A dual-emission fluorescent probe for discriminating cysteine from homocysteine and glutathione in living cells and zebrafish models

Article abstract of DOI:10.1016/j.bioorg.2019.103215

Cellular biothiols function crucially and differently in physiological and pathological processes. However, it is still challenging to detect and discriminate thiols within a single one molecule, especially for cysteine (Cys) and homocysteine (Hcy). In th

Full text of DOI:10.1016/j.bioorg.2019.103215

Bioorganic Chemistry 92 (2019) 103215
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
A dual-emission fluorescent probe for discriminating cysteine from
homocysteine and glutathione in living cells and zebrafish models
⁎
Zhengliang Lu, Yanan Lu, Xin Sun, Chunhua Fan , Zongyi Long, Liying Gao
School of Chemistry and Chemical Engineering, University of Jinan, Jinan 250022, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Cellular biothiols function crucially and differently in physiological and pathological processes. However, it is
still challenging to detect and discriminate thiols within a single one molecule, especially for cysteine (Cys) and
homocysteine (Hcy). In this study, a simple two-emission turn-on fluorescent biothiol probe (ICN-NBD) was
rationally designed and synthesized through a facile ether bond linking 7-nitro-1,2,3-benzoxadiazole (NBD) and
phenanthroimidazole containing a cyano tail. The probe in the presence of Cys elicited two fluorescence re-
sponses at 470 nm and 550 nm under excitation at 365 nm and 480 nm, respectively, because of the concomitant
generation of both the fluorophore and NBD-N-Cys. In contrast, addition of Hcy and glutathione (GSH) could
result in only a blue fluorescence enhancement at 470 nm. which was reasonably attributed to rearrangement
from NBD-S-Hcy/GSH to NBD-N-Hcy/GSH as a result of geometrical constraints or solvent effects. Therefore,
the fluorescent probe with the NBD scaffold could detect biothiols and simultaneously discriminate Cys from
Hcy/GSH in both blue and green channels. The probe has been successfully applied for visualizing biothiols in
living cells and zebrafish.
Dual-emission fluorescent probe
Discrimination of biothiols
NBD, Living cell
Zebrafish models
1
. Introduction
conveniently and sensitively detect and simultaneously discriminate
biothiols in clinic diagnostics and pathophysiology exploration.
The study of reactive sulfur species (RSS) in biological systems,
Compared with conventional detection methods including high-
performance liquid chromatography, electrochemical assay, and mass
spectrometry [15–17], fluorescence spectroscopy offers many ad-
vantages including simplification, high sensitivity and low cost, non-
invasiveness, high spatial resolution and notably, visualization of the
target of interest in a biological environment [18–25]. In this regard,
various mechanisms including Michael addition, cleavage of sulfona-
mide and sulfonate ester, cyclization with aldehyde, and others, have
been used to construct numerous fluorescent probes for biothiols
[26–38]. Due to their similar structure and reaction sites, most fluor-
escent probes could respond to only one or two of the biothiols.
Therefore, discrimination of biothiols is still a big challenge although
several probes have been developed for biothiols using multiple reac-
tion sites in one molecule [39–43]. It is worth noting that most probes
are single-emission or intensity-based fluorescent probes which could
usually suffer at least one of the drawbacks, such as low selectivity and
sensitivity, a high limit of detection, large volumes of organic solvent,
and/or short excitation/emission wavelengths. However, dual-emission
fluorescent probes would be more accurate because of the good built-in
self-calibration to effectively eliminate the interference from environ-
mental conditions and biological systems [44–48]. Unfortunately, few
especially cysteine (Cys), homocysteine (Hcy) and glutathione (GSH)
has been attracting much attention due to their crucial roles in phy-
siological and pathological processes [1–3]. Among them, Cys partici-
pates in cell growth and in the synthesis and increased rigidity of
proteins as well as chelation of metal ions in enzymes [4–6]. In the
presence of cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE),
and 3-mercaptopyruvate sulfur transferase (3-MST), Cys could be cat-
alytically transformed into the endogenous gaseous transmitter H S
2
which has been implicated in the induction of hippocampal long-term
potentiation, brain development, and blood pressure regulation [7].
With assistance of γ-glutamylcysteine synthetase (GSH I) and glutamine
synthetase (GSH II), Cys is involved in the formation of the antioxidant
GSH maintains cellular redox homeostasis and protects thiol-containing
proteins and enzymes from reactive oxygen species, such as free radi-
cals and peroxides, as well as heavy metals through reversible oxidi-
zation of glutathione disulfide (GSSG) [8]. Hcy can be catalytically
converted into Cys by CBS and CSE with the aid of Vitamin B [9].
6
Imbalance of biothiols is involved in edema, retarded growth rate, liver
damage, leucocyte loss, psoriasis, Alzheimer’s disease and down’s syn-
drome [10–14]. Therefore, it is meaningful and important to be able to
⁎
Corresponding author.
E-mail address: chm_fanch@ujn.edu.cn (C. Fan).
https://doi.org/10.1016/j.bioorg.2019.103215
Received 25 March 2019; Received in revised form 9 July 2019; Accepted 21 August 2019
Available online 23 August 2019
0045-2068/ © 2019 Elsevier Inc. All rights reserved.

Z. Lu, et al.
Bioorganic Chemistry 92 (2019) 103215
dual-emission fluorescent probes have been reported for discriminating
biothiols [49–53]. For instance, Lin and co-workers developed a dual-
suppliers. NMR spectra were carried out on a Bruker spectrometer. ESI-
HRMS were recorded on a Bruker Daltonics APEX II 47e FT-ICR spec-
trometer. UV-vis and Fluorescence spectra were measured on a TU-
1901 spectrometer and F-380 luminescence spectrometer, respectively.
The cells were imaged by a confocal laser scanning microscope.
Phenanthroimidazole fluorophore was obtained according to the lit-
erature [59].
emission fluorescent probe for discrimination of biothiols and H S in
2
phosphate-buffered saline (PBS) containing 20% acetonitrile [49]. Wu
and co-workers reported a hemicyanine fluorescent probe for distin-
guishing Cys/Hcy from GSH in 5% DMSO/PBS [50]. Very recently,
Song and co-workers designed a multi-emission fluorescent probe for
discriminatory detection of Cys/Hcy, GSH/H S, and thiophenol [51].
2
However, these fluorescent probes failed to distinguish between Cys
and Hcy. It is clear that the selectivity of a probe toward the target of
interest can be significantly affected by minor changes of a moiety
within the probe [54–57]. Thus, we envisioned that introduction of a
suitable electron-withdrawing group within a dual-emission fluorescent
probe could induce a remarkable shift of emission wavelength after
reaction with biothiols. Based on this hypothesis and our former re-
search [58], we synthesized a simple dual-emission fluorescent probe
for biothiols by anchoring 7-nitro-1,2,3-benzoxadiazole (NBD) to a
cyano-phenanthroimidazole fluorophore. The initial nucleophilic attack
of biothiols toward the ether bond of the probe would release the
phenanthroimidazole fluorophore with emission at 470 nm upon ex-
citation at 365 nm. Due to the solvent impact and/or geometrical dif-
ference between Cys and Hcy/GSH, an intramolecular cyclization-re-
arrangement cascade reaction took place in the presence of only Cys,
eliciting a remarkable green fluorescence at 550 nm (Ex 480 nm) (see
Scheme 1). Therefore, the probe ICN-NBD could detect biothiols and
further discriminate Cys from Hcy/GSH in both blue and green chan-
nels. Moreover, the probe was successfully applied for visualizing bio-
thiols in living HeLa cells and zebrafish models.
2.2. Synthesis of ICN-NBD
Compound ICN-OH (0.41 g, 1 mmol) and NBD-Cl (0.40 g, 2 mmol)
was dissolved in dry dichloromethane (10 mL) under a nitrogen atmo-
sphere. Triethylamine (0.33 mL) was added and the mixture was stirred
ambiently overnight. The residue was purified by flash chromatography
using EtOAc/pet. ether (v/v 1:4) to afford the pure compound ICN-NBD
1
as a blue solid (0.28 g, yield 49%). H NMR (400 MHz, CDCl ) δ 8.94 –
3
8.84 (m, 2H), 8.59 (d, J = 7.8 Hz, 1H), 8.46 (d, J = 2.4 Hz, 1H), 8.40
(d, J = 8.3 Hz, 1H), 8.26 (d, J = 7.7 Hz, 1H), 8.07 (dd, J = 8.7, 2.5 Hz,
1H), 8.01 (d, J = 5.0 Hz, 3H), 7.79 (t, J = 7.4 Hz, 1H), 7.73–7.70 (m,
1H), 7.66 (dt, J = 5.7, 3.6 Hz, 1H), 7.56 (t, J = 7.7 Hz, 1H), 7.47 (d,
J = 8.7 Hz, 1H), 7.22 (t, J = 7.6 Hz, 1H), 7.10 (d, J = 8.3 Hz, 1H), 6.66
1
3
(d, J = 8.4 Hz, 1H). C NMR (101 MHz, CDCl ) δ 151.66, 150.57,
3
148.40, 146.25, 144.54, 143.78, 143.36, 142.54, 139.09, 138.67,
137.37, 133.13, 132.99, 132.76, 132.59, 131.55, 130.90, 130.41,
130.08, 129.82, 128.83, 128.68, 127.82, 126.95, 126.75, 126.24,
124.68, 124.46, 123.29, 122.60, 121.89, 121.41, 120.42, 118.37,
+
109.56. MS (ESI, m/z) Calcd for [C
3
4 18 6 4
H
N O -H] , 573.1311; found,
573.1350.
2
. Experimental
2.3. HeLa cells and zebrafish imaging
2
.1. Materials and instruments
HeLa cells were seeded on glass-bottomed dishes in culture media
for 24 h. The cultured cells were washed twice by PBS before sub-
sequent experiments and divided into four groups. In a control group,
All reagents and chemicals were commercially obtained from
Scheme 1. Proposed recognition mechanism of ICN-NBD towards biothiols.
2

Z. Lu, et al.
Bioorganic Chemistry 92 (2019) 103215
HeLa cells were incubated with 100 μM N-ethylmaleimide (NEM) for
3
0 min and then ICN-NBD (20 μM) at 37 ℃ for another 30 min. In the
second group, the HeLa cells were treated with the probe (20 μM) for
0 min after incubation with NEM (100 μM) for 30 min and then Cys
20 μM) for another 30 min. In the third group, HeLa cells pretreated
with 100 μM NEM for 30 min and then with Hcy (50 μM) for another
3
(
3
0 min were incubated with the probe (20 μM) for 30 min. In the fourth
group, HeLa cells were cultured with NEM (100 μM) for 30 min, and
subsequently incubated with GSH (50 μM) for 30 min, and finally with
the probe (20 μM) for 30 min. After washing, HeLa cell images were
taken using a confocal laser scanning microscope. Zebrafish were im-
aged after incubation with NEM (500 μM), the probe (20 μM), or bio-
thiols (50 μM) for 30 min, respectively.
3
. Results and discussion
3.1. Rational design and synthesis of ICN-NBD
Although several probes relying on multiple reaction sites have been
developed for discriminating biothiols, it is still challenging to si-
multaneously discriminate between them [60]. In contrast, dual-emis-
sion fluorescent probes due to the build-in self-calibration pave a more
promising way for detecting biothiols and even distinguishing Cys and
Hcy [50–51]. These previous studies containing the NBD moiety de-
monstrated a similar cyclization rearrangement cascade reaction with
Cys and Hcy. We imagined that the cyano group of the probe could
affect the emission wavelength of the fluorophore. Considering that
NBD acts as both a good indicator and biothiol-sensing group, the probe
ICN-NBD with a cyano group tail was synthesized through a one-step
reaction between NBD-Cl and ICN-OH in the presence of Et N. ICN-
3
1
13
NBD was fully characterized by H NMR, C NMR and HRMS (Fig.
S1–S3).
3.2. Sensing properties of ICN-NBD toward biothiols
With compound ICN-NBD in hand, the fluorescence responses of the
probe were initially investigated in the presence and absence of bio-
thiols in DMF-H
2
O (v/v, 4:6, 20 mM PBS buffer; pH 7.4). Free ICN-NBD
(
10 μM) displayed no fluorescence upon excitation at both 365 nm and
4
80 nm. However, addition of biothiols to media containing ICN-NBD
induced strong fluorescence changes. As shown in Fig. 1a, in the pre-
sence of Cys, the probe demonstrated two independent fluorescence
enhancements at 470 and 550 nm when excited at 365 and 480 nm,
respectively. The fluorescence in blue and green channels increased and
reached a maximum plateau at 470 and 550 nm, respectively, in the
presence of 10 equiv. of Cys. In contrast, only blue fluorescence at
Fig. 1. Fluorescence spectra of ICN-NBD (10 μM) with gradual addition of Cys
4
70 nm was observed upon excitation at 365 nm in the presence of Hcy
(
a), Hcy (b) and GSH (c), respectively, in DMF-PBS (v/v, 4:6, 20 mM PBS, pH
and GSH (Fig. 1b and c). The strong blue fluorescence confirmed that
the nucleophilic substitution reaction was successfully initiated, which
emitted fluorescence at 470 nm due to released ICN-OH. The probe
showed very weak fluorescence at 550 nm with Hcy and GSH upon
excitation at 480 nm. The maximum emission intensities at 470 nm
were obtained with addition of 10 equiv. of Hcy and 18 equiv. of GSH.
Compared with Cys, the weak green fluorescence of ICN-NBD with Hcy
and GSH was probably attributed to the unfavorable transformation
from NBD-S-Hcy and NBD-S-GSH to NBD-N-Hcy and NBD-N-GSH,
respectively. This is likely due to the influence of steric or solvent in-
teractions as has been observed in previous work for similar probes
7
.4).
3.3. Selectivity of ICN-NBD toward biothiols
In order to prove excellent selectivity toward biothiols, the fluor-
escence responses of ICN-NBD (10 μM) were investigated with biolo-
gically relevant species in DMF-buffer solution (4:6, v/v, PBS 20 mM,
pH 7.4). As shown in Fig. 2a, the probe showed strong fluorescence at
470 nm in the presence of biothiols (Cys, Hcy, and GSH). Related in-
[
61]. Moreover, good linear relationships were obtained between the
terfering species, such as thiophenol (PhSH), Na S, other amino acids
2
fluorescence intensities at 470 nm of the probe and the concentration of
Cys, Hcy, and GSH in the range of 0–100 μM, 0–100 μM, and 0–180 μM,
respectively (Fig. S4). The corresponding limits of detection were esti-
mated to be 22.6 nM for Cys, 31.2 nM for Hcy, and 17.7 nM for GSH,
respectively. These results indicate that the probe could quantitatively
detect biothiols and further discriminate Cys from Hcy and GSH.
(200 μM) (Asp, l-Try, Gly, Val, Ile, Glu, Arp, Leu, Ala, His, Lys, Met, Phe,
–
2+
–
–
2+
2–
2+
Tyr, Ser, Pro, Try and Thr) and common species (H
2
O
2
, F , I , Cl , SO
4
,
,
2
–
–
+
+
3+
2+
2+
3+
SO
3
, AcO , K , Na , Al , Mg , Zn , Cr , Hg , Ba , Cu
2
+ 2+ 2+ 3+ 2+
Fe , Mn , Cd , Fe
and Ca ), did not induce significant fluor-
escence emission. It is to be noted that only Cys led to a strong emission
intensity at 550 nm although Hcy and GSH resulted in weak green
3

Z. Lu, et al.
Bioorganic Chemistry 92 (2019) 103215
3.4. The proposed sensing mechanism of ICN-NBD towards biothiols
At present, numerous single-emission fluorescent probes for bio-
thiols have been developed whose further potential application was
limited due to a lack of built-in self-calibration, although a few probes
have been designed for discriminating biothiols based on multi reaction
sites [62–64]. Fortunately, several groups have shown that dual-emis-
sion fluorescent probes are a promising tool due to high accuracy from
their built-in self-calibration, when they followed a popular strategy of
combining a good indicator and recognition group NBD with other
fluorophores [50,52]. An aromatic nucleophilic substitution reaction
can readily break the ether bond of the probes and release the fluor-
ophore ICN-OH and NBD-containing intermediates thereby emitting
blue fluorescence at 470 nm (ex 365 nm) by switching on in-
tramolecular charge transfer (ICT). It should be noted that these
fluorescent probes could discriminate Cys/Hcy from GSH because both
NBD-containing intermediates (NBD-S-Cys and NBD-S-Hcy) can un-
dergo a cyclization and substitution cascade reaction that is not ob-
served for NBD-S-GSH. The probe ICN-NBD was synthesized by an-
choring NBD to the cyano-fluorophore (ICN-OH). To gain insight into
the sensing mechanism, the HRMS spectra of ICN-NBD in the presence
of Cys, Hcy, and GSH, were performed. As shown in Fig. S7, the spectra
of ICN-NBD with GSH presented a peak at m/z = 410.13026 assigned
+
to the fluorophore ([ICN-OH-H ]: Calcd 410.12934) and a peak at m/
z = 469.07870 corresponding to the species NBD-S-GSH ([NBD-S-
+
GSH-H ]: Cald 469.07777), an expected compound regardless of steric
factor. Besides the peak at 410.12967 from the fluorophore ([ICN-OH-
+
H ]: Calcd 410.12989), HRMS spectra of ICN-NBD in the presence of
Cys also demonstrated a new peak at 283.01419 reasonably attributed
+
to NBD-N-Cys ([NBD-N-Cys-H ]: Calcd 283.03172) which is good
agreement with literatures (Fig. S8) [51,65]. Surprisingly, addition of
Hcy to the probe can produce the fluorophore and the nonfluorescent
species NBD-S-Hcy which was scarcely transformed into florescent
NBD-N-Hcy as observed in Fig. 1b. This phenomenon was reasonably
attributed to the influence from geometrical constraints and/or solvent
interaction [61]. As shown in Fig. S9, addition of all three biothiols to
the probe led to an absorption peak at 365 nm, which was assigned to
ICN-OH. Notably, reaction between ICN-NBD and GSH resulted in a
broad absorption shoulder at about 420 nm corresponding to the non-
fluorescent NBD-S-GSH. Addition of Hcy to the probe produced a new
broad absorption band at 446 nm from nonfluorescent NBD-S-Hcy.
This designation was further confirmed by a Cys-induced absorption
peak at 470 nm corresponding to fluorescent NBD-N-Cys. Thus, these
results strongly support the proposed recognition mechanism of the
probe toward biothiols.
Fig. 2. Fluorescence intensity at 470 nm (a) and 550 nm (b) of ICN-NBD
(
10 μM) upon addition of biothiols and other interfering species upon excitation
–
at 365 nm and 480 nm, respectively. From left to right, 1. Blank, 2·H
2
O
2
, 3. F ,
–
–
2–
2–
–
+
+
3+
2+
4
. I , 5. Cl , 6. SO
4
, 7. SO
3
, 8. AcO , 9. K , 10. Na , 11. Al , 12. Mg , 13.
2
+
3+
2+
2+ 2+ 2+ 2+ 2+
Zn , 14. Cr , 15. Hg , 16. Ba , 17. Cu , 18. Fe , 19. Mn , 20. Cd
,
3
+
2+
2
1. Fe , 22. Ca , 23. Asp, 24. l-Try, 25. Gly, 26. Val, 27. Ile, 28. Glu, 29. Arp,
0. Leu, 31. Ala, 32. His, 33. Lys, 34. Met, 35. Phe, 36. Tyr, 37. Ser, 38. Pro, 39.
3
Try, 40. Thr, 41. PhSH, 42. Na
2
S, 43. Hcy, 44. Cys, 45. GSH.
fluorescence (Fig. 2b). These results indicate that the probe could not
only detect biothiols over the other related interfering species but also
discriminate Cys from Hcy/GSH.
The effect of pH on the fluorescence response at 470 nm of the probe
in the absence or presence of 20 equiv. of biothiols was explored in 40%
DMF aqueous solution in order to extend potential applications in
biological systems (Fig. S5). It was found that free probe ICN-NBD did
not elicit any significant fluorescence at 470 nm in the pH range from
3.5. Imaging biothiols in living cells and zebrafish
The excellent selectivity and sensitivity of the probe toward bio-
thiols in vitro under simulated physiological conditions encouraged us
to further explore its potential application in biological systems. The
cytotoxicity effect of probe ICN-NBD was initially checked by a stan-
dard MTT assay in living HeLa cells. After incubation with ICN-NBD at
different concentration ranging from 0 to 20 μM for 24 h, HeLa cells did
not show any major loss of viability (Fig. S10). Indeed, the cell viability
is still more than 90% after incubation with up to 20 μM of probe. These
results show the low cytotoxicity and good permeability of ICN-NBD in
living HeLa cells.
2
.0 to 12.0, which demonstrated the stability of the probe. As expected,
addition of 20 equiv. of biothiols resulted in strong blue fluorescence at
4
70 nm of the probe in the pH range from 5.0 to 10.0. However, the
fluorescence of the probe at 470 nm was silent with biothiols in more
acidic (pH < 5.0) or basic environment (pH greater than 10.0) which
probably inhibited the aromatic substitution reaction between the
probe and biothiols. This wide working pH range of 5.0–10.0 indicates
that the probe can work well under physiological conditions.
As a crucial parameter of reaction-based probes, response time was
checked by investigating the fluorescence of the probe at 470 nm in the
presence of biothiols at different concentration over time. As shown in
Fig. S6, addition of biothiols at different concentration such as Cys (50
and 100 μM), Hcy (50 and 100 μM) and GSH (100 and 200 μM) induced
a gradual fluorescence enhancement of the probe and finally reached a
plateau in approximately 30 min. These time-course fluorescence
measurements of the probe evidenced that response time is almost in-
dependent of the biothiol concentration.
Next, we carried out a series of cell experiments to check whether
the probe could visualize biothiols in HeLa cells by utilizing a confocal
laser scanning microscope (Leica Microsystems). In a control group,
HeLa cells were incubated with NEM, a well-known thiol-specific sca-
venger, for 30 min, and subsequently with the probe (20 μM) for an-
other 30 min. As shown in Fig. 3a–d, there was not any obvious fluor-
escence in both blue and green channels observed. This indicated that
NEM successfully deplete the endogenous biothiols. Subsequently, HeLa
4

Z. Lu, et al.
Bioorganic Chemistry 92 (2019) 103215
Fig. 3. Confocal Fluorescence images of living
HeLa cells in blue and green channels. (a–c)
HeLa cells incubated with NEM (100 μM) and
then ICN-NBD (20 μM). (d) Overlay of a–c. (e–g)
HeLa cells incubated with NEM (100 μM), then
Cys (50 μM), and then ICN-NBD (20 μM). (h)
Overlay of e–g. (i–k) HeLa cells incubated with
NEM (500 μM), then Hcy (50 μM), and then ICN-
NBD (20 μM). (l) Overlay of i–k. (m–o) HeLa
cells incubated with NEM (100 μM), then GSH
(
50 μM), and then ICN-NBD (20 μM). (p) Overlay
of m-o. Blue and green channels correspond to
the emission windows of 420–470 and
5
4
00–550 nm, respectively. Excitation at 405 and
88 nm, respectively. Scale bar = 20 μm.
cells were incubated with the probe for 30 min after treatment with
NEM (100 μM) and then Cys (50 μM). The cells showed strong fluor-
escence in both blue and green channels (Fig. 3e–h). In subsequent
groups (Fig. 3i–p), the cells demonstrated strong fluorescence at
4. Conclusion
In summary, this study has developed a simple dual-emission fluor-
escent probe for biothiols, based on combining two fluorophores ni-
trobenzofurazan (NBD) and phenanthroimidazole with a facile ether
bond. In the presence of Cys, the probe emitted remarkable fluorescence
in both blue (470 nm) and green (550 nm) channels. However, the ad-
dition of Hcy and GSH could only induce strong blue fluorescence, which
was reasonably assigned to the influence from geometrical constraints
and/or weak solvent interaction. These interesting findings demonstrate
the probe could be a promising and effective tool to detect biothiols and
further discriminate Cys from Hcy/GSH through a dual-emission mode.
Moreover, the probe has been successfully applied for visualizing en-
dogenous and exogenous biothiols in living HeLa cells and zebrafish
models with low cytotoxicity and good membrane permeability.
4
70 nm when HeLa cells were treated with the probe (20 μM) after
incubation with NEM and then Hcy (50 μM) or GSH (50 μM). this group
of cells presented very weak green fluorescence. These results indicate
that ICN-NBD can selectively bioimage biothiols based on the blue
fluorescence in living cells and could also differentiate Cys from Hcy/
GSH depending on the green fluorescence.
Inspired by the prominent above-mentioned fluorescence imaging
in living HeLa cells, the feasibility of ICN-NBD for bioimaging biothiols
was further checked in living zebrafish models. In a control group, 2-
day-old zebrafish were incubated with NEM (500 μM) for 30 min and
then with the probe (20 μM) for another 30 min. As show in
Fig. 4a1–a4, zebrafish did not emit any significant fluorescence in both
blue and green channels. In the experimental group, zebrafish were first
incubated with NEM (500 μM) for 30 min and then treated with Cys
Acknowledgements
(
50 μM) for another 30 min followed by the probe (20 μM) for 30 min.
We thank for the supporting from Shandong Provincial Natural
Science Foundation of China (grant No. ZR2019MB040), the Science
and Technology Project of University of Jinan (Grant No. XKY1823 and
XBS1320). We also thank Dr. Cory A. Black at BDG Synthesis (PO Box
38627, Wellington Mail Centre, Wellington 5045, New Zealand) for his
suggestions and comments which allowed us to largely improve the
manuscript.
As shown in Fig. 4b1–b4, both strong blue and green fluorescence was
detected in zebrafish incubated with Cys. Zebrafish incubated with the
probe after pretreatment with NEM and Hcy or GSH demonstrated
strong blue fluorescence (Fig. 4c1–c4 and d1–d4). Only weak green
fluorescence was detected. These results suggested that ICN-NBD could
be successfully applied for visualizing biothiols in zebrafish.
5

Z. Lu, et al.
Bioorganic Chemistry 92 (2019) 103215
Fig. 4. Fluorescence images of zebrafish. a1–a4) Zebrafish only for comparison. (B1–b4) Zebrafish incubated with NEM (500 μM) and then ICN-NBD (20 μM). (c1–c4)
Zebrafish with Cys (50 μM). d1–d4) Zebrafish with Hcy (50 μM). (e1–e4) Zebrafish incubated with GSH (50 μM). Scale bar = 500 μm.
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