A lysosome-targetable fluorescent probe for real-time imaging cysteine under oxidative stress in living cells

Article abstract of DOI:10.1016/j.saa.2019.117175

As an effective lysosomal biomarker for oxidative stress status, cysteine (Cys)plays an important role in lysosomal proteolysis. Herein, we report the first lysosome-targetable fluorescence probe (MCAB)for Cys-selective detection based on nucleophilic add

Full text of DOI:10.1016/j.saa.2019.117175

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 221 (2019) 117175
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Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
journal homepage: www.elsevier.com/locate/saa
A lysosome-targetable fluorescent probe for real-time imaging cysteine
under oxidative stress in living cells
Xiao-dong Wang a,1, Li Fan a, ,1, Jin-yin Ge a, Feng Li b, Cai-hong Zhang a, Juan-juan Wang c,
⁎
Shao-min Shuang a, Chuan Dong a,
⁎
a
Institute of Environmental Science, College of Chemistry and Chemical Engineering, Shanxi University, Taiyuan 030006, PR China
Department of Chemistry, Centre for Biotechnology, Brock University, St. Catharines, Ontario L2S 3A1, Canada
Scientific Instrument Center, Shanxi University, Taiyuan 030006, PR China
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
As an effective lysosomal biomarker for oxidative stress status, cysteine (Cys) plays an important role in lyso-
somal proteolysis. Herein, we report the first lysosome-targetable fluorescence probe (MCAB) for Cys-selective
detection based on nucleophilic addition reaction of sulfhydryl toward a α, β-unsaturated ketone and demon-
strate its application to lysosomal-targetable imaging. MCAB is designed based on a α, β-unsaturated
ethanoylcarbazole as the fluorophore and the thiols reaction site, and a methylcarbitol unit as a lysosome-
targetable group. Upon reacting with Cys, this probe turns on highly specific fluorescence signals linearly propor-
tional to Cys concentrations over the range of 0–300 μM. MCAB detects Cys with a rapid response time (within
12 min) and low limit of detection (0.38 μM). MCAB is highly selective to Cys over other similar biothiols includ-
ing homocysteine (Hcy) and glutathione (GSH). Moreover, it also exhibits significant lysosomal-targetable abil-
ity, which is ideal for lysosomal Cys-selective imaging. Using MCAB, we have successfully visualized the
fluctuation endogenous Cys in lysosomes under oxidative stress status in real-time.
Received 11 January 2019
Received in revised form 22 April 2019
Accepted 26 May 2019
Available online xxxx
Keywords:
Fluorescent probe
Lysosome-targetable
Real-time imaging
Cysteine
Oxidative stress
© 2019 Elsevier B.V. All rights reserved.
1. Introduction
disulphide bonds [12]. For example, GSH is an effective stimulator of al-
bumin proteolysis in kidney lysosomes, whereas Cys can be degradated
Low molecular weight biothiols, like cysteine (Cys), homocysteine
(Hcy), and glutathione (GSH), play crucial roles in many physiological
and pathological processes [1–3]. Despite the similar structures of the
three biothiols, they perform various important biological functions, as-
sociated with different diseases. Among those, Cys within normal intra-
cellular concentration (30–200 μM) is the precursor of protein
synthesis, the source of sulfide in human metabolism, as well as the an-
tidote for toxic hepatitis [4,5]. Deficiency in Cys has involved in many
syndromes related to slowed growth in children, liver damage, lethargy,
psoriasis, as well as hair depigmentation [6–8]. On the other hand, ex-
cess amounts of Cys are associated with Parkinson's disease, and
Alzheimer's disease [9]. Therefore, it is highly desirable and urgently
needed to develop an effective tool for the specific monitoring Cys
over other biothiols (Hcy and GSH) in live cells.
in liver lysosomes [13]. Moreover, Cys is more effective to be oxidized
than GSH, making Cys as a more effective lysosomal biomarker for oxi-
dative stress [14,15]. The concentration level of Cys can be altered in ox-
idative stress, which may further lead to other diseases including
cancers and neurotoxicity-related diseases [15–17]. In order to have a
better understanding about the role of Cys within lysosomes, it is funda-
mentally important to develop Cys-selective detection methods to
monitor lysosomal Cys in living cells.
Thiols-selective fluorescent probes have been developed in the past
decade [18–42]. However, very few probes have been designed for
lysosomal-targetable biothiols imaging [43–49]. The first example of
lysosomal-targetable probe for biothiols detection was reported by
Kang and Kim's group through the thiol-induced disulfide cleavage of
a gemcitabine-coumarin-biotin conjugate [43]. Colocalization experi-
ments revealed that this response predominately occurred in the lyso-
some possibly via receptor-mediated endocytosis. Song's group
developed a series of acid-activatable Michael-type fluorescent probes
(QMAs), which have been used to imaging thiols within lysosomes in
live cells [44]. Fan and co-workers reported an efficient two-photon
1,8-naphthalimide based probe, enabling to signal thiols via a nucleo-
philic substitution reaction with deprotection of 2,4-dinitrobenzene-
sulfonyl (DNBS) [45]. Recently Wang and co-workers exploited a
Lysosomes are membrane-bound cytoplasmic organelles that serve
as a major degradative compartment in eukaryotic cells, due to the ap-
proximately 50 different degradative enzymes within lysosomes
[10,11]. Thiols facilitate proteolysis in the lysosome by reducing
⁎
Corresponding authors.
E-mail addresses: fanli128@sxu.edu.cn (L. Fan), dc@sxu.edu.cn (C. Dong).
These authors contributed equally to this work.
1
https://doi.org/10.1016/j.saa.2019.117175
1386-1425/© 2019 Elsevier B.V. All rights reserved.

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X. Wang et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 221 (2019) 117175
naphthalimide-based fluorescent probe for lysosomal thiols sensing via
conjugation and removal of the specific trigger, acrylate moiety [47]. Nev-
ertheless, these probes were either failed in discriminating lysosomal Cys
over Hcy and GSH, or sense GSH from Cys/Hcy. Till 2018, Liu's group re-
ported a coumarinocoumarin-based probe (CCy) employing an acrylate
group as the thiol recognition unit and the N, N-diethyl group as
lysosome-targeting group, for specifically detecting lysosomal Cys over
Hcy and GSH [49]. As we know this probe is the only one report about ly-
sosomal fluorescent biosensor for Cys-selective imaging. Unfortunately,
these probes bearing nitrogen-containing side chains including 4-(2-
hydroxyethyl)morpholine or N,N-dimethylethylenediamine as
lysosome-targeting groups would exhibit an “alkalizing effect” on lyso-
somes, such that longer incubation with these probes could induce an in-
crease in lysosomal pH [50,51]. Therefore, it is of great significance and
practical value to develop novel lysosomal-targetable fluorescent probe
for Cys-selective detection.
In this respect, an excellent fluorescent probes for real-time imaging
lysosomal Cys should meet at least two prerequisites: [1] appropriate
mechanism making the reaction between the probe and Cys reversible
and fast to promote real-time imaging, [2] the lysosome-targeting group
should have no nitrogen atoms to avoid the “alkalizing effect” on lyso-
somes. With these criteria in mind, we selected α, β-unsaturated
ethanoylcarbazole as the fluorophore and the thiols reaction site
(Scheme 1), which may feature reversible responses toward thiols with
the addition of N-ethylmaleimide (NEM), a thiol-scavenging reagent
[24]. On the other hand, a methylcarbitol was introduced into carbazole
to avoid the “alkalizing effect” on lysosomes [50–52]. Carbazole dyes
have been widely exploited as fluorescence sensing or imaging probes be-
cause of their desirable photophysical properties and low-toxicity
[52–58]. By integrating methylcarbitol-substituted ethanoylcarbazole
and terephthalaldehyde, we have successfully synthesized MCAB
(Scheme 1), a lysosomal-targetable probe with fast response and highly
selectivity to imaging Cys in living cells. As expected, MCAB displays sig-
nificant lysosome-targetable ability. We also demonstrated the applicabil-
ity of MCAB for the real-time imaging of lysosomal Cys fluctuation in H2O2
or lipopolysaccharide (LPS)-induced oxidative stress in living cells. Fur-
thermore, as far as we know, MCAB is the first reported probe based on
nucleophilic addition reaction of sulfhydryl toward a α, β-unsaturated ke-
tone to image lysosomal Cys over other thiols.
2. Results and discussion
2.1. Materials and apparatus
All chemicals and solvents were of analytical grade and were used
without further purifications. 2-(2-Methoxyethoxy)ethanol, Carbazole,
acetyl chloride, aluminium chloride, terephthalaldehyde and tosyl chlo-
ride were purchased from Shanghai Macklin Biochemical Technology
Co., Ltd. LysoTrackerTM Green DND-26 were obtained from ThermoFisher
Scientific company. N-ethylmaleimide were purchased from Sigma-
Aldrich company. Lipopolysaccharides was commercially available from
Dalian Meilun Biotechnology Co., Ltd. 3-(4, 5-Dimethylthiazol-2-yl)-2,
5-diphenyltetrazolium bromide was obtained from Thermo-Fisher Bio-
chemical Products (Beijing) Co., Ltd. All other chemicals and solvents
were bought from Tianjin chemical reagent company.
NMR spectra were recorded on a Bruker instrument (AVANCE DRX,
Fälanden, Switzerland) using tetramethylsilane (TMS) as the internal
standard in the solvent deuterated dimethyl sulfoxide (DMSO d6), deu-
terated chloroform (CDCl3) or deuteriumoxide (D2O) with 600 MHz for
1H NMR and 150 MHz for 13C NMR, respectively. LC-MS was obtained by
an Agilent Accurate-Mass-Q-TOF MS 6520 system equipped with an
electrospray ionization (ESI) source (Agilent, USA). Deionized water
was measured with a Milli-Q water purification system (Millipore).
The pH value was acquired with a Beckman pH-3c digital meter (Shang-
hai LeiCi Device Works, Shanghai, China). The UV–visible spectra were
performed on a UV–vis spectrophotometer (U-2910, Hitachi Co., Ltd.
(Beijing, China). Fluorescence spectra were taken on a FLS-920 Edin-
burgh Fluorescence Spectrophotometer (Edinburgh Co., Ltd., England)
equipped with a xenon discharge lamp using 1 mL Fluor Micro Cell.
Fluorescence images were carried out on a confocal laser scanning mi-
croscope (Zeiss, LSM880, Germany).
2.2. Synthesis and characterization
2.2.1. Synthesis of (2-methoxyethoxy) methyl 4-methylbenzenesulfonate (1)
2-(2-Methoxyethoxy)ethanol (6.01 g, 50 mmol) was dissolved in an
aqueous solution of sodium hydroxide (7.00 g, 75 mmol), and the mix-
ture was stirred for 1 h under ice-water bath. Then THF solution (50 mL)
of tosyl chloride (11.4 g, 60 mmol) was added dropwise to the above
Scheme 1. Synthetic scheme for probe MCAB and the proposed sensing mechanism of MCAB with Cys.

X. Wang et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 221 (2019) 117175
3
solution mixture over 2 h, and the reaction mixture was stirred for 12 h
at room temperature. The solution was poured into ice water, and the
product was extracted with dichloromethane three times. The organic
layer was dried over anhydrous magnesium sulfate filtered and evapo-
rated. The crude colorless oil product was used for the subsequent syn-
thesis without purification. The yield was 82%.
2.0 nm, and the excitation wavelength was fixed at 370 nm. All spectro-
scopic experiments were measured at room temperature.
2.4. Determination of detection limit
The limit of detection (DL) of MCAB for Cys was calculated based on
the fluorescence titration data and determined from the following equa-
tion [59]:
2.2.2. Synthesis of 9-(2-(2-methoxyethoxy)ethyl)-9H-carbazole (2)
Carbazole (3.34 g, 20 mmol) and sodium hydrogen (0.72 g,
30 mmol) were first dissolved in THF (50 mL), then NaH (0.72 g,
30 mmol) was added. The solution was stirred for 30 min, compound
1 (8.23 g, 30 mmol) was added with constant stirring. The resulting
mixture was stirred at room temperature for 5 h. The solution was
poured into ice water, and the product was extracted with ethyl acetate
three times. The organic layer was dried over anhydrous magnesium
sulfate filtered and evaporated. The crude product was purified by col-
umn chromatography (ethyl acetate/petroleum ether, 1/5, v/v) to afford
the desired compound 2 in 74% yield as yellow oily product. 1H NMR
(DMSO d6, 600 MHz) δ (ppm): 8.07 (d, 2H), 7.44 (m, 4H), 7.23 (m,
2H), 4.47–4.49 (t, 2H), 3.83–3.85 (t, 2H), 3.49 (m, 2H), 3.40 (m, 2H),
3.29 (s, 3H).
DL ¼ 3σ=K
where σ is the standard deviation of the blank solution; K is the slope of
the calibration curve.
2.5. Fluorescent imaging in live cells
SMMC-7721 cells were cultured in RPMI1640 medium supple-
mented with 10% fetal bovine serum 37 °C in a 5% CO2 atmosphere.
For colocalization experiments, MCAB dissolved in DMSO (10 μL,
1 mM) are added to the cells medium (1 mL) at 10 μM final concentra-
tions for 10 min, and then cells were washed with PBS (pH 7.4) three
times. 10 μM LysoTracker Green was then added and co-incubated for
additional 30 min, and cell imaging was carried out after washing cells
with PBS three times. Then fluorescence images are carried out on a
confocal laser scanning microscope (Zeiss, LSM880) with blue channel
(Ex = 405 nm, Em = 410–500 nm) for MCAB, Green channel (Ex =
488 nm, Em = 493–588 nm) for LysoTracker Green, respectively.
For fluorescent imaging experiments, the SMMC-7721 cells were
first incubated with MCAB (10 μM) for 10 min. Then excess MCAB
were removed by rinsing with PBS (pH 7.4) three times, and incubated
with NEM (0.5 mM) for 40 min, thiols (Cys, Hcy or GSH, 100 μM) for
20 min, H2O2 (200 μM) for 30 min or LPS (50 μL in saline, 1 mg/mL)
for 120 min, respectively. The fluorescence images are carried out on a
confocal laser scanning microscope (Zeiss, LSM880) with blue channel
(Ex = 405 nm, Em = 410–500 nm).
2.2.3. Synthesis of 9-(2-(2-methoxyethoxy)ethyl)-9H-carbazole-3-yl)ace-
tyl (3)
Compound 2 (5.38 g, 20 mmol) and aluminium chloride (2.67 g,
20 mmol) were dissolved in 50 mL dichloromethane (DCM) in a
100 mL round bottom flask before acetyl chloride (1.73 g, 22 mmol)
was added. The reaction mixture was stirred for 15 h at room tempera-
ture. Then the solution was poured into ice water, and extracted with
dichloromethane three times. The organic layer was dried over anhy-
drous magnesium sulfate filtered and evaporated. The crude product
was purified by column chromatography (dichloromethane/methanol,
20/1, v/v) to afford the desired compound 3 in 65% yield as white needle
solid. 1H NMR (CDCl3, 600 MHz) δ (ppm): 8.73 (s, 1H), 8.13 (m, 2H),
7.49 (m, 3H), 7.31 (m, 1H), 4.52 (t, 2H), 3.88 (t, 2H), 3.51 (t, 2H), 3.41
(t, 2H), 3.29 (s, 3H), 2.72 (s, 3H).
3. Results and discussion
2.2.4. Synthesis of 4-(3-(9-(2-(2-methoxyethoxy)ethyl)-9H-carbazol-3-
yl)-3-acryloyl) benzaldehyde (MCAB)
3.1. Synthesis of the probe MCAB
A mixture of compound 3 (3.11 g, 10 mmol), terephthalaldehyde
(2.01 g, 15 mmol) and piperidine (1 mL) were dissolved anhydrous eth-
anol (50 mL) and then stirred overnigth under 85 °C. The reaction was
cooled to room temperature, and then poured into ice water, then ex-
tracted with dichloromethane three times. The organic layer was dried
over anhydrous magnesium sulfate filtered and evaporated. The crude
product was purified by column chromatography (chloroform/metha-
nol, 20/1, v/v) to afford the desired probe in 53% yield as dark green
solid. 1H NMR (600 MHz CDCl3) δ (ppm): 10.08 (s, 1H), 8.93 (s, 1H),
8.29–8.28 (d, 1H), 7.97–7.92 (m, 3H), 7.90–7.88 (m, 5H), 7.62–7.60 (d,
2H), 7.43 (m, 1H), 4.60 (s, 2H), 3.96 (s, 2H), 3.56 (s, 2H) 3.43 (m, 2H),
3.30 (s, 3H). 13C NMR (150 MHz, CDCl3) δ (ppm): 191.53, 188.71,
144.45, 142.27, 140.87, 137.23, 128.91, 124.84, 123.23, 122.24, 109.84,
71.99, 70.96, 69.38, 59.10, 29.71. HR-MS m/z: [M H] calclated for
The synthesis route of MCAB is displayed in Scheme 1. Compound 1
and 2 were synthesized from 2-(2-methoxyethoxy)ethanol and carba-
zole according to our previous report [52]. Compound 2 was then
refluxed with acetyl chloride to give the corresponding acelylcarbazole
derivative, compound 3 in 65% yield. Subsequent condensation of Com-
pound 3 with terephthalaldehyde by piperidine-catalyzing afforded the
desired product. After being purified by silica gel column chromatogra-
phy, MCAB was obtained as a dark green solid in 53% yield, and was fully
characterized by 1H NMR, 13C NMR (Fig. S1), and HR-MS analysis
(Fig. S2). The data obtained were consistent with the proposed
structures.
3.2. UV–vis and fluorescence measurements
C27H25NO4, 428.1856; measured, 428.1855.
The time-dependent sensing behavior of MCAB to Cys was primarily
investigated in DMSO/PBS (1/1, v/v, pH 7.4). As shown in the fluores-
cence spectra (Fig. 1a and Fig. S3), MCAB (25 μM) displayed almost no
fluorescence under excitation at 370 nm. Upon treating with 500 μM
Cys, MCAB exhibited a significantly time-dependent turn-on fluores-
cence signal at 440 nm with blue fluorescent color observed (inset of
Fig. 1a). Moreover, the emission intensity attained saturation within
12 min, indicating a faster response than that of Liu's report [49]. Mean-
while, the maximum absorption peak in the UV–vis spectra changed
from 300 nm (ε = 2.735 × 104·M−1·cm−1), 388 nm (ε = 1.414
× 104·M−1·cm−1) to 255 nm (ε = 3.042 × 104·M−1·cm−1) and 338 nm
(ε = 1.367 × 104·M−1·cm−1) (Fig. 1b).
2.3. UV–vis and fluorescence measurements
Doubly distilled water was used to prepare all the aqueous solutions.
Stock solution of MCAB (1.0 mM) was prepared in DMSO, and the solu-
tions for spectroscopic determination were obtained by diluting the
stock solution to a final concentration of 25 μM with DMSO/PBS (1/1,
v/v) at pH 7.4. Stock solutions (0.1 M) of the analytes (including
biothiols, amino acids and ions) were prepared in doubly distilled
water. For optical measurements, 2 mL of MCAB and compound 1 solu-
tion were poured into a quartz optical cell of 1 cm optical path length
each time. Excitation and emission bandwidths were both set at

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X. Wang et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 221 (2019) 117175
Fig. 1. (a) Fluorescence spectral changes of MCAB (25 μM) against time of 40 min in the presence of Cys (500 μM) in DMSO/PBS (1/1, v/v, pH 7.4). Inset: The photograph of MCAB solution
in the absence or presence of Cys under a UV light. (b) UV−vis spectral changes of MCAB (25 μM) against time of 332 min in the presence of Cys (500 μM) in DMSO/PBS (1/1, v/v, pH 7.4).
(c) Fluorescent kinetic of MCAB (25 μM) with 500 μM of Cys, Hcy and GSH in DMSO/PBS (1/1, v/v, pH 7.4) within 180 min. (d) A linear relationship of the fluorescence emission with Cys
concentration in the range of 0–300 μM, and the calculated detection limit was 0.38 μM.
In order to study the reactivity characteristics, the time-dependent
fluorescence spectra change of MCAB with other thiols including Cys,
Hcy and GSH, were evaluated, respectively. As seen in Fig. 1c and
Fig. S3, under the same reaction conditions, MCAB displayed a more
than 24-fold enhancement within 12 min in the presence of 20 equiv.
of Cys, while no more than 8-fold fluorescence enhancement was ob-
served in other biothiols. It is obvious that MCAB exhibited a much
higher reactivity resulting in the potential application for rapidly dis-
criminating Cys from Hcy and GSH in physiological conditions. In addi-
tion, the capability of MCAB for quantitative detection of Cys was
investigated. As depicted in Fig. S4, there was a distinct enhancement
in the fluorescence emission intensity of MCAB along with the concen-
tration of Cys increasing from 0 to 500 μM. Meanwhile, the fluorescence
intensity was found to be linearly proportional to Cys concentration in
the range of 0–300 μM, with a good linear coefficient (R2) of 0.9958
(Fig. 1d), and the calculated detection limit (DL) of MCAB for Cys was
as low as 0.38 μM.
Comparison of the 1H NMR spectra of MCAB and MCAB-adduct, the
original signals at 8.14 and 7.20 ppm, which belong to the α, β-
unsaturated ketone protons, disappeared, and new signals appeared at
5.76, 5.57 and 4.26 ppm. The results indicated that −SH-induced fluores-
cence enhancement was resulted from the nucleophilic addition reac-
tion of sulfhydryl toward the α, β-unsaturatedketone in MCAB.
Meanwhile, in order to obtain more detailed insights into the mech-
anism, time-dependent density functional theory (TDDFT) calculations
were conducted for MCAB with the B3LYP exchange functional
employing 6-31G (d) basis sets using a suite of Gaussian 09 programs.
Shown in Fig. S6 are the optimized structures and the lowest unoccu-
pied and the highest occupied molecular orbital (LUMO and HOMO)
plots of MCAB and MCAB-adduct in the ground state. The energy gap
between the HOMO and the LUMO of MCAB (3.22 eV) was larger than
that of MCAB-adduct (4.37 eV), which was in good agreement with
the blue shift in the absorption spectra of MCAB upon binding with Cys.
3.4. Effect of pH and selectivity of probe MCAB toward Cys
3.3. Mechanism of probe DNEPI responding to Cys
Considering the further application in live cells, the effect of pH
(from 3 to 11) on the fluorescence of MCAB in the absence or presence
of Cys (500 μM) was evaluated. As shown in Fig. S7, the fluorescence
emission of MCAB at 440 nm showed no significant change within the
To explore the fluorescence response mechanism, the interaction
between MCAB and Cys was monitored by 1H NMR titration experi-
ments after an addition of Cys over a period of 10 min (Fig. S5).

X. Wang et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 221 (2019) 117175
5
Furthermore, selectivity experiments were also conducted in the
presence of biological thiols such as Cys, Hcy, GSH, or other different
amino acids (Try, Glu, Asn, His, Arg, Pro, Phe, Leu, Gln, Ser, Val, Met,
Ile, Asp, Thr, Tyr, Ala, Iso, Lys, Gly), sulfureted anions (S2−, SO32−, SO42−
,
HSO3−), and other common ions (K , Na , Ca2 , Mg2 , Al3 , Zn2 , Cu2 , Fe3 ,
Fe2 , Cl−, Br -, CO23−, AcO−, NO3−, NO−2). As depicted in Fig. 2, MCAB
displayed remarkable fluorescence enhancement in the presence of
Cys but weak response toward Hcy or GSH, indicating that MCAB
could separate Cys from Hcy and GSH. Under the same condition, the re-
sults showed that other interfering species also scarcely had any influ-
ence on the detection of Cys. Therefore, the probe MCAB is highly
selective toward Cys without any interference from other biologically
relevant analytes.
3.5. Fluorescence imaging of Cys in live cells
Fig. 2. Fluorescence responses of MCAB (25 μM) toward thiols (500 μM), different amino
acids (500 μM) and essential ions (500 μM) in DMSO/PBS (1/1, v/v, pH 7.4). (1) blank;
(2) Cys; (3) Hcy; (4) GSH; (5) Try; (6) Glu; (7) Asn; (8) His; (9) Arg; (10) Pro; (11)
Phe; (12) Leu; (13) Gln; (14) Ser; (15) Val; (16) Met; (17) Ile; (18) Asp; (19) Thr; (20)
Tyr; (21) Ala; (22) Iso; (23) Lys; (24) Gly; (25) S2−; (26) SO23−; (27) SO24−; (28) HSO3−;
Before cell imaging, we use a MTT assay to evaluate the cytotoxicity
of MCAB at various probe concentrations (from 0 to 20 μM). As shown in
Fig. S8, more than 89% of cells are viable, suggesting the low toxicity of
MCAB under the experimental conditions (10 μM), and inferring their
potential application in intracellular imaging of live cells.
As reported that methylcarbitol has the capability of targeting the ly-
sosomes of living cells [50–52]. We next investigated the specificity of
MCAB toward lysosomes by a colocalization imaging experiments,
where live SMMC-7721 cells were stained with both MCAB (10 μM)
(29) K ; (30) Na ; (31) Ca2 ; (32) Mg2 ; (33) Al3 ; (34) Zn2 ; (35) Cu2 ; (36) Fe3 ; (37) Fe2
(38) Cl−; (39) Br−; (40) CO32−; (41) AcO−; (42) NO3−; (43) NO−2.
;
pH range of 3.0–9.0, indicating that free probe was very stable over a rel-
atively wide pH range. Interestingly, after treating with Cys, the emis-
sion intensity was kept on a high level with pH value, indicating
MCAB could work well as a probe both in cytoplasm and lysosome.
and
a
commercial lysosome-specific green fluorescent dye,
(LysoTracker Green DND-26, 10 μM). As demonstrated in Fig. 3c, the
blue fluorescence image of MCAB (Fig. 3a) overlapped well with the
green fluorescence image of LysoTracker Green (Fig. 3b, c). Most
Fig. 3. Confocal microscopy fluorescence images of 10 μM MCAB (a) and LysoTracker Green DND-26 (10 μM) (b) costained in HeLa cells. (c) Brightened field image. (d) Merged image.
(e) The correlation of MCAB and LysoTracker Green DND-26 intensities (A = 0.90). (f) Intensity profile of linear regions across SMMC-7721 cells. The blue channel image was
collected at 410–500 nm (λex = 405 nm) for MCAB. The green channel image was collected at 510–550 nm (λex = 488 nm) for LysoTracker Green DND-26. Scale bar: 5 μm. (For
interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

6
X. Wang et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 221 (2019) 117175
Fig. 4. Confocal microscopy fluorescence images of biothiols in SMMC-7721 cells. (a) SMMC-7721 cells stained with MCAB (10 μM) for 10 min. (b) SMMC-7721 cells pretreated with NEM
(0.5 mM) for 40 min and then stained with MCAB (10 μM) for 10 min. (c-e) SMMC-7721 cells pretreated with NEM (0.5 mM) for 30 min and then stained with Cys, Hcy, or GSH (100 μM)
for 20 min, respectively, and finally stained with MCAB (10 μM) for 10 min. The fluorescence images were carried out on a confocal laser scanning microscope (Zeiss, LSM880) with blue
channel (Ex = 405 nm, Em = 410–500 nm). The right two images represent the corresponding bright field and overlay images, respectively. Scale bar: 10 μm. (For interpretation of the
references to color in this figure legend, the reader is referred to the web version of this article.)
notably, the changes in the intensity profile of linear regions of interest
(MCAB and LysoTracker Green co-staining) tended toward synchroni-
zation (Fig. 3e). Moreover, the high average Pearson's co-localization
coefficient (A) is evaluated to be 0.90 (Fig. 3f). There results indicated
that MCAB can specifically target to lysosomes in live cells.
3.6. Real-time imaging of Cys in live cells under oxidative stress status
As known that the oxidative stress status with enhanced production
of reactive oxygen species (ROS) could trigger a decreasing of Cys con-
centration level, which impairs the function of the cellular antioxidant
defence system [15,60,61]. In order to explore the relationship between
lysosomal Cys concentration level and oxidative stress, MCAB-stained
SMMC-7721 cells were first challenged with 200 μM H2O2 for 30 min.
Fig. 5 a, c shows that MCAB-stained SMMC-7721 cells featured stable
fluorescent emission in the blue channel within 30 min, indicating
that MCAB could image lysosomal Cys with good photostability. Then
H2O2 was added, no apparent fluorescent changes were observed in
the initial 20 min (Fig. 5b, c). This might be attributed to the consump-
tion of H2O2 by the originally existing intracellular GSH [24]. After equi-
librium, a weak fluorescence emission quenching occurred in the
following 10 min, implying that the decrease of the lysosomal Cys con-
centration induced by H2O2 oxidation of Cys.
With its intrinsic ability to target lysosomes confirmed, MCAB
was further studied for its Cys-selective fluorescence response. As
can be seen from Fig. 4a, MCAB-stained SMMC-7721 cells exhibited
bright blue fluorescence, resulting from the presence of endogenous
Cys in live cells. Subsequently, SMMC-7721 cells were pretreated
with N-ethylmaleimide (NEM, 0.5 mM) for 40 min to remove endog-
enous thiols, and then 100 μM Cys, Hcy or GSH were added for an-
other 20 min, respectively. Almost no fluorescence imaging was
observed in the NEM-pretreated SMMC-7721 cells (Fig. 4b), whereas
blue fluorescence imaging enhanced after an addition of Cys
(Fig. 4c). As another control experiment, the treatment of Hcy or
GSH hardly afford any fluorescence emission (Fig. 4d, e), indicating
that MCAB could detect lysosomal Cys with negligible interference
from Hcy and GSH.
In addition, the oxidative stress of SMMC-7721 cells was mediated
by lipopolysaccharide (LPS, 50 μg/mL) [15,62]. As shown in Fig. 6, the

X. Wang et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 221 (2019) 117175
7
Fig. 5. Time-dependent Cys fluorescence imaging with MCAB in SMMC-7721 cells under oxidative stress status mediated by H2O2. (a) SMMC-7721 cells were treated with MCAB (10 μM)
for 10 min and washed by PBS (10 mM, pH = 7.4) three times. (b) SMMC-7721 cells were treated with MCAB (10 μM) for 10 min and washed by PBS (pH = 7.4) three times, and then
H2O2 (200 μM) was added. The fluorescence images were carried out on a confocal laser scanning microscope (Zeiss, LSM880) with blue channel (Ex = 405 nm, Em = 410–500 nm). The
right images represent the corresponding bright field images. Scale bar: 10 μm. (b) Corresponding time-dependent arithmetic mean intensity changes of the PBS or H2O2-treated cell
images. Data are expressed as mean SD of three parallel experiments. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of
this article.)
mean fluorescence intensity of MCAB decreased in LPS-treated cells,
indicating a gradual decline of lysosomal Cys concentration along
with the time. Taken together, the above results confirmed that the
probe MCAB possessed the satisfactory ability in real-time imaging
of endogenous Cys level fluctuation in live cells under oxidative
stress status.
oxidative stress status. As a result, our probe would be practically
useful and greatly potential powerful tool for elucidation of the intri-
cate roles of Cys in oxidative stress responses.
Acknowledgements
4. Conclusion
Financial supports from the National Natural Science Foundation of
China (No. 21475080, 21575084 and 21874087), Natural Science Foun-
dation of Shanxi Province (No. 2016021036), Scientific and Technolog-
ical Innovation Programs of Higher Education Institutions in Shanxi and
the Shanxi Province Hundred Talents Project. We appreciate Scientific
Instrument Center (Shanxi University, Taiyuan, China) for us to identify
samples and technician and Modern Research Center for Tradition Chi-
nese (Shanxi University, Taiyuan, China) for kindly providing us with
SMMC-7721 cells.
In summary, a novel fluorescent probe (MCAB) was developed for
lysosomal Cys imaging in live cells, by introducing a α, β-
unsaturated ethanoylcarbazole as the fluorophore and the thiols re-
action site, and a methylcarbitol unit as a lysosome-targetable group.
The probe exhibited a selectively marked turn-on emission upon
reacting with Cys over Hcy and GSH. More importantly, the probe
could detect Cys with a fast response time (within 12 min) and the
detection limit was quantitatively calculated as 0.38 μM. Meanwhile,
with good biocompatibility and low cell cytotoxicity, MCAB was fur-
ther used for fluorescence imaging in live cells. We revealed that
MCAB can selectively diffuse into lysosomes and permit the real-
time imaging of endogenous Cys level fluctuation in live cells under
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.saa.2019.117175.

8
X. Wang et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 221 (2019) 117175
Fig. 6. Time-dependent Cys fluorescence imaging with MCAB in SMMC-7721 cells under oxidative stress status mediated by lipopolysaccharide (LPS). (a) SMMC-7721 cells were treated
with MCAB (10 μM) for 10 min. (b-d) SMMC-7721 cells were mediated by LPS (50 μg/mL) for 30 min, 60 min or 120 min, respectively, and then stained with MCAB (10 μM) for another
10 min. The fluorescence images were carried out on a confocal laser scanning microscope (Zeiss, LSM880) with blue channel (Ex = 405 nm, Em = 410–500 nm). The lower images
represent the corresponding bright field images. Scale bar: 10 μm. (e) Corresponding time-dependent arithmetic mean intensity changes of the LPS-treated cell images. Data are
expressed as mean SD of three parallel experiments. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
M.M. Matzuk, Growth retardation and cysteine deficiency in gamma-glutamyl
transpeptidase-deficient mice, Proc. Natl. Acad. Sci. U. S. A. 93 (1996)
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