A fluorescent probe for intracellular cysteine overcoming the interference by glutathione

Article abstract of DOI:10.1039/c4ob00382a

Cysteine (Cys) plays important roles in many physiological processes of eukaryotic cells and its detection in cells is of fundamental significance. However, glutathione (GSH), homocysteine, N-acetyl-l-cysteine and other thiols greatly hamper the detection

Full text of DOI:10.1039/c4ob00382a

Organic &
Biomolecular Chemistry
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A fluorescent probe for intracellular cysteine
overcoming the interference by glutathione†
Minggang Tian,‡a Fuqiang Guo,‡a,e Yuming Sun,b Weijia Zhang,a Fang Miao,a
Yong Liu,a Guofen Song,a Cheuk-Lam Ho,c Xiaoqiang Yu,*a Jing Zhi Sun*d and
Wai-Yeung Wong*c,f
Cite this: Org. Biomol. Chem., 2014,
12, 6128
Cysteine (Cys) plays important roles in many physiological processes of eukaryotic cells and its detection
in cells is of fundamental significance. However, glutathione (GSH), homocysteine, N-acetyl-L-cysteine
and other thiols greatly hamper the detection of Cys. In particular, GSH strongly interferes with the
detection of cellular Cys (30–200 μM) due to its high intracellular concentration (1–10 mM). In this work,
an off–on fluorescent probe (HOTA) for the detection of Cys is presented. This probe possesses both
excellent sensitivity and satisfactory selectivity for cellular Cys detection: with the addition of 200 μM Cys,
the fluorescence intensity of the probe (10 μM) enhanced 117-fold and the detection limit was calculated
to be 13.47 μM, which is lower than the cellular Cys concentration; the probe also selectively detected
30–200 μM cysteine over 1–10 mM glutathione. Consequently, cell imaging experiments were performed
with probe HOTA. Furthermore, the results of the thiol-blocking and GSH synthesis inhibiting experiments
confirmed that the intracellular emission mainly originates from the interaction between Cys and HOTA.
Received 19th February 2014,
Accepted 28th May 2014
DOI: 10.1039/c4ob00382a
www.rsc.org/obc
organisms. For instance, abnormal levels of physiological Cys
are associated with many diseases, such as slow growth, hair
Introduction
Cysteine (Cys) bears a thiol group on its residue, which is depigmentation, edema, lethargy, liver damage, leucocyte loss,
nucleophilic and easily oxidized, and plays numerous roles in skin lesions and Alzheimer’s disease.2 Therefore, the exclusive
biological processes.1 For example, Cys is a crucial amino acid detection of intracellular Cys is a fundamental and vital work
of eukaryotic cells that constitutes various proteins and exhi- for biochemistry and biomedicine.
bits a high affinity to mercury, lead, cadmium and other heavy
Up to now, various methods, including high performance
metals. Cys is also related to the maintenance of the intra- liquid chromatography, gas chromatography-mass spectrometry,
cellular redox homeostasis, together with other biothiols including capillary electrophoresis separations, and electro-chemical
glutathione (GSH). More specifically, Cys exhibits distinctive assays, have been utilized for the analysis of physiological
and significant physiological functions and is indispensable to Cys.3 Optical assays with fluorescent probes exhibit distinct
advantages, such as facile operation, high sensitivity and
little damage to intact cells, and thus various fluorescent dyes
for the detection of Cys have been extensively investigated.4
Since Strongin et al. originally developed a fluorescent probe
containing aldehyde for the detection of Cys and homocysteine
(Hcy) over other thiols,5 fluorescent probes with aldehyde have
been studied intensively and fluorescence images of cells have
been obtained by some of the probes reported.6 For example,
Ma et al. have developed a probe for which adding Cys/Hcy
resulted in a significant fluorescence enhancement while
adding other thiols caused little interference.6 In addition, our
group has also reported three Cys-selective probes, CA1, AM1
and CB1, and applied them for cell imaging.7 CA1 and AM1
react with Cys immediately with high selectivity and CB1
enables the discrimination of Cys in 40 min.
aCentre of Bio and Micro/Nano Functional Materials, State Key Lab of
Crystal Materials, Shandong University, Jinan 250100, P. R. China.
E-mail: yuxq@sdu.edu.cn
bSchool of Information Science and Engineering, Shandong University, P. R. China
cInstitute of Molecular Functional Materials and Department of Chemistry and
Partner State Key Laboratory of Environmental and Biological Analysis,
Hong Kong Baptist University, Hong Kong, P. R. China.
E-mail: rwywong@hkbu.edu.hk
dMoE Key Laboratory of Macromolecule Synthesis and Functionalization,
Department of Polymer Science and Engineering, Zhejiang University,
Hangzhou 310027, P. R. China. E-mail: sunjz@zju.edu.cn
eDepartment of Physics, Changji University, Xinjiang, Changji 831100, P. R. China
fInstitute of Polymer Optoelectronic Materials and Devices, State Key Laboratory of
Luminescent Materials and Devices, South China University of Technology,
Guangzhou 510640, P. R. China
†Electronic supplementary information (ESI) available: Details of the synthetic
process and spectroscopic results. See DOI: 10.1039/c4ob00382a
‡Tian and Guo made equal contributions.
With the development of more and more probes for Cys, it
has been gradually recognized that the actual intracellular
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concentration of various biothiols has to be considered higher than the minimum concentration of intracellular Cys).
when discriminating intracellular Cys. In general, to certify the This slightly lower off–on ratio may be attributed to the
selectivity of a probe with equimolar bio-analytes in vitro is pyridine moiety. The electron withdrawing property of the pyri-
feasible in many cases. However, equimolar measurements dine moiety hampers the photo-induced electron transfer (PET)
would sometimes result in the deviation of the actual infor- process and thus results in a higher fluorescence intensity of
mation. Particularly, as intracellular concentrations of GSH CB1. Therefore, HOTA was designed by keeping the α,β-unsatu-
and Cys are 1–10 mM and 30–200 μM, respectively,8 abundant rated aldehyde group of CB1 intact but replacing the electron-
cellular GSH may greatly hamper Cys discrimination. To test withdrawing pyridine moiety with an electron-releasing phenolic
whether GSH will interfere with Cys detection at a cellular con- group. HOTA exhibited a similar reaction with Cys, as shown
1
centration, we have chosen an intermediate compound 3 in Scheme 1, and the mechanism was confirmed by H NMR
(Fig. S1†) as a probe. The results show that 3 selectively and HRMS data (Fig. S2 and S3†). Accordingly, HOTA is
detected Cys over equimolar GSH. However, at a cellular con- expected to exhibit both excellent selectivity and off–on
centration, the interference of GSH could not be ignored, responses to Cys with a lower detection limit, suitable for
while highly concentrated glycine never enhanced emission intracellular applications.
(Fig. S1†). Thus, in some cases, equimolar measurements might
be unreliable and it is extremely necessary to test the selectivity
to Cys with actual cellular concentrations of other thiols.
Results and discussion
Jung and co-workers firstly considered the effect of highly In vitro testing
concentrated GSH on cellular Cys detection and tried cou-
In order to evaluate the sensitivity of HOTA to Cys, the fluo-
rescence response of the probe to different amounts of Cys
was investigated. With the addition of 200 μM Cys, the fluo-
marin derivatives to detect Cys over GSH at intracellular con-
centrations.9 They still tested the selectivity by measuring the
fluorescence intensity of their probes in the presence of
rescence response at 450 nm increased by 117-fold immedi-
equally concentrated bio-analytes. To supplement the study, a
ately. The fluorescence response to Cys at 450 nm is linearly
related to the concentration of Cys in a range of 0–100 μM and
DFT calculation and kinetic analysis were carried out to prove
the selectivity. To date, developing a fluorescent probe for the
then sharply enhances with an increase in Cys concentration
discrimination of Cys over GSH at intracellular concentrations
(Fig. 1a). Therefore, these intensities were linearly fitted in a
is challenging work,10 because the molecular design of a probe
concentration range of 0–100 μM and the detection limit was
exhibiting such high selectivity is still lacking.
calculated to be 13.5 μM (Fig. 1b),11 which is lower than the
Here, we show a molecular improvement based on our pre-
minimum concentration of intracellular Cys, indicating that
viously reported probe CB1 and attempt to solve this tough
the sensitivity of HOTA is suitable for the intracellular detec-
tion of Cys.
problem using our novel probe, HOTA. Fortunately, this probe
discriminated Cys from GSH at intracellular concentrations
As stated above, the most abundant intracellular thiol GSH
with a large enhancement in fluorescence. According to our
may hamper the discrimination of cellular Cys due to its high
previous work, CB1 can react with Cys by forming a 7-mem-
concentration. Thus, fluorescence responses of the probe to
Cys and GSH were tested at intracellular concentrations (at
both the maximum and minimum concentrations) and the
results are displayed in Fig. 2. It can be clearly seen that the
interference from GSH is negligible at both the maximum and
minimum intracellular GSH concentrations. Particularly, the
bered ring, which is impossible for GSH. Hence, CB1 exhibits
high selectivity for Cys detection, but its sensitivity to Cys
needs to be improved: the fluorescence intensity of CB1 was
enhanced 13-fold in the presence of 600 μM Cys and the detec-
tion limit was calculated to be 59.8 μM using 10 μM probe (little
emission of HOTA with 200 μM Cys at 450 nm is much higher
Fig. 1 (a) Fluorescence intensity of HOTA (10 μM) in the presence of
Cys with concentrations of 0 μM, 10 μM, 20 μM, 30 μM, 40 μM, 50 μM,
60 μM, 80 μM, 100 μM, 130 μM, 150 μM, 170 μM and 200 μM. (b) Fluore-
scence response at 450 nm of HOTA (10 μM) to Cys with different
concentrations: 0 μM, 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 60 μM,
80 μM and 100 μM. Insert: linearly fitted regression equation and corres-
Scheme 1 Structures and sensing mechanisms of CB1 and HOTA.
ponding relevancy score.
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Fig. 4 (a) Fluorescence spectra of HOTA and its fluorescence responses
to various analytes in an EtOH–PBS buffer solution (v/v 1 : 1, 20 mM,
pH 7.4); (b) the histogram of the relative fluorescence intensities (450 nm)
shown in (a). Inset of (b): photo taken under UV light. Concentration of
HOTA: 10 μM; concentration of analytes: 20 equiv. (2–26), 1000 equiv.
(27, 28); analytes 1 to 28: 1: Blank, 2: Cys, 3: Hcy, 4: GSH, 5: NAC, 6: Ala,
7: β-ala, 8: Arg, 9: Asn, 10: Asp, 11: Gln, 12: Glu, 13: Gly, 14: His, 15: Ile,
16: Lys, 17: Leu, 18: Mpa, 19: Met, 20: Pro, 21: Phe, 22: Ser, 23: Tyr,
24: Thr, 25: Trp, 26: Val, 27: H2O2, 28: Gly; λex = 365 nm for all
measurements.
Fig. 2 Fluorescence responses of HOTA (10 μM) with the addition of
(a) 200 μM Cys and 10 mM GSH, and (b) 30 μM Cys and 1 mM GSH. Inset
of (a): photo of HOTA (10 μM) in the presence of 200 μM Cys (left) and
10 mM GSH (right) under UV light (λex = 365 nm).
than that with 10 mM GSH, and this selectivity could be
observed clearly with the naked eye under 365 nm UV light
(Fig. 2a).
For a more detailed evaluation of the selectivity, fluo-
rescence titration experiments of GSH in a concentration range
of 1–10 mM were performed (Fig. 3). The fluorescence inten-
sity of HOTA with GSH at 450 nm linearly increased with
increasing GSH concentration. It was found that the intensity
could be linearly fitted in this concentration range, with an R
value of 0.9876 (Fig. 3b). According to the two fitting
equations, when the concentrations of Cys and GSH were in
the blue-marked area above the red line (Fig. S4†), the interfer-
ence of GSH with Cys can be ignored.
Apart from GSH, there are many other intracellular bio-ana-
lytes that may cause interference. Therefore, fluorescence and
absorbance spectra of HOTA in the presence of 26 chosen bio-
analytes including biothiols and amino acids were investigated
(Fig. 4 and S5†). As depicted in Fig. S5,† the absorbance
spectra of HOTA exhibited little change with the addition of
different analytes, including Cys. However, the fluorescence of
the probe was greatly enhanced with the addition of Cys, while
adding other bio-analytes induced no significant enhancement
in fluorescence. This strong emission caused by Cys was
observed with the naked eye (Fig. 4b, inset).
group, Cys/Hcy could have special cyclization reactions with
the probe, unlike other thiols. These cyclization reactions have
a more stable product and may have a lower reaction barrier
than normal addition reactions of other thiols and therefore
lead to higher reaction rates of Cys/Hcy. Secondly, the thiol
group of Cys was reported to be more reactive than that of
Hcy.12 Moreover, the corresponding 7-membered ring (Cys) is
generally more stable than the 8-membered ring (Hcy). Accord-
ingly, the reaction of HOTA and Cys is much quicker than that
of HOTA and Hcy or other thiols.
Before using HOTA to image living cells, we performed
competition experiments (Fig. S6†) and investigated the influ-
ences of the pH value (Fig. S7†) and viscosity (Fig. S8†) on bio-
imaging. In the competition experiments, the fluorescence
intensity at 450 nm in the presence of Cys and each bio-
analyte was obtained. The experimental results show that the
addition of other bio-analytes hardly resulted in any signifi-
cant changes to the fluorescence of HOTA with Cys. Mean-
while, fluorescence spectra of HOTA in the presence of Cys
using buffer solutions with a series of pH values were also
examined. The fluorescence spectra of HOTA at different pH
values are almost identical. The system of HOTA and Cys
exhibited a strong emission at the pH range of the cytoplasm
(6.8–7.4),13 indicating that the intracellular pH would not
cause any interference. The fluorescence responses of HOTA in
the mixed solvents of water and glycerol with a series of vis-
cosity values were also tested. The results demonstrated that
the emission spectra of HOTA in a wide viscosity range
(1.005–219 cp) are also identical, thus intracellular viscosity
(1–2 cp in the cytosol and about 140 cp in the membrane) also
results in little interference.14
The high selectivity of probe HOTA for Cys over other
biothiols could be reasonably interpreted. Firstly, as men-
tioned above, due to the existence of an additional amino
Fluorescence imaging results
Fig. 3 (a) Fluorescence intensity of HOTA (10 μM) in the presence of
GSH with concentrations of 0 mM, 1 mM, 3 mM, 5 mM, 7 mM and
10 mM in an EtOH–PBS buffer solution (v/v, 1 : 1, pH 7.4). (b) Fluore-
scence response at 450 nm of HOTA to Cys with different concen-
trations: 0 mM, 1 mM, 3 mM, 5 mM, 7 mM, 10 mM. Insert: linearly fitted
regression equation and corresponding relevancy score.
As discussed above, the probe HOTA exhibited super-high
selectivity for Cys over GSH and many other bio-analytes,
while intracellular pH values and intracellular viscosities
showed little interference. Thus, HOTA should be a suitable
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Conclusions
In summary, we have reported a novel fluorescent probe, HOTA,
that afforded Cys discrimination with both excellent sensitivity
and high selectivity over GSH and other thiols. Moreover, 30 μM
Cys, corresponding to its lowest intracellular concentration, can
still induce the detectable fluorescence of HOTA over GSH.
According to the experimental results, intracellular Hcy and
NAC do not distort the responsivity of HOTA to Cys. We suggest
that the following points are rational: (1) N-terminal Cys pep-
tides/proteins and coenzyme A do not disturb the selectivity of
HOTA due to their low content within cells;18 (2) the intracellular
concentration of Cys and GSH should be mutually checked
because Cys is one of the three component amino acids of GSH.
Furthermore, NEM- and BSO-treated cell experiments confirmed
that the intracellular emission probably came from the reaction
of Cys and the probe. Although we still cannot certify that we
have exactly imaged intracellular Cys, the interference from
GSH and other biothiols has been considered and eliminated to
a great extent. Probe HOTA offers a possible method to detect
intracellular Cys over the most abundant intracellular thiol, GSH.
Fig. 5 Fluorescence DIC images and their merged pictures of SiHa cells
after being stained with probe HOTA (5 μM) for 30 min: (a) the cells were
stained with probe HOTA directly without any treatment; (b) the cells
were pre-treated with 5 mM NEM for 30 min; (c) the cells were
pre-treated with 1 mM BSO for 3 h.
Experimental
Materials
fluorescent probe for the detection of cellular Cys. The fluo-
rescence imaging experiments were carried out with SiHa cells
by wide-field microscopy, as shown in Fig. 5. The differential
interference contrast (DIC) images confirmed that the cells
were viable throughout the imaging process.15 A strong intra-
cellular emission demonstrated an excellent plasma mem-
brane permeability of the probe.
All chemicals used are of analytical grade. Triphenylamine and
N-bromosuccinimide (NBS) were purchased from Sinopharm
Chemical Reagent Co., Ltd (Shanghai, China). Palladium(II)
acetate, tri-o-tolylphosphine, 4-acetoxystyrene and triphenyl-
phosphoranylidene were purchased from J&K Chemical
(Beijing, China). GSH, N-ethylmaleimide (NEM), DL-buthionine
sulfoximine (BSO), amino acids and PBS were purchased from
Seikagaku Corporation (Japan). The solvents used in the spectral
measurements are of chromatographic grade. All spectroscopic
measurements of HOTA were performed in an ethanol–
phosphate buffer solution with pH = 7.4 and v/v of 1 : 1. TLC
analyses were performed on silica gel plates and column
chromatography was conducted over silica gel (mesh 200–300),
both of which were obtained from Qingdao Ocean Chemicals.
In order to confirm the source of intracellular fluorescence,
control experiments were performed (Fig. 5b). The cells were
pre-treated with N-ethylmaleimide (NEM, a well-known thiol-
blocking agent) for 30 min, and then incubated with HOTA.
With the same exposure time as for Fig. 5a, the emission from
these cells became undetectable. Thus, the intracellular fluo-
rescence is associated with the intracellular thiols. Since GSH
is the most abundant cellular thiol and might strongly inter-
fere, further verification of the fluorescence source was
needed. We then pre-treated the cells with DL-buthionine sul-
foximine (BSO, an inhibitor of the GSH biosynthesis) for 3 h
before incubating the cells with the probe.16 As a result, the
intracellular emission was still strong with only a slight
decrease in intensity. This decrease may be ascribed to the oxi-
dation of Cys, because deficient GSH would inevitably lead to
more oxidative cellular conditions.17 In spite of this fluo-
rescence decrease, the BSO-treated cell experiments proved
that a strong intracellular fluorescence still occurred without
GSH. In a nutshell, the absence of thiols led to an undetect-
able intracellular emission, while a strong fluorescence was
detected without GSH. Thus, the intracellular emission was
caused by thiols other than GSH. Based on the in vitro experi-
mental results, we can conclude that the intracellular emission
mainly comes from the reaction of Cys and probe HOTA.
Apparatus
The UV-visible-near-IR absorption spectra of the dilute solu-
tions were recorded on a U2910 spectrophotometer using a
quartz cuvette with a 1 cm path length. IR spectra were
measured on a Nexus 670. One-photon fluorescence spectra
were obtained on
a HITACH F-2700 spectrofluorimeter
equipped with a 450 W Xe lamp.
Wide-field fluorescence microscopy images were acquired
with an Olympus IX71 inverted microscope coupled with a
CCD and display controller software. The fluorescence of
HOTA was excited and collected through U-MNIBA3 and
U-MWU2, respectively.
Spectroscopic measurements
Stock solutions of probe HOTA (5 × 10−3 M) and amino acids
(1 × 10−2 M, and 5 × 10−2 M of GSH) were prepared in DMSO
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and PBS buffer solutions, respectively. 2 mL EtOH–PBS buffer
solution (v/v, 1 : 1) was firstly added to a 5 mL volumetric flask,
and then 10 μL of probe stock solutions and 100 μL of amino
acid stock solutions were added (for GSH, 1 mL stock solution
was added together with 1 mL EtOH). After that, the 5 mL volu-
metric flask was brought to volume with the EtOH–PBS buffer
solution (v/v, 1 : 1). The resulting solution was thoroughly
shaken before recording the spectra.
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SiHa cells were grown in H-DMEM (Dulbecco’s Modified
Eagle’s Medium, high glucose) supplemented with 10% FBS
(fetal bovine serum) in a 5% CO2 incubator at 37 °C. The cells
(1 × 105 mL−1) were placed on glass coverslips and allowed to
adhere for 24 h. For the living cell imaging experiment of
HOTA, the cells were incubated with 5 μM HOTA in DMSO–
PBS (v/v, 1 : 100, pH 7.4) for 30 min at 37 °C. After rinsing
them with PBS three times, the cells were imaged immediately.
For the NEM-treated cell experiment, the SiHa cells were pre-
treated with a DMSO–PBS (v/v, 1 : 499, v/v, pH 7.4) solution of
5 mM NEM for 30 min in a 5% CO2 incubator at 37 °C and
then incubated with 5 μM HOTA in DMSO–PBS (v/v, 1 : 100,
v/v, pH 7.4) for 30 min at 37 °C.
Fluorescence imaging was then carried out after washing
the cells with the PBS buffer. For the BSO-treated cell exper-
iments, the SiHa cells were pre-treated with a PBS (pH 7.4)
solution of 1 mM BSO for 3 h in a 5% CO2 incubator at 37 °C
and then incubated with 5 μM HOTA in DMSO–PBS (v/v,
1 : 100, v/v, pH 7.4) for 30 min at 37 °C. Fluorescence imaging
was then carried out after washing the cells with the PBS
buffer. All the cell images were captured using wide-field fluo-
rescence microscopy. When the microscopy imaging exper-
iments were performed, all exposure times and sensitivity
conditions were set the same for comparison.
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Acknowledgements
For financial support, we thank the National Natural Science
Foundation of China (51273107, 51273175), Natural Science
Foundation of Shandong Province, China (ZR2012EMZ001),
Shandong University 2011JC006, and the Open Project of State
Key Laboratory for Supermolecular Structure and Materials
(SKLSSM201419), 2013CB834704. W.-Y.W. acknowledges the
financial support from the National Basic Research Program of
China (973 Project 2013CB834702), the Areas of Excellence
Scheme from University Grants Committee of HKSAR
(AoE/P-03/08), Hong Kong Research Grants Council
(HKBU203011 and HKUST2/CRF/10) and Hong Kong Baptist
University (FRG2/11-12/156).
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