Simultaneous visualization of cysteine/homocysteine and glutathione in living cells and Daphnia magna via dual-signaling fluorescent chemosensor

Article abstract of DOI:10.1016/j.dyepig.2019.04.056

Biological thiols, including cysteine (Cys), homocysteine (Hcy) and glutathione (GSH), play crucial roles in living organisms, such as maintaining the cellular redox equilibrium, and are closely related to many diseases. The development of efficient fluor

Full text of DOI:10.1016/j.dyepig.2019.04.056

Accepted Manuscript
Simultaneous visualization of cysteine/homocysteine and glutathione in living cells
and Daphnia magna via dual-signaling fluorescent chemosensor
Mingwang Yang, Jiangli Fan, Wen Sun, Jianjun Du, Saran Long, Xiaojun Peng
PII:
S0143-7208(19)30775-2
https://doi.org/10.1016/j.dyepig.2019.04.056
DYPI 7511
DOI:
Reference:
To appear in: Dyes and Pigments
Received Date: 9 April 2019
Revised Date: 22 April 2019
Accepted Date: 23 April 2019
Please cite this article as: Yang M, Fan J, Sun W, Du J, Long S, Peng X, Simultaneous visualization of
cysteine/homocysteine and glutathione in living cells and Daphnia magna via dual-signaling fluorescent
chemosensor, Dyes and Pigments (2019), doi: https://doi.org/10.1016/j.dyepig.2019.04.056.
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Graphical abstract
A fluorescent probe for simultaneous discrimination of cysteine/homocysteine and glutathione was
developed to visualize biothiols in living cells and Daphnia magna.

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Simultaneous visualization of cysteine/homocysteine and
glutathione in living cells and Daphnia magna via dual-signaling
fluorescent chemosensor
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Mingwang Yang1, Jiangli Fan*1,2, Wen Sun1,2, Jianjun Du1,2, Saran Long1,2, Xiaojun
Peng1,2
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1State Key Laboratory of Fine Chemicals, Dalian University of Technology, 2
Linggong Road, Dalian, 116024, P.R. China.
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2Research Institute of Dalian University of Technology in Shenzhen, Gaoxin South
fourth Road, Nanshan District, Shenzhen 518057, China
*Corresponding Author:
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Jiangli Fan, Email: fanjl@dlut.edu.cn
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Abstract
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Biological thiols, including cysteine (Cys), homocysteine (Hcy) and glutathione
(GSH), play crucial roles in living organisms, such as maintaining the cellular redox
equilibrium, and are closely related to many diseases. The development of efficient
fluorescent probes that can diacritically detect biothiols with similar reactivity are of
great desirable for the study of biological processes. The conventional single-emission
fluorescent probes may be disturbed by surrounding environment and biological
systems, which is greatly lose the accuracy of its detection. Herein, we have
constructed a fluorescent probe (TCF-NBD) by connecting two fluorophores with a
biothiol-reactive linker, which has two separated emissions at 547 nm and 610 nm via
different excitations (470 nm and 570 nm, respectively), for simultaneously fast
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detecting Cys/Hcy and GSH with high selectivity and sensitivity. Furthermore, the
probe TCF-NBD exhibits low cytotoxicity and was successfully applied to visualize
intracellular biothiols in living cells and Daphnia magna by fluorescence imaging. We
believe that TCF-NBD can be a powerful tool for conducting the research of
thiol-related diseases.
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Keywords: Fluorescent probe; Biothiols; Bioimaging; Separated emissions; Daphnia
magna.
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1. Introduction
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Intracellular biothiols, such as cysteine (Cys), homocysteine (Hcy) and glutathione
(GSH), play essential roles in many physiological processes, especially in maintaining
cellular redox homeostasis [1-3]. Much studies have shown that abnormal levels of
cellular thiols are closely related with a various of diseases including liver damage,
Parkinson’s disease, leucocyte loss, psoriasis, cardiovascular diseases, Alzheimer’s
and hair depigmentation [4-9]. Consequently, the effective detection of biothiols is
significant for intensive understanding the relationship between diseases and biothiols
and evaluating the disease progression.
Fluorometry have attracted more attention due to its high sensitivity, fast-response,
good biocompatibility, high spatiotemporal resolution and simple operation [10-18],
which are conducive to be applied in biological systems. According, various of
fluorescence probes based on the strong nucleophilicity or the high binding affinity
towards metal ions of the biothiols have been developed to visualize the thiols in vitro
and vivo [19-32]. Most of the optical probes involve specific reactions between thiols
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and probes including Michael addition, cyclization or cleavage reaction [33-56]. Yao
and co-workers reported a coumarin-benzothiazolium fluorescent probe (BCC) for
selectively detect Cys, Hcy and GSH in DMSO/PBS (pH 7.4, 10 mM, v/v 6:4) [54].
Sun et al identified a commercial compound, 2,3,5,6-tetrafluoroterephthalonitrile
(4F-2CN), which was used for simultaneously distinguish Cys, Hcy and GSH with
the addition of CTAB and a detection time up to 2h [55] (Table S1). However, many
of them also suffer from various of deficiencies, such as longer response time, the
inability to distinguish between Cys/Hcy and GSH, the need for surfactants or higher
ration of organic solvent (Table S1). Since the concentration of Cys (30-200 µM) and
Hcy (5-12 µM) are much lower than GSH (1-10 mM) in cells as well as the similar
structures and reaction activities of Cys/Hcy and GSH, it would be highly desirable to
construct fluorescent sensor that could simultaneous distinguish Cys/Hcy and GSH
with few interferences and a rapid response time, which is helpful to understand their
generation and metabolism mechanisms [57,58].
The single-emission fluorescent probes may be disturbed by surrounding
environment and biological systems, but, by contrast, the two-emissions channels
fluorescent probes could effectively avoid these interferences due to its good
self-calibration of two build-in excitation and emission wavelengths [59,60]. In this
work, we develop a fluorescent probe for simultaneously detecting Cys/Hcy and GSH
with different emissions via two independent excitation wavelengths, as shown in
Scheme
1.
The
probe
TCF-NBD
consists
of
two
parts:
a
dicyanomethylenedifuran-based fluorophore (TCF-OH) and 7-Nitrobenzofurazan
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(NBD), connecting by an ether linker which serves as the recognitive group for
Cys/Hcy and GSH and allow the probe exhibits two separated emission signals upon
reaction with biothiols. The probe NR-HNO is non-fluorescent state due to the
intramolecular electron transfer (ICT) blocked quenching [61-63]. Since Cys/Hcy
would induce the occurrence of intramolecular rearrangement cascade reaction
[64-67], in the presence of Cys/Hcy, there were two separated different fluorescence
emissions, including NBD moiety in 547 nm and TCF-OH in 610 nm with two
independent excitations (470 nm and 570 nm, separately). Due to the lack of a
proximal amine group, however, GSH was unable to induce intramolecular
rearrangement reaction, which resulting that only one emission of TCF-OH (610 nm)
was observed in the presence of GSH [61, 63]. The probe TCF-NBD exhibited high
sensitivity and selectivity to distinguish between Cys/Hcy and GSH toward others
amino acids by dual fluorescence signals in vitro and vivo and a rapid response time
(about four minutes). Moreover, the probe is low cytotoxicity and was successfully
applied to visualize intracellular Cys/Hcy and GSH in living HeLa cells and Daphnia
magna by fluorescence imaging.
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Scheme 1 Chemical structure and response mechanism of TCF-NBD for Cys/Hcy
and GSH
85 2. Experimental section
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2.1. Materials and instruments
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All chemicals and solvents were purchased from commercial suppliers and used
without further purification unless otherwise stated. The solvents were purified by
traditional methods before used. 4-chloro-7-nitrobenzo[c], [1,2,5] oxadiazole was
purchased from TCI chemical. Silica gel (200-300 mesh) used for flash column
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chromatography was purchased from Qingdao Haiyang Chemical Co., Ltd. HNMR
and 13CNMR spectra were determined by 400 MHz and 100 MHz using Bruker NMR
spectrometers. Chemical shifts (δ) were expressed as parts per million (ppm, in CDCl3
or DMSO). Meanwhile, high-resolution mass spectrometry was achieved with
ESI-TOF instrument. The absorption spectra and fluorescence spectra were measured
on a PerkinElmer Lambda 35 UV−vis spectrophotometer and Agilent Technologies
CARY Eclipse fluorescence spectrophotometer respectively. The pH values of
detection solutions were determined with a precise pH-meter pHS-3C. MTT assays
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experiments were conducted on the Varioskan LUX Multimode Microplate Reader.
The Olympus FV 1000 confocal microscopy was used for live HeLa cells
fluorescence imaging
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2.2. The preparation of TCF-NBD
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Compound 1: NaOEt (0.9 g, 13 mmol) was added to EtOH (10 mL) solution which
include 3-hydroxy-3-methyl-2-butanone (9 g, 88 mmol) and malonitrile (12 g, 181
mmol) and then stirred for 1 h. After that, 30 mL EtOH was added and the mixture was
then refluxed for 1 h, which was then cooled in refrigerator. The solid precipitate was
filtered and washed with minimal amount of cold EtOH, affording the compound 1 as
off-white crystalline solid (8.8 g, 44 mmol, 50.0 %). 1H NMR (400 MHz, DMSO-d6) δ
(ppm): 2.37 (s, 3H), 1.60 (s, 6H). MS (ESI-TOF): calculated for C11H8N3O-, [M-H]-,
m/z, 198.07, found: 198.42.
TCF-OH: The 4-hydroxybenzaldehyde (0.122 g, 1 mmol) and compound 1 (0.3 g,
1.15 mmol) were mixed in EtOH (10 mL), then two drops of piperidine were added to
the mixture. The reaction was conducted in the microwave reactor for 20 min at 100oC,
which was then cooled to room temperature. After he solvent was removed, the residue
was purified by column chromatography with CH2Cl2: MeOH (50:1) to afford the
desire product as a deep brown solid (0.197 g, yield 65.0%).1H NMR (400 MHz,
DMSO-d6) δ (ppm): 10.61 (s, 1H), 7.90 (d, J = 16.2 Hz, 2H), 7.80 (d, J = 8.5 Hz, 2H),
7.02 (d, J = 16.3 Hz, 1H), 6.90 (d, J = 8.6 Hz, 2H), 1.77 (s, 6H). MS (ESI-TOF):
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calculated for C18H12N3O2 , [M-H]-, m/z, 302.09, found: 302.36.
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TCF-NBD: The Et3N (0.056 mL, 0.4 mmol) was added to a mixture of TCF-OH
(60.4 mg, 0.2 mmol) and NBD-Cl (80 mg, 0.4 mmol) in 5 mL of DMF. The mixture
was stirred overnight at 25 oC under argon atmosphere. After the reaction completed,
50 mL of water was added to the mixture and extracted with dichloromethane for three
times, dried over anhydrous Na2SO4. After he solvent was removed, the residue was
purified by silica gel column chromatography with CH2Cl2: MeOH (20:1) as the eluent
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to afford the desired product as yellow solid (59 mg, 63.3%). H NMR (400 MHz,
DMSO-d6) δ (ppm): 8.68 (d, J = 8.4 Hz, 1H), 8.14 (d, J = 8.5 Hz, 2H), 7.98 (d, J = 16.3
Hz, 1H), 7.55 (d, J = 8.5 Hz, 2H), 7.29 (d, J = 16.8 Hz, 1H), 6.93 (d, J = 8.6 Hz, 1H),
1.82 (s, 1H). 13C NMR (101 MHz, DMSO-d6) δ (ppm): 177.52, 175.46, 156.36, 152.50,
146.17, 145.92, 144.93, 135.71, 133.26, 132.40, 131.46, 121.77, 116.48, 113.08,
112.24, 111.82, 111.22, 100.46, 99.97, 55.21, 25.55. HRMS (ESI-TOF): calculated for
C24H13N6O5 , [M-H]-, m/z, 465.0953, found: 465.0959.
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2.3. Spectral studies
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The probe TCF-NBD was dissolved in DMSO to form the stock solution at a
concentration of 2 mM. The stock solutions (10 mM) of Cys, Hcy and GSH were
prepared in ultrapure water. The following solutions (50 mM) were prepared in
deionized water: amino acids (His, Asp, Val, Phe, Tyr, Ala, Ser, Leu, Arg, Pro, Thr,
Trp, Lys), and inorganic salts (NaHCO3, NaClO4, NaOAc, AgNO3, NaSCN, PdCl2,
MgCl2, NaF, KBr, KI, Na2SO4, Na2SO3, Na2S, FeCl3, CaCl2, ZnCl2, CuCl2). For the
spectral test, the probe stock solution was diluted to 10 µM in 10 mM PBS buffer
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(containing 10% DMF) solutions and then the various of analytes were added and
reacted at 37 °C for 10 min. Unless otherwise stated, the excitation wavelength is 470
nm and 570 nm for all measurements.
2.4. Determination of detection limits
The calibration curve was determined by the relationship between the specific
wavelength fluorescence intensity and the analyte concentration (Cys, Hcy or GSH).
The probe detection limit was calculated using the equation: Detection limit = 3σ/k.
Where σ is the standard deviation of the blank sample (n=10), k is the slop between
the fluorescence intensity versus the concentrations of biothiols.
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2.5. Cell culture and cell viability assay
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Human cervical cancer cells (HeLa) were purchased form Institute of Basic
Medical Sciences (IBMS) of the Chinese Academy of Medical Sciences. HeLa cells
were cultured in 90% Dulbecco's Modified Eagle Medium (DMEM) including with
10% FBS and 1% antibiotics (100 U/mL penicillin and 100 µg/mL streptomycin) in
an atmosphere of 37°C and 5% CO2. The MTT method was employed to evaluate the
cytotoxicity of probe TCF-NBD. Before experiments, HeLa cells at a density of
1×104 cells/well were seeded into 96-well plates and cultured for 24 h. Then the fresh
culture with different concentrations of TCF-NBD (0-30 µM) (n = 6) were used to
replace the previous media, and further incubation for 24 h. After that, 10 µL of MTT
(5 mg/mL in PBS) was added into per well and incubated for another 4 h. Finally, 100
µL of DMSO was then added to dissolve formazan. The absorbance at 490 nm was
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measured, and the cell viability (%) was calculated according to the following
equation: Viability (%) = [OD490 (sample) - OD490 (blank)] / [OD490 (control) -
OD490 (blank)] × 100
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2.6. Cell imaging experiments
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One day before imaging, the HeLa cells were replanted separately on
glass-bottomed dishes and incubated for 24h. In the first control experiment, the cells
were pretreated with or without NEM (500 µM) at 37 °C for 30 min, then washing
cells with PBS, the cells were further incubated with the probe TCF-NBD (10 µM) at
37°C for 30 min before imaging. In the second experiments, the HeLa cells were
pretreated with NEM (500 µM) at 37°C for 30 min and washed three times with PBS,
then the cells were further incubated with 100 µM Cys, Hcy or GSH respectively for
30 min, then incubated with the probe TCF-NBD (10 µM) at 37 °C for 30 min before
imaging.
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2.7. Fluorescence imaging experiment in Daphnia magna
The Daphnia magna (age < 72 h) were cultured in clean non-chlorinated tap water
under cool-white light (14 h)-dark (10 h) photoperiod. The animals were pretreated
with or without 500 µM NEM for 30 min, and then incubated with 10 µM TCF-NBD
for another 30 min, followed by washing twice with PBS before imaging.
180 3. Results and discussion
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3.1. Synthesis of TCF-NBD
TCF-NBD was readily
synthesized
by
coupling
TCF-OH
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4-chloro-7-nitro-1,2,3-benzoxadiazole in the presence of Et3N at room temperature
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(Scheme S1). The target compound was well characterized by H NMR, C NMR,
HR-MS (Figs. S6-S12).
3.2. Spectral response of TCF-NBD to Cys/Hcy and GSH
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The optical spectra evaluation of probe and its reaction with Cys, Hcy and GSH were
initially measured in PBS (10 mM, pH 7.4, 10% DMF). As shown in Fig. 1A, the
probe TCF-NBD shows the obvious absorption peak at 400 nm. When the Cys and
Hcy were added respectively, significant absorption peak appeared at both around 470
nm and 570 nm. While in case of GSH, the main absorption peak appeared only at
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570 nm.
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Fig.1 The absorption spectra (A) and fluorescence spectra (B) changes of probe
TCF-NBD (10 µM) prior and after addition of Cys, Hcy, GSH (100 µM each) in PBS
(10 mM, pH 7.4, 10% DMF) at 37oC for 10 min. For fluorescence measurements, λex
=470 nm and 570 nm for NBD moiety and TCF–OH moiety respectively.
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Due to the presence of intramolecular electron transfer (ICT) blocked quenching, the
probe is non-fluorescent when the excitation wavelength is 470 nm or 570 nm. The
fluorescence responses of the probe TCF-NBD (10 µM) to different concentration of
Cys/Hcy or GSH are summarized in Figs. 2A-I. Upon increasing the concentrations
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of Cys or Hcy, both the fluorescence intensity at 547 nm and 610 nm gradually
increased when excitation wavelengths were 470 nm and 570 nm, respectively (Figs.
2A-F). The fluorescence intensity at 547 nm and 610 nm reached plateau when 100
µM analytes were added (Fig. S1). And the detection limit of Cys and Hcy were
calculated to be 0.015 µM and 0.034 µM respectively (Table S2). In the case of GSH,
the fluorescence intensity at 610 nm increased gradually and the fluorescence
emission signal at 547 nm did not change significantly (Figs. 2G-I). The detection
limit for GSH were determined to be 0.030 µM (Table S2). In order to make a deeper
understanding of the reaction mechanism, we monitored the reaction process of the
probe TCF-NBD and biothiols by HRMS. The new peak appeared at m/z=302.0937
[M]- was attributed to TCH-OH and another peak appear at m/z=283.0150 [M]- was
assigned to TCF-Cys (Fig. S4); similarly, in Fig. S5, the peak at m/z= 469.0783 was
the product TCF-GSH. Thus, the probe TCF-NBD can simultaneous differentiate
Cys/Hcy and GSH with different excitation wavelengths.
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Fig. 2 Fluorescence spectra of TCF-NBD (10 µM) upon addition of varied
concentration of Cys (A), Hcy (D) and GSH (G) respectively in PBS (10 mM, pH 7.4,
10% DMF). Fluorescence intensity changes at 547 nm as a function of concentrations
of Cys (B), Hcy (E) and GSH (H) respectively and at 610 nm as a function of
concentrations of Cys (C), Hcy (F) and GSH (I) respectively. For fluorescence
measurements, λex =470 nm and 570 nm for NBD moiety and TCF–OH moiety
respectively.
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3.3. Time-dependent and pH-dependent fluorescence changes of probe
TCF-NBD
We then investigated the time-dependent fluorescence response of the probe
TCF-NBD in the presence of 100 µM Cys/Hcy and GSH (Fig. 3). Upon addition of
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Cys and Hcy, the fluorescence intensity of probe at 547 nm and 610 nm increased
rapidly and reached plateau within 10 min. When GSH was added, the fluorescence
intensity at 610 nm could reach its strongest after 10 min of reaction, while there was
almost no significant fluorescence change at 547 nm with the extended response time.
Meanwhile, the effect of pH from pH 5.0-9.0 on the fluorescence detection of
Cys/Hcy and GSH were carried out. As shown in Fig. S2, in the absence of thiols, the
probe TCF-NBD shows little fluorescence change but displays significant
fluorescence enhancement at 610 nm in the pH range from 6.0-8.0 with 100 µM Cys,
Hcy and GSH, respectively. Meanwhile, Cys/Hcy can also increase the fluorescence
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intensity at 547 nm in the pH range of 7.0-9.0. These data show that the probe is
capable of detecting biothiol under physiological conditions.
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Fig. 3 Time-dependent changes of fluorescence intensity at 547 nm (A) and 610 nm
(B) of probe TCF-NBD (10 µM) upon addition of 100 µM Cys, Hcy, GSH in PBS (10
mM, pH 7.4, 10% DMF). λex =470 nm and 570 nm.
3.4. Selectivity of probe TCF-NBD
High selectivity is a necessary and crucial indicator for the application of the
fluorescent probe to biological system. We then used various analytes, such as amino
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acids (His, Asp, Val, Phe, Tyr, Ala, Ser, Leu, Arg, Pro, Thr, Trp, Lys,) and ions (HCO3 ,
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2-
ClO4 , OAc-, NO3 , SCN-, F-, Cl-, Br-, I-, SO3 , SO4 , S2-, Pd2+, Mg2+, Fe3+, Ca2+, Zn2+,
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Cu2+) and others (H2O2, NaClO, NO) to investigate the selectivity of the probe
TCF-NBD towards Cys/Hcy and GSH. As shown in Fig. 4 and S3, the fluorescence
intensity of probe TCF-NBD at 547 nm and 610 nm are significantly enhanced after
the addition of 100 µM Cys or Hcy; and the fluorescence turn-on response only at 610
nm after addition of 100 µM GSH. However, there was no obvious change in
fluorescence response can be observed when the probe reacted with other analytes
with the excitation at 470 nm or 570 nm. These results indicated that the ability of
TCF-NBD to specifically recognize Cys/Hcy and GSH over others relative analytes
in complexed biosystem.
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Fig. 4 (A) Fluorescence spectra of TCF-NBD (10 µM) in the presence of Cys, Hcy or
GSH (100 µM) and some relevant species (500 µM): (1) Probe, (2) His, (3) Asp, (4)
Val, (5) Phe, (6) Tyr, (7) Ala, (8) Ser, (9) Leu, (10) Arg, (11)Pro, (12)Thr, (13)Trp,
(14)Lys, (15) H2O2, (16) NaClO, (17) NO, (18) Cys, (19) Hcy, (20) GSH in PBS (10
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mM, pH 7.4, 10% DMF). Fluorescence intensity of TCF-NBD (10 µM) upon addition
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of different analytes: (B) λex =470 nm, λem =547 nm (D) λex =570 nm, λem =610 nm.
(C) The cell cytotoxicity of various concentration of TCF-NBD in HeLa cells for 24
h.
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3.5. Fluorescence imaging of probe TCF-NBD in living cells
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Fig.5 Fluorescence imaging for HeLa cells. (A1-A5): HeLa cells were incubated with
10 µM TCF-NBD only for 30 min; (B1-B5): The cells were pretreated with 500 µM
NEM for 30 min, and then incubated with 10 µM TCF-NBD for 30 min. The cells
were pretreated with 500 µM NEM for 30 min, and then treated with 100 µM Cys
(C1-C5), Hcy (D1-D5) and GSH (E1-E5) respectively and further incubated with 10
µM TCF-NBD for 30 min. Green channel: λex =488 nm, collected: 500-550 nm; Red
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channel: λex =559 nm, collected: 570-630 nm. Scaler bar: 20 µm.
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In order to verify the bioavailability of the probe TCF-NBD, we evaluated its
capability to selectively visualize intracellular Cys/Hcy and GSH with fluorescence
imaging. Accordingly, the cytotoxicity of TCF-NBD was established using MTT
assays with HeLa cells. It was found that the cell viabilities exceed 94% when
incubated cells for 24 h with10 µM TCF-NBD, demonstrating that the probe
TCF-NBD is of low cytotoxicity and is suitable for fluorescence confocal biological
imaging (Fig. 4C).
As shown in Fig. 5A, when the HeLa cells were only incubated with probe
TCF-NBD (10 µM), it shows fluorescence signals in green and red channels
simultaneously, which were caused by the endogenous biothiols in HeLa cells. We
then performed a series of control experiments so that to evaluate selective responses
of probe TCF-NBD toward Cys/Hcy and GSH in living cells. When
N-ethylmaleimide (NEM, a thiol-consumptive agent)-pretreated cells were further
incubated with TCF-NBD for 30 min, however, an obvious fluorescence reduction in
both green and red channels can be observed (Fig. 5B), indicating that thiols were
completely reacted with NEM [68]. In contrast, when NEM-pretreated HeLa cells
were incubated with Cys/Hcy and then treated with probe TCF-NBD for 30 min,
strong fluorescence signals occurred in green and red channels (Figs. 5C and 5D).
Upon the addition of 100 µM GSH to NEM-pretreated cells followed by incubation
with probe, only obvious red fluorescence signal was observed (Fig. 5E). These
above experiments indicated that TCF-NBD could discriminate intracellular Cys/Hcy
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and GSH by different emission signals and serve as a potential tool for investigating
the metabolism and mechanism of biothiols.
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3.6. Fluorescence imaging of probe TCF-NBD in Daphnia magna
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Fig.6 Fluorescence images of endogenous biothiols in Daphnia magna. (A1-A5)
Daphnia magna were incubated with 10 µM TCF-NBD only for 30 min. (B1-B5)
Daphnia magna were pretreated with 500 µM NEM for 30 min, and then incubated
with 10 µM TCF-NBD for 30 min. Scale bar: 300 µm.
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The ability of TCF-NBD for visualizing biothiols in vivo was also evaluated in
living Daphnia magna, a widely used animal as a standard Environmental Protection
Agency test organism [69], using fluorescence imaging. As shown in Fig. 6A, the gut
of Daphnia magna exhibited strong fluorescence signals in green channel and red
channel when the Daphnia magna were incubated with 10 µM TCF-NBD for 30 min,
indicating that there were abundant endogenous biothiols in the gut of Daphnia
magna. To verify this result, Daphnia magna were pretreated with 500 µM NEM for
30 min, and then incubated with 10 µM TCF-NBD for further 30 min before
fluorescence imaging. The Daphnia magna displays almost unobservable
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fluorescence signals in two channels (Fig. 6B), suggesting the signals observed in Fig.
6A were induced by endogenous biothiols. These results indicating that TCF-NBD
could be used for tracking biothiols in complex living bodies.
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4. Conclusions
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In summary, an efficient fluorescent probe, TCF-NBD, for simultaneously
discriminating Cys/Hcy and GSH by separated emissions via independent excitations
was designed and synthesized. The strategy is to couple two fluorophores, TCF-OH
and NBD with a biothiol-sensitive ether linkage. This probe emits a dual emission
signal toward Cys and Hcy through a substitution- rearrangement cascade reaction;
however, a single emission response toward GSH, which can be used to
simultaneously distinguish Cys/Hcy and GSH. Taking the merits of low cytotoxicity
and good selectivity of probe TCF-NBD into consideration, we have successfully
applied it for discriminative detecting of Cys/Hcy and GSH in HeLa cells through
fluorescence confocal imaging. More importantly, the in vivo ability of TCF-NBD
was proved by the visualization of endogenous biothiols in Daphnia magna.
Therefore, the probe TCF-NBD would serve as a powerful tool for further elucidating
the physiological role of biothiols and conducting pathological analysis of
thiol-related diseases.
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330
Acknowledgements
This work was financially supported by National Science Foundation of China
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(21576037, 21676047, 21421005, 21703025), NSFC-Liaoning United Fund
(U1608222).
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Supporting Information
Simultaneous visualization of cysteine/homocysteine and
glutathione in living cells and Daphnia magna via dual-signaling
fluorescent chemosensor
Mingwang Yang1, Jiangli Fan*1,2, Wen Sun1,2, Jianjun Du1,2, Saran Long1,2, Xiaojun
Peng1,2
1State Key Laboratory of Fine Chemicals, Dalian University of Technology, 2
Linggong Road, Dalian, 116024, P.R. China.
2Research Institute of Dalian University of Technology in Shenzhen, Gaoxin South
fourth Road, Nanshan District, Shenzhen 518057, China
Email: fanjl@dlut.edu.cn
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