Mitochondria-targeted fluorescent probe for rapid detection of thiols and its application in bioimaging

Article abstract of DOI:10.1016/j.dyepig.2021.109376

Biothiols are crucial reactive sulfur species in living cells, playing an essential role in maintaining the normal function of organisms. Effective detection of biothiols promotes the exploration of metabolic pathways and physiological functions of organisms. In this paper, a cyanine-based, mitochondria-targetable fluorescent probe, Cy-DNBS, was designed and synthesized. This probe uses a lipophilic cation unit as mitochondrial targeting site and DNBS group as the fluorescence quencher as well as the recognition site. The probe shows a distinct fluorescence emission at 604 nm upon rapid interaction with intracellular biothiols, owing to the removal of the 2,4-dinitrobenzene-sulfonyl (DNBS) group and the blockage of PET process. Moreover, the probe can not only image endogenous thiols, but also efficiently target mitochondria and evaluate thiol fluctuations under mitochondrial oxidative stress induced by H₂O₂. Collectively, the new low cytotoxic probe can serve as a promising tool for monitoring cellular thiols and further exploring elevant disease processes.

Full text of DOI:10.1016/j.dyepig.2021.109376

Dyes and Pigments 191 (2021) 109376
Contents lists available at ScienceDirect
Dyes and Pigments
journal homepage: http://www.elsevier.com/locate/dyepig
Mitochondria-targeted fluorescent probe for rapid detection of thiols and its
application in bioimaging
Yuedong Zhu, Haiting Pan, Yanyan Song, Chao Jing, Jia-An Gan, Junji Zhang*
Key Laboratory for Advanced Materials and Joint International Research Laboratory of Precision Chemistry and Molecular Engineering, Feringa Nobel Prize Scientist
Joint Research Center, School of Chemistry and Molecular Engineering, Frontiers Center for Materio biology and Dynamic Chemistry, East China University of Science and
Technology, Shanghai, 200237, PR China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Biothiols are crucial reactive sulfur species in living cells, playing an essential role in maintaining the normal
function of organisms. Effective detection of biothiols promotes the exploration of metabolic pathways and
physiological functions of organisms. In this paper, a cyanine-based, mitochondria-targetable fluorescent probe,
Cy-DNBS, was designed and synthesized. This probe uses a lipophilic cation unit as mitochondrial targeting site
and DNBS group as the fluorescence quencher as well as the recognition site. The probe shows a distinct fluo-
rescence emission at 604 nm upon rapid interaction with intracellular biothiols, owing to the removal of the 2,4-
dinitrobenzene-sulfonyl (DNBS) group and the blockage of PET process. Moreover, the probe can not only image
endogenous thiols, but also efficiently target mitochondria and evaluate thiol fluctuations under mitochondrial
oxidative stress induced by H2O2. Collectively, the new low cytotoxic probe can serve as a promising tool for
monitoring cellular thiols and further exploring elevant disease processes.
Cyanine dyes
Biothiols
Mitochondria-targetable
Oxidative stress
PET
1. Introduction
the most abundant intracellular biothiol, maintains redox homeostasis
through protecting cells from injury caused by reactive oxygen species
Mitochondria, which are considered as the cellular power packs, play
vital roles in energy metabolism and apoptosis of cells, organs and tis-
sues[1,2]. Moreover, mitochondria are the main source of intracellular
reactive oxygen species (ROS) and related with various physiological
processes[3,4]. However, excessive ROS can induce mitochondrial
oxidative damage and eventually lead to numerous pathological con-
ditions, including neurodegenerative disorders, cardiovascular diseases,
diabetes, cancers and so on[5–8]. In order to protect cells from oxidative
injury, mitochondria simultaneously possess a large number of free
radical scavengers and mitochondria thiols, including homocysteine
(Hcy), cysteine (Cys) and glutathione (GSH)[9–11], which are closely
related to antioxidation processes in biological systems through the
equilibrium of free thiols and oxidized disulfides forms[12–15], thus
playing an essential role in complicated physiological and pathological
processes[16–19]. The normal intracellular concentrations of Hcy, Cys
(ROS) and free radicals, which is quite significant for normal cell func-
tion[27,28]. Deviation from normal GSH concentration could lead to
severe diseases as cancers. Many studies indicate that the above three
mercaptan are related to each other and significantly decreased level of
these thiols can cause mitochondrial damage and dysfunction[29–31].
In order to gain deeper understanding of the physiological mechanism
and diagnosis of various related diseases, it is of essential importance to
explore methods for real-time monitoring mitochondrial thiols fluctua-
tions in living cells.
Fluorescence technique has aroused increasing interest due to the
advantages of non-invasion, operational simplicity, high sensitivity/
selectivity and fast response, and has witnessed great progress in the
past decade[32]. Owing to the high reactivity of thiol groups, various
fluorescent probes employing different mechanisms, such as Michael
addition, disulfide exchange reaction, cyclization with aldehyde,
nucleophilic substitution etc., have been developed for biothiol detec-
tion[33–38]. Maeda et al. [39] have reported that 2,4-dinitrobenzene--
sulfonyl (DNBS) group has high sensitivity and responsiveness towards
thiols, being useful to design thiol sensors. Inspired by this strategy, a
new mitochondria-targeting probe Cy-DNBS, cyanine decorated with
and GSH are approximately 5–12
μM, 30–200 μM and 1–10 mM,
respectively [20–23]. Large fluctuations in intracellular thiol concen-
trations will cause many diseases. For instance, low level of Cys has been
reported to cause liver damage and lethargy[24,25]. Overexpression of
Hcy results in neural tube defect and coronary heart disease[26]. GSH,
* Corresponding author.
E-mail address: zhangjunji@ecust.edu.cn (J. Zhang).
https://doi.org/10.1016/j.dyepig.2021.109376
Received 15 March 2021; Received in revised form 3 April 2021; Accepted 3 April 2021
Available online 17 April 2021
0143-7208/© 2021 Elsevier Ltd. All rights reserved.

Y. Zhu et al.
Dyes and Pigments 191 (2021) 109376
DNBS group, was designed and synthesized by our group for selective
thiols detection and imaging in vitro and in living cell (Scheme 1). This
probe uses a lipophilic cation unit as mitochondrial targeting site and
DNBS group as the fluorescence quencher as well as the recognition site.
Cy-DNBS shows no fluorescence in the absence of thiols due to the
photo-induced electron transfer (PET) between fluorophore and DNBS
group. Upon addition of thiols, distinct fluorescence emission at 604 nm
can be detected owing to the removal of the DNBS group and the
blockage of PET process. We investigated the spectral response of the
probe in aqueous solution, and further in living cells. The results illus-
trated that the sensor displays high sensitivity and selectivity towards
thiols and possesses excellent mitochondria-targeted ability.
bromo-4-hydroxybenzaldehyde (3.00 g, 14.92 mmoL) were added.
Then distilled toluene and triethylamine were injected under argon at-
mosphere after dehydration. After freezing and filtrating of oxygen, 4-
vinylpyridine (2.0 mL, 18.81 mmoL) was added. Then the mixture was
refluxed at 100 ◦C overnight and monitored by TLC. The reaction
mixture was poured into CH2Cl2 after cooling to room temperature and
filtered. The filtrate was washed with weak acid water and the aqueous
layer was extracted with CH2Cl2 for three times. The combined organic
layers were dried over anhydrous Mg2SO4 and purified by column
chromatography on silica gel with CH2Cl2/MeOH (50:1, v/v) as eluent
to get compound 2 as a pale yellow solid (1.20 g, yield 35.7%). 1H NMR
(400 MHz, DMSO‑d6) δ (ppm) 11.22 (s, 1H), 9.87 (s, 1H), 8.56 (d, J =
5.90 Hz, 2H), 8.21 (d, J = 2.01 Hz, 1H), 7.75 (dd, J = 10.46 Hz, 1H),
7.69 (d, J = 16.57 Hz, 1H), 7.57 (d, J = 6.16 Hz, 2H), 7.38 (d, J = 16.57
Hz, 1H), 7.06 (d, J = 8.42 Hz, 1H). 13C NMR (101 MHz, DMSO‑d6) δ
(ppm) 191.24, 161.21, 150.14, 144.45, 131.09, 129.95, 128.59, 127.08,
123.51, 120.82, 116.46. HRMS-ESI (m/z): Calcd. for (C14H11NO2):
226.0863 (M + H)+, found 226.0862.
2. Experiment
2.1. Materials and apparatus
All reagents and chemicals used for synthesis were purchased from
commercial suppliers and used without further purification unless
otherwise indicated. All oxygen or water sensitive reactions were per-
formed under argon atmosphere using the standard Schlenk method.
Mitotracker® Green FM and Hochest 33342 were purchased from
YEASEN Biological Company. HeLa, Du 145, PC-12 and HUVEV cells
were purchased from ATCC (American Type Culture Collection).
The 1H NMR and 13C NMR spectra of compounds were recorded by
Bruker AM-400 spectrometer, using tetramethylsilane (TMS) as an in-
ternal reference and CDCl3 or DMSO‑d6 as solvent. MS spectra of com-
Synthesis of compound 3. To a 100 mL three-neck round-bottom
flask, compound 2 (0.30 g, 1.33 mmoL) in dry CH2Cl2 and NEt3 was
added. The mixture was stirred at 0 ◦C in ice-water bath for 60 min and
2,4-dinitrobenzenesulfonyl chloride (0.53 g, 2.00 mmoL) in CH2Cl2 was
added dropwise. Then the mixture was stirred at room temperature and
monitored by TLC. After completion, the resulting mixture was filtered
and the filter cake was dried in vacuum to get compound 3 as a pale
yellow solid (0.54 g, yield 89.1%). 1H NMR (400 MHz, DMSO‑d6) δ
(ppm) 10.08 (s, 1H), 8.95 (d, J = 2.25 Hz, 1H), 8.55 (d, J = 5.96 Hz, 2H),
8.47 (dd, J = 11.03 Hz, 1H), 8.42 (d, J = 1.77 Hz, 1H), 8.25 (d, J = 8.54
Hz, 1H), 8.00 (dd, J = 10.39 Hz, 1H), 7.60 (d, J = 8.33 Hz, 1H), 7.35 (dd,
J = 18.15 Hz, 3H), 7.23 (d, J = 16.45 Hz, 1H). 13C NMR (101 MHz,
DMSO‑d6) δ (ppm) 191.19, 151.22, 150.25, 149.59, 147.93, 142.69,
135.78, 133.54, 131.01, 130.79, 130.56, 129.12, 127.43, 124.76,
123.74, 120.76. HRMS-ESI (m/z): Calcd. for (C20H13N3O8S): 454.0351
(M ꢀ H)-, found 454.0356.
pounds were measured on
a
Micromass LCTTM (HREI-TOF)
spectrometer. PBS buffers with different pH ranges were prepared using
FiveEasy Plus pH meter. The absorption and emission spectra were
recorded by Varian Cary 500 and Varian Cary Eclipse, respectively. Cell
viability data were recorded by Synergy H4 Hybrid Reader (BioTek,
USA). The cell confocal fluorescence imaging experiments were con-
ducted on a Nikon (Japan) laser confocal scanning microscope.
Synthesis of compound Cy-DNBS. To a 50 mL Schlenk tube, com-
pound 1 (0.20 g, 0.66 mmoL), compound 3 (0.30 g, 0.66 mmoL) and
NaOAc (0.05 g, 0.66 mmoL) were added and acetic anhydride (10 mL)
was injected under argon atmosphere after dehydration. Then the
mixture was refluxed at 80 ◦C for 6 h. After cooling to room temperature,
the reactive mixture was suspended in diethyl ether and filtered to yield
2.2. Synthesis and characterization
Synthesis of compound 1.2,3,3-Trimethyl-3H-indole (4.0 mL, 25
mmoL) was dissolved in CH3CN (15 mL). To the mixture was added CH3I
(4.0 mL, 60 mmoL). Then the mixture was refluxed at 80 ◦C overnight
under argon atmosphere. After cooling to room temperature, the pre-
cipitate was filtered and washed with n-hexane and dried in vacuum to
obtain compound 1 as a pale pink solid powder (6.68 g, yield 85.8%). 1H
NMR (400 MHz, DMSO‑d6) δ (ppm) 7.92 (dd, J = 8.81 Hz, 1H), 7.84 (m,
J = 8.57 Hz, 1H), 7.63 (m, J = 22.25 Hz, 2H), 3.98 (s, 3H), 2.77 (s, 3H),
1.53 (s, 6H).
1
Cy-DNBS as a brown solid (0.20 g, yield 42.2%). H NMR (400 MHz,
DMSO‑d6) δ (ppm) 8.94 (d, J = 2.26 Hz, 1H), 8.77 (d, J = 6.34 Hz, 2H),
8.74 (d, J = 1.39 Hz, 1H), 8.50 (d, J = 2.41 Hz, 1H), 8.47 (m, J = 10.53
Hz, 1H), 8.43 (dd, J = 10.62 Hz, 1H), 8.26 (d, J = 8.72 Hz, 1H), 7.98 (dd,
J = 9.06 Hz, 1H), 7.94 (dd, J = 8.73 Hz, 1H), 7.83 (d, J = 16.52 Hz, 1H),
7.77 (d, J = 6.36 Hz, 2H), 7.68 (dd, J = 8.84 Hz, 2H), 7.64 (d, J = 8.81
Hz, 1H), 7.56 (d, J = 16.30 Hz, 1H), 7.37 (d, J = 16.38 Hz, 1H), 4.25 (s,
3H), 1.85 (s, 6H). 13C NMR (101 MHz, DMSO‑d6) δ (ppm) 181.71,
151.28, 150.00, 148.55, 147.97, 145.94, 143.79, 141.80, 134.77,
133.65, 131.77, 130.47, 130.37, 129.85, 129.42, 129.05, 127.45,
124.77, 122.96, 122.24, 120.69, 115.60, 115.27, 52.48, 25.04, 21.02.
HRMS-ESI (m/z): Calcd. for (C32H27N4O7S+): 611.1595 (M)+, found
611.1641.
Synthesis of compound 2. To a 200 mL Schlenk tube, Pd(OAc)2
(0.60 g, 2.67 mmoL), tri-o-tolylphosphane (1.64 g, 5.37 mmoL) and 3-
Synthesis of compound Cy-OH. To a 50 mL Schlenk tube, com-
pound 1 (0.15 g, 0.49 mmoL) and compound 2 (0.10 g, 0.44 mmoL)
were added. After dehydration, EtOH (3 mL) and pyridine (0.4 mL) were
injected successively under argon atmosphere. Then the mixture was
refluxed at 80 ◦C for 4 h. The reactive mixture was filtered after cooling
to room temperature. The filter cake was washed with EtOH and dried in
vacuum to yield Cy-OH as a rufous solid (74 mg, yield 32.8%). 1H NMR
(400 MHz, DMSO‑d6) δ (ppm) 11.44 (s, 1H), 8.59 (d, J = 6.07 Hz, 2H),
8.54 (d, J = 1.81 Hz, 1H), 8.41 (d, J = 16.29 Hz, 1H), 8.14 (dd, J =
10.58 Hz, 1H), 7.87 (m, J = 11.55 Hz, 2H), 7.70 (d, J = 16.46 Hz, 1H),
7.65-7.53 (m, 5H), 7.41 (d, J = 16.40 Hz, 1H), 7.09 (d, J = 8.52 Hz, 1H),
4.13 (s, 3H), 1.81 (s, 6H). 13C NMR (101 MHz, DMSO‑d6) δ (ppm)
181.39, 161.22, 153.46, 150.15, 144.56, 143.23, 141.87, 132.53,
Scheme 1. Proposed sensing mechanism of Cy-DNBS to thiols.
2

Y. Zhu et al.
Dyes and Pigments 191 (2021) 109376
131.31, 128.85, 128.78, 127.10, 126.18, 124.18, 122.79, 120.83,
117.05, 114.68, 109.77, 51.79, 34.57, 25.66. HRMS-ESI (m/z): Calcd.
for (C26H25N2O+): 381.1961 (M)+, found 381.1959.
from MitoTracker® Green FM channel, λex = 488 nm, λem = 500–550
nm; emission from Hochest 33342 channel, λex = 404 nm, λem
425–475 nm; emission from Cy-DNBS channel, λex = 561 nm, λem
570–620 nm.
=
=
2.3. Limit of detection (LOD)
3. Results and discussion
The limit of detection was calculated according to the fluorescence
titration curve of probe Cy-DNBS in the presence of thiols. We measured
the fluorescence intensity of Cy-DNBS for 10 times and then calculated
the standard deviation of blank measurement. The limit of detection was
calculated according to the following equation:
3.1. Design and synthesis
Cyanine derivative was selected as the fluorophore due to its excel-
lent photophysical properties and the positively charged moiety of
indole ammonium cation for mitochondria targeting. 2,4-dinitrobenze-
nesulfonyl (DNBS) group was modified at the hydroxyl site of the fluo-
rophore as both a thiol responsive group and quencher. Cy-DNBS probe
was synthesized through a three-step synthesis shown in Scheme 2.
Structure of all new compounds were characterized by 1H NMR, 13C
NMR and HRMS (see SI for details). After recognizing thiols, distinct
fluorescence emission at 604 nm can be detected due to the removal of
the DNBS group and blockage of PET process. ESI-MS was carried out to
verify the reaction mechanism. As demonstrated in Fig. S1, Cy-DNBS
shows a characteristic peak of m/z 611.1594 ([M]+ calcd 611.1595).
The new peak at m/z 381.1955 assigned to cleavable product Cy-OH
([M]+ calcd 381.1961) was observed after adding Cys.
LOD = 3σ/k
where
σ
is the standard deviation of the blank measurement, k is the
slope between the fluorescence intensity versus thiol concentrations.
2.4. Cell culture and fluorescence imaging
HeLa, PC-12 and HUVEC cells were cultured at 37 ◦C in DMEM
(Gibco, Gland Island, NY, USA) while Du 145 cells were cultured at
37 ◦C in MEM (Gibco, Gland Island, NY, USA) supplemented with 10%
fetal bovine serum (Gibco, Gland Island, NY, USA) and 1% penicillin-
streptomycin in humidified environment of 5% CO2.
Cytotoxicity was estimated by cell counting kit-8 (CCK-8) assay.
Firstly, cells were seeded into 96-well plates at 1 × 104/well and
cultured at 37 ◦C in humidified environment of 5% CO2 for 24 h. Sec-
3.2. Spectral responses of Cy-DNBS toward thiols
ondly, different concentrations of probe (0, 1, 2.5, 5, 10, 20
μ
M) were
With the probe in hand, we first investigated the optical response of
Cy-DNBS toward thiols in phosphate buffered saline (PBS, 0.01 mM, pH
7.4 containing 10% DMSO). As shown in Fig. 1A, free Cy-DNBS
exhibited two absorption maxima around 300 nm and 380 nm belonging
to the DNBS and cyanine moieties, respectively. The absorption band at
380 nm disappeared with the addition of thiols, accompanied with the
appearance of a new absorption band at 545 nm. Cy-DNBS alone dis-
played extremely minimized fluorescence and an obvious fluorescence
emission signal at 604 nm appeared after interacting with thiols, which
became stronger with the increasing concentration of thiols (Fig. 1B).
The fluorescence intensity at 604 nm was linearly proportional to the
added to wells and cells were incubated for 12 h. Finally, CCK-8 was
added to each well and incubated for 30 min. The OD450 (absorbance
value) was recorded by Synergy H4 Hybrid reader (BioTek, USA).
Cells were seeded on confocal cuvette and allowed to adhere for 12 h.
For colocalization experiment, cells were incubated with 5 μM Cy-DNBS
for 30 min. After washed with PBS (2 mL × 2 times), cells were incu-
bated with 250 nM MitoTracker® Green FM for 30 min (or 5 g/mL
μ
Hochest 33342 for 10 min). Finally, cell imaging was carried by laser
confocal scanning microscope (Nikon, Japan) after washed with PBS (2
mL × 2 times). For endogenous thiol imaging experiment and H2O2
induce oxidative stress experiment, cells were divided into three groups.
The first group was untreated. The second group was incubated with 5
GSH concentration in the range of 0–4 μM with a limit of detection
(LOD) of 1.38 × 10ꢀ 7 M (Fig. 1C). Under the same conditions, Cy-DNBS
exhibited similar fluorescence responses to Cys and Hcy with LODs of
1.34 × 10ꢀ 7 M and 2.29 × 10ꢀ 7 M, respectively (Fig. S2–S3). These
results illustrated that Cy-DNBS shows excellent sensitivity towards
thiols.
μ
M Cy-DNBS for 30 min. The last group was pre-treated with 500
phenylmaleimide (or 1 mM H2O2) for 30 min and then incubated with 5
M Cy-DNBS for 30 min after washed with PBS (2 mL × 2 times). All
group were washed with PBS (2 mL × 2 times) before imaging. Emission
μM N-
μ
Scheme 2. The synthetic route of Cy-DNBS and Cy-OH.
3

Y. Zhu et al.
Dyes and Pigments 191 (2021) 109376
Fig. 1. Photophysical properties of Cy-DNBS. (A) Absorption spectra of Cy-DNBS (10
μ
M) and Cy-OH (10
μ
M) in phosphate buffered saline (PBS, 0.01 mM, pH 7.4
containing 10% DMSO) before and after addition of thiols (100 μM). (B) Fluorescence spectra changes of Cy-DNBS (10 μM) upon addition of different concentrations
of GSH in phosphate buffered saline (PBS, 0.01 mM, pH 7.4 containing 10% DMSO). (C) Fluorescence intensity at 604 nm as a function of GSH concentration. Insert:
Linear relationship of fluorescence intensity at 604 nm as a function of GSH concentration, y = 52.758 + 55.524[GSH], with R2 = 0.9932. (D) Changes in fluo-
rescence intensity at 604 nm in the absence and presence of GSH, Cys and Hcy (100 μM) as a function of reaction time, respectively. (E) Fluorescence responses of Cy-
DNBS (10
μ
M) to various biological species. 0–31: blank, Naþ (1 mM), Cl¡ (1 mM), HCO3¡ (1 mM), CH3COO¡ (1 mM), NO2¡ (1 mM), HPO42¡ (1 mM), H2PO¡4 (1 mM),
CO23¡ (1 mM), Ca2þ (1 mM), SO32¡ (1 mM), S2O23¡ (1 mM), HSO¡4 (1 mM), Pro (100
μ
M), Tyr (100
M), Arg (100 M), Phe (100
M) at 604 nm before and after addition of GSH, Cys and Hcy (100 μ
μM), Met (100
μ
M), His (100
μ
M), Trp (100
M), GSH (100
M) in phosphate
μ
M), Ile (100
μ
M), Gly
(100
(100
μ
M), Asn (100
M), Hcy (100
μM), Ala (100
μM), Val (100
μM), Thr (100
μ
M), Ser (100
μ
μ
μ
M), Leu (100
μ
M), Lys (100
μ
μM), Cys
μ
μ
M). (F) Fluorescence intensity changes of Cy-DNBS (10
μ
buffered saline at different pH.
Furthermore, we tested time-dependent fluorescence spectra of Cy-
DNBS with GSH, Cys and Hcy in phosphate buffered saline (PBS, 0.01
mM, pH 7.4 containing 10% DMSO). As illustrated in Fig. 1D and Fig. S4,
the fluorescence intensity displays a time-dependent enhancement upon
addition of 10 eq. GSH/Cys/Hcy. Within the first 2 min, the fluorescence
intensity enhanced obviously and it took about 8 min to reach equilib-
rium. This indicated that the probe was capable to rapidly respond to
thiols.
could be observed in pH range of 2.0–5.0. However, in pH range of
5.0–11.0, the fluorescence signal was obviously increased. All these
demonstrated that Cy-DNBS was suitable for use under physiological
conditions.
3.5. Cytotoxicity assay
Before the application of probe Cy-DNBS in fluorescent imaging in
living cells, the cellular cytotoxicity of Cy-DNBS to HeLa cells was
3.3. Selectivity
Selectivity is another crucial parameter of fluorescence probe[12].
To evaluate the selectivity of Cy-DNBS towards biothiols, we tested the
response of Cy-DNBS to different biological species, including various
amino acids and essential ions. Fig. 1E and Fig. S5 show the fluorescence
intensity of Cy-DNBS towards the tested species. Upon the addition of
GSH, Cys and Hcy, the fluorescence intensity of Cy-DNBS obviously
enhanced. However, the emission exhibits a negligible change in the
presence of other species. The UV–Vis spectra and color change of
Cy-DNBS after addition of various species were shown in Fig. S6–S7,
respectively. All these results demonstrated that Cy-DNBS has excellent
selectivity towards thiols.
3.4. Effect of pH on response performance
It was found that the pH value of solution was crucial to the cleavage
reaction[37]. To explore the effect of pH on our probe, we examined the
fluorescence responses of 10
presence of 100
μM Cy-DNBS at 604 nm in the absence and
μ
M thiols at different pH. Fig. 1F showed that the probe
itself was stable and underwent negligible change in emission from pH
2.0 to 11.0. Upon the addition of thiols, no fluorescence enhancement
Fig. 2. Cell viability values (%) of HeLa cells estimated by cell counting kit-8
(CCK-8) assay. HeLa cells were cultured with Cy-DNBS and Cy-OH for 12 h.
4

Y. Zhu et al.
Dyes and Pigments 191 (2021) 109376
estimated using the standard cell counting kit-8 (CCK-8) assay. As shown
fluorogenic performance of Cy-DNBS is indeed attributed to the thiol
interaction. To show the versatile live cell-imaging application of Cy-
DNBS, fluorescence imaging experiments were also conducted in living
Du 145 and PC-12 cells (Fig. S11–S12), both of which exhibited similar
results as HeLa. These results confirmed that Cy-DNBS can be used as a
powerful tool for bioimaging in various cancer cells.
in Fig. 2, after cultured with different concentrations (0–20 μM) of Cy-
DNBS and Cy-OH for 12 h, more than 80% of cells remained alive, the
results exhibited the low cytotoxicity of Cy-DNBS. The CCK-8 assays
were also conducted in other cells, including Du 145, PC-12 and HUVEC
cells (Fig. S8).
Many evidences suggest that mitochondria maintain the cellular
redox homeostasis and the intracellular thiol levels are related to
mitochondria oxidative stress[13]. In order to evaluate whether the
probe Cy-DNBS can detect the fluctuations in thiol levels caused by
oxidative stress in living cells, we first evaluated the effect of H2O2 on
responses of Cy-DNBS toward thiols in vitro (Fig. S13). We then
employed Cy-DNBS to image thiols in living HeLa cells stimulated with
H2O2. As shown in Fig. 5, compared with those untreated with H2O2,
HeLa cells pre-treated with H2O2 (1 mM) for 30 min displayed a distinct
decrease reduced fluorescence emission. These results suggested that the
reduced thiols concentration is caused by H2O2 consumption or oxida-
tion. Thus, Cy-DNBS can be used as a powerful probe for monitoring
intracellular thiols fluctuation induced by oxidative stress.
3.6. Cell imaging
Since the indole ammonium moiety of Cy-DNBS owns a positive
charge, it tends to accumulate in mitochondria through electrostatic
interactions due to the mitochondrial membrane negative potential[11].
To confirm the subcellular distribution of Cy-DNBS, fluorescence
co-localization experiments were conducted using MitoTracker® Green
FM, a commercially available mitochondrial specific fluorescent dye. As
illustrated in Fig. 3, the fluorescence of Cy-DNBS in red channel
responding to thiols overlaps perfectly with that of the MitoTracker®
Green FM, with a high Pearson’s correlation coefficient of 0.91. In
contrast, the fluorescence of probe in red channel overlaps poorly with
that of Hochest 33342, a commercial blue-fluorescent nucleus dye, with
a low Pearson’s correlation coefficient of 0.26. In addition, to further
confirm the mitochondrial targeting ability of the probe, co-localization
experiments were also conducted in other cancer cells, including Du 145
(Human prostate cancer cell) and PC-12 cells (rat adrenal chromaffin
cells) (Fig. S9–S10). Finally, all results are similar with those of HeLa.
These results strongly indicate that Cy-DNBS has excellent ability to
target mitochondrial in living cells.
4. Conclusions
In summary, we successfully designed and synthesized a cyanine-
based, mitochondria-targetable fluorescent offꢀ on probe, Cy-DNBS,
for thiols imaging. The probe used indole ammonium moiety as the
mitochondrial target site and DNBS group as the fluorescence quencher
as well as the recognition site. This probe rapidly and selectively
responded to thiols with high sensitivity over other related biological
species. In addition, Cy-DNBS demonstrated low cytotoxicity and great
membrane permeability to living cells, which allowed it to be success-
fully applied in bioimaging. Most importantly, the probe was able to
image endogenous thiols and efficiently target mitochondria to evaluate
mitochondrial oxidative stress level induced by H2O2. Consequently, the
probe has a potential to serve as a powerful tool for monitoring cellular
thiols and providing insight into biological behavior.
Inspired by the above advantages, we further validated the ability of
Cy-DNBS for imaging endogenous thiols in living HeLa cells. As shown
in Fig. 4, the HeLa cells alone showed no fluorescence. After cultured
with Cy-DNBS (5 μ
M) at 37 ◦C for 30 min, the cells displayed distinct red
fluorescence, owing to the formation of Cy-OH. The results indicated
that Cy-DNBS could efficiently permeate into cells and react with the
thiols to produce fluorescence. However, when the cells were pre-
treated with 500 μM N-phenylmaleimide (NMM, a well-known thiol
scavenger) for 30 min, followed by incubation with Cy-DNBS (5
μM) for
30 min, quite weak fluorescence was observed, indicating the
Fig. 3. Colocalization imaging of living Hela cells. (A) The cells were cultured with MitoTracker® Green FM (250 nM) for 30 min and then cultured with 5
μ
M Cy-
DNBS for 30 min. (B) The cells were cultured with Hochest 33342 (5 μg/mL) for 30 min and then cultured with 5 μM Cy-DNBS for 30 min (A1) Emission from
MitoTracker® Green FM channel, λex = 488 nm, λem = 500–550 nm. (B1) Emission from Hochest 33342 channel, λex = 404 nm, λem = 425–475 nm. (A2, B2)
Emission from Cy-DNBS channel, λex = 561 nm, λem = 570–620 nm. (A3, B3) Bright-Field image. (A4, B4) Merged image of A1-A3, B1–B3, respectively. (A5, B5) Co-
localization coefficient (Pearson’s coefficient) of (A1) and (A2), (B1) and (B2), respectively. Scale bar: 50 μm.
5

Y. Zhu et al.
Dyes and Pigments 191 (2021) 109376
Fig. 4. Confocal fluorescence images of Cy-
DNBS responding to endogenous biothiols in
living HeLa cells. (A) Fluorescence image of
HeLa cells; (B) Fluorescence image of HeLa
cells cultured with Cy-DNBS (5 μ
M) at 37 ◦C
for 30 min; (C) Fluorescence image of HeLa
cells pre-treated with N-methylmaleimide
(NMM, 500 μM) for 30 min followed by in-
cubation with Cy-DNBS (5 μ
M) at 37 ◦C for
30 min. First row: bright-field image; second
row: red channel of 570–620 nm (λex = 561
nm); third row: merged bright field images
with red channel images. Scale bar: 50 μm.
Fig. 5. Confocal fluorescence images of Cy-
DNBS in living HeLa cells. (A) Fluorescence
image of HeLa cells; (B) Fluorescence image
of HeLa cells cultured with Cy-DNBS (5 μM)
at 37 ◦C for 30 min; (C) Fluorescence image
of HeLa cells pre-treated with H2O2 (1 mM)
for 30 min followed by incubation with Cy-
DNBS (5 μ
M) at 37 ◦C for 30 min. First row:
bright-field image; second row: red channel
of 570–620 nm (λex = 561 nm); third row:
merged bright field images with red channel
images. (D) The quantification of fluores-
cence intensity of the (B) and (C) group.
Scale bar: 50 μm.
6

Y. Zhu et al.
Dyes and Pigments 191 (2021) 109376
addition of mercapto group to maleimide. Biosens Bioelectron 2017;91:553–9.
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CRediT authorship contribution statement
[13] Yin J, Kwon Y, Kim D, Lee D, Kim G, Hu Y, Ryu JH, Yoon J. Cyanine-based
fluorescent probe for highly selective detection of glutathione in cell cultures and
live mouse tissues. J Am Chem Soc 2014;136(14):5351–8. https://doi.org/
10.1021/ja412628z.
Yuedong Zhu: Investigation, Synthesis, Spectral testing and cell
imaging, Data curation, Writing – original draft. Haiting Pan: Spectral
testing and cell imaging. Yanyan Song: Cell imaging. Chao Jing:
Writing – review & editing. Jia-An Gan: Writing – review & editing.
Junji Zhang: Conceptualization, Resources, Supervision, Writing – re-
view & editing.
[14] Li P, Shi XH, Xiao HB, Ding Q, Bai XY, Wu CC, Zhang W, Tang B. Two-photon
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introducing tellurium to mimetic glutathione peroxidase for monitoring the redox
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Declaration of competing interest
[16] Yang XP, Liu WY, Tang J, Li P, Weng HB, Ye Y, Xian M, Tang B, Zhao YF. A multi-
signal mitochondria-targeted fluorescent probe for real-time visualization of
cysteine metabolism in living cells and animals. Chem Commun 2018;54(81):
11387–90. https://doi.org/10.1039/c8cc05418e.
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
[17] Lim CS, Masanta G, Kim HJ, Han JH, Kim HM, Cho BR. Ratiometric detection of
mitochondrial thiols with a two-photon fluorescent probe. J Am Chem Soc 2011;
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Acknowledgments
[18] Chen XQ, Zhou Y, Peng XJ, Yoon J. Fluorescent and colorimetric probes for
detection of thiols. Chem Soc Rev 2010;39(6):2120–35. https://doi.org/10.1039/
b925092a.
The project was supported by NSFC (21878086), the Shanghai
Rising-Star Program (19QA1402500 to J. Z.), Shanghai Sailing Program
(18YF1405700 to C. J.) and the Fundamental Research Funds for the
Central Universities (222201717003). The authors acknowledge also
acknowledge the Shanghai Municipal Science and Technology Major
Project (Grant No.2018SHZDZX03) and the international cooperation
program of Shanghai Science and Technology Committee
(17520750100).
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[22] Lee HY, Choi YP, Kim S, Yoon T, Guo ZQ, Lee S, Swamy KMK, Kim G, Lee JY, Shin I,
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Appendix A. Supplementary data
[23] Hwang C, Sinskey AJ, Lodish HF. Oxidized redox state of glutathione in the
endoplasmic reticulum. Science 1992;257(5076):1496–502. https://doi.org/
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Supplementary data to this article can be found online at https://doi.
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