 10 mM, pH=7.4)
2
-
solution is added to the scale line of the volumetric flask. The HSO with different
3
concentrations was added into 2 mL of the above solution, which was used to record the
-
-
3
fluorescence signals at 643 nm. The measured concentration and recovery of HS /HSO
were calculated using the standard curve and the standard additions method.
Cell viability assays
After the addition of different concentrations of TCF-DNs (1, 5, 10, 30, 50 μM), the
incubated MCF-7 cells were cultured for 24 h. The 10 μL CCK-8 (Cell Counting Kit-8,

Dojindo, Japan) was added into TCF-DNs-pretreated cells, and the cells were further
incubated for another 4 hours. After being washed three times with PBS, the absorbance
at 600 nm of cells was recorded, and cell viability was calculated using the reported
methods of literature.
Cell imaging
The cultured MCF-7 cells were placed in confocal culture dishes, then 2 μM
TCF-DNs was transferred into each dish and cultured for 30 min. Subsequently, MCF-7
cells treated by TCF-DNs were washed twice using PBS buffer, and further cultured for
-
-
3
0 min upon addition of HS (0, 1 and 10 µM) or 100 µM HSO . After being washed
3
three times with PBS solution, the cells were used for fluorescence imaging.
Results and discussion
Synthesis of TCF-DNs
Probe TCF-DNs was synthesized through a simple two-step reaction (Scheme 1).
Compound TCF-1 was firstly prepared by condensation of 4-diethylamino
salicylaldehyde and 2-(3-cyano-4,5,5-trimethylfuran-2(5H)-ylidene)-malononitrile.
Then
TCF-1
underwent
a
nucleophilic
substitution
reaction
with
2
,4-dinitrobenzenesulfonyl chloride to obtain the target probe TCF-DNs under weak
1
13
alkaline condition. The structure of TCF-DNs was confirmed by H and C NMR and
HRMS (Fig. S1-S3, ESI†).

Scheme 2. Synthesis of TCF-DNs.
-
Absorption properties of TCF-DNs to H S and HSO
2
3
-
-
To investigate the colorimetric selectivity of TCF-DNs to HS and HSO , we
3
accomplished the UV absorption of TCF-DNs (20 μM) in EtOH/H O (2/8, v/v, HEPES
2
1
0 mM, pH=7.4). As shown in Fig. 1, the maximum absorption wavelength of
-
2-
-
TCF-DNs is exhibited at 600 nm. After adding various anions (Br , CrO , CH COO ,
4
3
2
-
-
-
2-
-
-
-
4-
7
3-
4
2-
3
-
2-
-
-
CO , H PO , HCO , HPO , I , N , NO , P O , PO , S O , SCN , SO , F , CN )
3
2
4
3
4
3
2
2
2
4
-
-
and biothiols (Cys, Hcy, GSH), HS can cause a remarkable enhancement, and HSO
3
can lead to a significant decrease for the absorption of TCF-DNs. The color of
TCF-DNs changed from lilac to light-blue response to saturated concentrations of H S,
2
-
and a visible color change from lilac to colorless occurred on treatment with HSO (Fig.
3
1
inset). The other anions and biothiols did not cause any significant changes, indicating
-
-
TCF-DNs can recognize HS and HSO by the UV spectrum as well as colorimetric
3
sense through naked eye under ambient light.

Fig. 1 Change in UV spectra of TCF-DNs (20 µM) after the addition of various anions
and biothiols in EtOH/H O (2/8, v/v, pH=7.4). Inset: photograph of TCF-DNs before
2
-
-
and after the addition of HS and HSO under visible light.
3
-
-
To detect the sensitivity of the UV absorption sense for HS and HSO by TCF-DNs,
3
we tested the absorption spectra of TCF-DNs (20 μM) with different concentrations of
-
-
-
HS and HSO . As the increase of concentration of HS , the absorption intensity at 600
3
nm of TCF-DNs gradually enhanced and subsequently reached an equilibration when
-
the concentration of HS is 64 μM (Fig. 2a). On the contrary, continuous addition of
-
HSO (0-720 μM) cause the decrease of absorption intensity of TCF-DNs until the
3
-
concentration of HSO3 reaches 720 μM. These results indicate that the saturated
-
-
concentration of HS is 3.2 equity. of TCF-DNs, and that of HSO is 36 equivalent of
3
TCF-DNs.

Fig. 2 The absorption change of TCF-DNs (20 μM) in EtOH/H O (2/8, v/v, pH=7.4)
2
-
-
after adding different concentrations of (a) HS (0-3.2 equity.) and (b) HSO (0-36
3
equiv.).
To further investigate whether other anions and bioethics have interference on UV
-
-
recognition of HS and HSO by TCF-DNs, we detected anti-interference properties of
3
-
TCF-DNs. When HS was added separately into TCF-DNs (20 μM) containing various
anions and biothiols (3.2 equiv.), the absorption intensity of TCF-DNs was significantly
-
strengthened, illustrating that TCF-DNs recognize HS without any interference
-
-
including the existing HSO (Fig. 3). Similarly, it can be seen that HSO (36 equiv.)
3
3
caused a significant decrease in absorption intensity of TCF-DNs even in the presence
-
-
of HS , manifesting that the presence of HS , other anions and biothiols does not affect
-
-
-
the detection of HSO . These results show that TCF-DNs recognize HS and HSO by
3
3
UV spectrum with good anti-interference ability.

Fig. 3 Absorption intensity changes of TCF-DNs (20 μM) after the addition of various
-
anions and biothiols, (a) after 5 min followed by addition of HS (3.2 equiv.) and (b)
-
after 70 min followed by addition of HSO (36 equiv.).
3
-
Fluorescence response of TCF-DNs to H S and HSO
2
3
-
A fluorescence selectivity experiment of TCF-DNs to H S and HSO was performed
2
3
through addition of various anions and biothols to TCF-DNs solution (10 μM,
EtOH/H O, v/v=2/8, pH=7.4), and the result is shown in Fig. 4a. The maximum
2
wavelength of fluorescence emission of TCF-DNs is 643 nm in the near-infrared region
under the excitation of 560 nm. Fluorescence intensity at 643 nm of TCF-DNs can be
-
-
significantly enhanced upon the addition of HS , and be obviously quenched by HSO .
3
-
These results indicate that TCF-DNs is capable of recognition for HS with
-
fluorescence turn-on mode and near-infrared emission, and detection of HSO with
3

fluorescence “on-off” responding. In addition, we tested the fluorescence intensity of
TCF-DNs after adding various cations (Fig. 4b). The probe TCF-DNs shows a
negligible response to most cations, and the fluorescence intensity of TCF-DNs was
2
+
+
quenched when Cu and Ag were added into the solution of TCF-DNs. These results
indicate that probe TCF-DNs can recognize H S with excellent selectivity among
2
2
+
+
various anions, cations and biothols. It is worth noting that the presence of Cu or Ag
can greatly quench the fluorescence of TCF-DNs. However, they cannot coexist with
the anions that can be detected by TCF-DNs, so they will not have a significant impact
on the application of TCF-DNs.
Fig. 4 Changes in fluorescence spectra of TCF-DNs (10 μM, EtOH/H O, v/v=2/8,
2
pH=7.4) after the addition of various anions, biothols (a) and cations (b) solution.

-
-
The sensitivity of TCF-DNs detects HS and HSO was evaluated through adding
3
-
-
-
different concentration of HS and HSO . When HS (48 μM) were added, fluorescence
3
intensity (643 nm) of TCF-DNs showed the strongest emission and reaches an
2
equilibration (Fig. 5a). At the same time, an excellent linear relationship (R >0.99) was
-
obtained between the fluorescence intensity at 643 nm and the HS concentration in the
range of 2-14 μM (Fig. 5b). The detection limit can be calculated to be 13.5 nM based
on the equation LOD=3S/K (where S is the standard deviation of the blank solution and
K is the slope of the standard curve).[42] These results indicate that TCF-DNs
recognize H S with good sensitivity, which can serve as a good quantitative detection
2
tool for H S. Similarly, the fluorescence intensity at 643 nm of TCF-DNs gradually
2
-
declined as the concentration of HSO3 increased, and kept constant when the
-
concentration of HSO was 600 μM (Fig. S4). The calculated detection limit is 22.3 μM
3
-
(
Fig. S5), manifesting that TCF-DNs sense HSO with good sensitivity.
3

Fig. 5 Fluorescence emission changes of TCF-DNs (10 μM) in EtOH/H O (2/8, v/v,
2
-
pH=7.4) after adding different concentrations of (a) HS (0-4.8 equiv.) and (b) the
-
calibration plot of emission intensity and HS concentration in TCF-DNs. Calculation
formula of detection limit: 3S/K.
We further evaluated the anti-interference property of TCF-DNs for fluorescence
-
-
recognition of HS and HSO3 through competition experiments. Probe TCF-DNs
showed a significant increase in fluorescence toward H S and H S+analyte except
2
2
-
HSO (Fig. S6a). This indicates that the other competitive ions do not affect H S
3
2
−
-
detection without the presence of HSO . By contrast, when HSO was added
3
3
separately into TCF-DNs containing competitive analyte, the fluorescence intensity of
−
TCF-DNs decreased for all the analyte including HS (Fig. S6b), indicating that
-
TCF-DNs for HSO recognition possess good anti-interference ability.
3
Since the response speed is an important factor for probe application, time-dependent
-
-
3
emission spectra of TCF-DNs in the presence of 4.8 equiv. HS and 60 equiv. HSO
were then examined. As shown in Fig. S7a, the fluorescence intensity at 643 nm of
TCF-DNs gradually increased against with time and reached a plateau within 2.5 min,
indicating that TCF-DNs has a fast response for H S recognition. Furthermore, Fig. S7b
2
-
showed that probe TCF-DNs reacted completely with HSO within ~70 min.
3
The pH adaptability is very vital for the applications of a newly developed probe, so
-
-
we tested the effect of pH on the recognition of HS and HSO by TCF-DNs (Fig. 6).
3
The fluorescence emission at 643 nm of TCF-DNs keeps it constant in the range of pH

-
3
-9. The addition of HS to TCF-DNs caused a significant increase in fluorescence at
pH 5-8. In contrast, the significant fluorescence quenching of TCF-DNs can be induced
-
by HSO in the pH range of 3-9. These results indicate that TCF-DNs can detect H S
3
2
-
and HSO at pH 5-8 and show a wide pH range.
3
-
Fig. 6 Fluorescence intensity at 643 nm changes of TCF-DNs, TCF-DNs+HS and
-
TCF-DNs+HSO at different pH.
3
-
Sensing mechanism of TCF-DNs to H S and HSO
2
3
−
The fluorescence spectrum of TCF-DNs after the addition of HS shows nearly the
-
same emission pattern as that of TCF-1 (Fig. S8), which initially confirms HS attacks
directly on the DNs ester moiety to release TCF-1 and 2,4-dinitrobenzenethiol. This
-
reaction process was further verified through HRMS of TCF-DNs+HS . The remarkable
-
peak observed at m/z=373.1947 and 230.9743 can be identified as TCF-1([M-H] ) and
-
2
,4-dinitrobenzenethiol ([M-H+CH OH] , Fig. S9), suggesting that thiolysis of DNP
3
-
ether triggered by HS to release TCF-1 really happens. The thiolysis product TCF-1
emits a red fluorescence on account of the strong ICT process from the
diethylaminophenol to the malononitrile moiety.[43]

1
-
H NMR and HRMS studies for TCF-DNs+HSO adduct were accomplished to
3
-
1
analyze the reaction mechanism. After the reaction of TCF-DNs with HSO , H NMR
3
signals at 7.93 ppm and 6.95 ppm assigned to the vinyl protons of TCF-DNs
disappeared, meanwhile, a new upfield signal appeared at 4.502 ppm (Fig. 7).
Combined with that the signals of the aromatic protons were shifted upfield, which
-
confirms the occurrence of nucleophilic addition, namely, HSO attacks directly on the
3
vinyl π-bond (Scheme 3). According to the HRMS data, a peak appeared at
-
-
m/z=685.1511, which exactly matched with addition-compound ([TCF-DNs-HSO -H] ,
3
1
Fig. S10). To sum up, HRMS and H NMR spectra support the nucleophilic addition of
−
HSO on the C=C bond of TCF-DNs. Consequently, the π-conjugation system of
3
TCF-DNs was interrupted, and the ICT process was weakened, which caused the
fluorescence quenching of TCF-DNs. Based on above results, the proposed mechanism
-
of TCF-DNs sense H S and HSO was shown in Scheme 3.[7, 44]
2
3
1
-
Fig. 7 H NMR spectra of TCF-DNs before and after addition of HSO in D O/DMSO
3
2
(3/1, v/v).

-
Scheme 3. Proposed mechanism for sensing H S and HSO by TCF-DNs.
2
3
Application of TCF-DNs in real samples
Real water samples, including lake water, river water and tap water, were used to the
preliminary evaluation of the practical application of TCF-DNs. The test solution of 10
μM was prepared using TCF-DNs stock solution (1 mM, DMSO) diluted by EtOH/H O
2
-
(
2/8, v/v) solution. The samples were spiked with HS , and then the fluorescence signals
at 643 nm were recorded. An excellent linear dependence of fluorescence intensity of
-
TCF-DNs versus HS concentrations in the range of 0~20 μМ indicates TCF-DNs can
-
be used for quantitative detection of HS within 0~20 μМ (Fig. 8).
Red wine samples were chosen to further assess the practical application of
TCF-DNs. The amounts of H S (50, 100 and 150 µM) were added into TCF-DNs (10
2
µM) mixed solution pre-configured by EtOH/wine (2/8, v/v), and the results of the test
are shown in Table 1 and Fig.S11-S12. The recoveries ranging from 88.33 to 118.42 %
show that TCF-DNs can detect H S levels in red wine samples.
2

-
Fig. 8 Linear dependence of fluorescence intensity (643 nm) of TCF-DNs versus HS
concentrations in the range of 0~35 μМ in EtOH/H O (tap water, lake water and river
2
water, 2/8, v/v).
Table 1. Results for detection of H S in red wine
2
Red Wine
sample
Added
(μM)
50
Found
(μM)
Recovery
(% )
Cv
(%, n=3)
2.43
Red wine 1
59.21
118.42
96.77
1
1
00
50
96.77
1.20
143.33
52.80
95.55
0.48
Red wine 2
Red wine 3
50
105.60
102.42
99.81
2.81
1
1
00
50
102.42
149.71
44.17
1.24
1.06
50
88.33
1.21
1
1
00
50
104.87
148.90
104.87
99.27
0.76
0.46
The experiments were repeated three times.
-
In view of the application of HSO in the food industry, the granulated sugar was
3
-
used to investigate the application of TCF-DNs detecting for HSO in food examples.
3
-
The different concentrations of HSO were added into TCF-DNs (10 μM) containing
3

sugar sample in EtOH/H O (2/8, v/v, HEPES 10 mM, pH=7.4) solution (Fig. S13). The
2
calculated recovery is shown in Table 2. The good recovery (102.6~121.4%) illustrates
−
that probe TCF-DNs is able to determine accurately the concentration of HSO in real
3
food samples.
-
Table 2. Results for detection of HSO in the sugar sample
3
Sugar sample
Added
Found
(μM)
6.55
6.64
6.75
6.45
6.50
6.67
Recovery
(%)
(μM)
Granulated sugar 1
5.5
119.1
110.7
103.9
121.4
108.3
102.6
6
6
.0
.5
Granulated sugar 2
5.5
6
6
.0
.5
The experiments were repeated three times.
Application of immobilized TCF-DNs
In order to prove the practicability of TCF-DNs, we made test strips and TLC plates
to detect H S. The six pieces of filter paper were immersed into TCF-DNs (20 μM,
2
EtOH/HEPES, 2/8, v/v, pH=7.4) solution and dried in air. Then five of them were
-
immersed into the deferent concentration of HS (20, 40, 60, 80, 100 μM) solution for 5
min and dried naturally. It can be seen from the daylight photos that the color of test
-
paper changed from purple to light-blue after adding different concentrations of HS
(Fig. 9). These results indicated that TCF-DNs could be used to prepare a cheap,
portable test paper strip for the rapid and qualitative detection of H S by the naked eye.
2

Fig. 9 Photograph of TCF-DNs (20μM) pretreated test strips immerged into H2S
solution (0, 20, 40, 60, 80, 100 μM) under daylight.
High-concentration hydrogen sulfide gas can cause the paralysis of the olfactory
nerve and make the poisoned person lose consciousness instantly, and will finally lead
to significant harm. Hence, we also prepared TLC plates for detecting H S gas to
2
develop TCF-DNs for more application. The moist TLC plates immerged by TCF-DNs
were exposed to saturated H S gas (produced by NaHS and dilute H SO ), and the color
2
2
4
of TLC plates changed obviously from light blue to dark green (Fig.10). The result
declares that TCF-DNs can be used as an indicator to detect H S gas in special
2
occasions such as leakage of H S gas or underground pipelines filled with H S gas.
2
2
Fig. 10 The color change of the moist TLC plates immersed by TCF-DNs in the
presence (left) and absence (right) H S gas.
2

Cell imaging
To corroborate the biological applications of TCF-DNs, the MCF-7 cells were used
for living cell imaging. The cytotoxicity of TCF-DNs was evaluated by CCK-8 kit and
showed that 50 μM TCF-DNs did not show any adverse effects on cell viability
(Fig.S14). The results indicate that TCF-DNs is nontoxic within the scope of 0-50 μM,
thus, the concentration of 2 μM for TCF-DNs was selected as a non-cytotoxic dose for
H S imaging.
2
Fig. 11 Dark field (red channel) and bright-field image of TCF-DNs treated MCF-7
-
cells after incubation with 1 μM and 10 μM HS for 30 min.
As displayed in Fig.11, TCF-DNs incubated living MCF-7 cells show a faint red
emission, which may be due to the weak fluorescence of TCF-DNs itself and the low
-
level of endogenous H S in cells. After that, an exogenous HS (1, 10 μM) was added to
2
MCF-7 cells pretreated by TCF-DNs, and then the cells were further incubated for 30
min. A substantial fluorescence enhancement in the red channel was observed with the

-
increase of HS concentration (Fig. 11). The morphology of MCF-7 cells in the bright
field images did not change, which suggested that the MCF-7 cells were still alive.
These results indicate that TCF-DNs is cell-permeable and can be used for exogenous
H S imaging in living cells.
2
Conclusions
In summary, we have successfully developed a novel NIR emitting fluorescent probe
-
-
TCF-DNs for distinguishable detection of HS and HSO in EtOH/H O (2/8, v/v,
3
2
HEPES 10 mM, pH=7.4). The distinct color change of TCF-DNs indicates that
-
-
TCF-DNs can recognize HS and HSO by the naked eye. The easy-to-synthesize
3
TCF-DNs can recognize H S with rapid fluorescence enhancementwitha detection limit
2
of 13.5 nM. The sensing mechanism of TCF-DNs is based on the thiolysis-triggered
-
removal of the 2,4-dinitrobenzenesulfonate unit by HS and the occurrence of a
-
-
nucleophilic addition for HSO , respectively. TCF-DNs can detect HSO in granulated
3
3
sugar, and sensitively detect H S in red wine, real water, living cells. Meanwhile,
2
TCF-DNs can be used to prepare test paper strips for the rapid and qualitative detection
of H S by the naked eye and to serve as an indicator to detect H S gas.
2
2
Acknowledgments
The project was supported by the Scientific Research Fund of Liaoning Provincial
Education Department (LJ2019005, LQ2019005), the National Natural Science
Foundation of China (Nos.21878023, U1608222 and 21304009), the Program for
Distinguished Professor of Liaoning Province, the Liaoning Revitalization Talents
Program (XLYC1807133).

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