A new chloro-substituted dicyanoisophorone-based near-infrared fluorophore with a larger Stokes shift and its application for detecting cysteine in cells and: In vivo

Article abstract of DOI:10.1039/d0nj02913k

Many dicyanoisophorone-based fluorophores with an optical hydroxyl group have been explored to meet different imaging needs along with the rapid and wide development of molecular fluorescence bioimaging in recent years. However, most of them used to need the addition of slight alkaline conditions to dissociate a proton for near-infrared emission and this limits their wider application in complex bio-systems. Aiming to modify the dicyanoisophorone-based fluorophore platform with an optically tunable hydroxyl group for better biocompatibility for molecular bioimaging in vivo, we developed a new chloro-substituted dicyanoisophorone-based near-infrared fluorophore (DCM-COH) which featured simple preparation (two steps), good optical properties and showed potential application prospects. As a proof-of-concept, DCM-COH was applied to design a new probe (CYS-1) for detecting cysteine. CYS-1 (λex/em = 490/655 nm) possessed a large Stokes shift (165 nm), low toxicity, good sensitivity (detection limit of ～173 nM) and selectivity for rapid cysteine detection. More importantly, CYS-1 successfully served as an indicator for imaging cysteine in cells and in vivo. Fluorophore DCM-COH may act as a potential platform to be extended as a capable tracking tool in biological chemistry and preclinical applications. This journal is

Full text of DOI:10.1039/d0nj02913k

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A new chloro-substituted dicyanoisophorone-based
near-infrared fluorophore with a larger Stokes shift
and its application for detecting cysteine in cells
and in vivo†
Cite this:DOI: 10.1039/d0nj02913k
Yu Wang,‡a Wenda Zhang, ‡*b Ting Ma,‡c Duolu Li,b Yubing Zhou,b
Xiaojian Zhang*bde and Jianbo Gao*a
Many dicyanoisophorone-based fluorophores with an optical hydroxyl group have been explored to
meet different imaging needs along with the rapid and wide development of molecular fluorescence
bioimaging in recent years. However, most of them used to need the addition of slight alkaline
conditions to dissociate a proton for near-infrared emission and this limits their wider application in
complex bio-systems. Aiming to modify the dicyanoisophorone-based fluorophore platform with an
optically tunable hydroxyl group for better biocompatibility for molecular bioimaging in vivo, we
developed a new chloro-substituted dicyanoisophorone-based near-infrared fluorophore (DCM-COH)
which featured simple preparation (two steps), good optical properties and showed potential application
prospects. As a proof-of-concept, DCM-COH was applied to design a new probe (CYS-1) for detecting
cysteine. CYS-1 (lex/em = 490/655 nm) possessed a large Stokes shift (165 nm), low toxicity, good
sensitivity (detection limit of B173 nM) and selectivity for rapid cysteine detection. More importantly,
CYS-1 successfully served as an indicator for imaging cysteine in cells and in vivo. Fluorophore
DCM-COH may act as a potential platform to be extended as a capable tracking tool in biological
chemistry and preclinical applications.
Received 10th June 2020,
Accepted 8th August 2020
DOI: 10.1039/d0nj02913k
rsc.li/njc
of dynamic life systems is difficult.1,3 In order to meet the
complex imaging needs, a variety of fluorophores have been
Introduction
In recent years, molecular fluorescence bio-imaging technology developed. Among them, the molecular fluorophores with an
has attracted more and more attention due to its advantages of optically tunable hydroxyl group play an important role in the
high temporal and spatial resolution, high sensitivity, strong development of activated fluorescent probes, the hydroxyl
specificity, simple operation, fast detection response and so group of which was regulated through the protection and
on.1–6 It has been used as a powerful strategy to detect enzyme deprotection of hydroxyl groups.10–12 The design rule is shown
activity or small biological species in pathophysiological eluci- in Fig. 1. When the characteristic hydroxyl group is protected by
dation and disease diagnosis of related diseases.7–9 However, to a trigger group, the fluorescence of the probe is ‘‘turned off’’ or
trace and visualize related biological species in the complexity
a Department of Radiology, The First Affiliated Hospital of Zhengzhou University,
No. 1, East Jianshe Road, Zhengzhou 450052, Henan, China.
E-mail: gaojianbo63@126.com
b Department of Pharmacy, The First Affiliated Hospital of Zhengzhou University,
No. 1, East Jianshe Road, Zhengzhou 450052, Henan, China.
E-mail: zhangwenda16@126.com, zhangxiaojian15@126.com
c School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou 450001,
China
d Henan Key Laboratory of Precision Clinical Pharmacy, Zhengzhou University,
Zhengzhou, China
e Henan Engineering Research Center of Clinical Mass Spectrometry for Precision
Medicine, Zhengzhou, China
Fig. 1 Design principle of molecular probes for biological analytes
through hydroxyl regulation.
† Electronic supplementary information (ESI) available. See DOI: 10.1039/d0nj02913k
‡ These authors contributed equally.
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the semichromatic displacement is displayed due to the weak- to design a new cysteine detection probe (CYS-1). CYS-1 (lex/em
=
ening of the electron donor capacity. The characteristic hydro- 490/655 nm) has a large Stokes shift (165 nm), low toxicity, high
xyl trigger group of the fluorophore is cleaved under the action sensitivity (detection limit is about 173 nM), and selectivity for
of a special analyst to produce a wide emission fluorescence the rapid detection of cysteine. CYS-1 can also be used as an
signal.
indicator for cysteine imaging in cells and in vivo under low
So far, many molecular fluorophores with optical hydroxyl background interference.
groups have been constructed, such as coumarin,13–16 naphthal-
imide,17,18 indanone,19 rhodol,12,20 dicyanomethylene-4H-pyran
(DCM),21 BODIPY,10 and HD dye,22,23 which are widely used due
to their photophysical properties (Fig. 2). However, the application
of these fluorophores in biological systems is still challenging, due
to the short emission wavelength (o600 nm) of coumarin,
naphthalimide and rhodol, the short Stokes shift of fluorescein
and HD dyes, and the complex preparation of BODIPY. Interest-
ingly, dicyanophosphorone fluorophores exhibit short wavelength
emission under physiological conditions, while they exhibit near-
infrared red emission in dimethyl sulfoxide or other organic
solvents.11,24–29 Although dicyanophosphorone fluorophores have
the advantages of simple synthesis, good water solubility, a large
Stokes shift and good biocompatibility, adding more dimethyl
sulfoxide or other organic solvents in the detection system will
cause enzyme degradation or damage the organisms, and this
Experimental section
Materials and general experimental methods
Unless specially stated, all chemicals and solvents were supplied
by Sinopharm chemical reagent Co. Ltd. Cysteine was obtained
from Sigma-Aldrich Co. Ltd. 1H NMR spectra were measured
using a Bruker spectrometer (500 MHz) and 13C NMR spectra
were measured using a Bruker spectrometer (125 MHz). The
fluorescence spectra were recorded on a Spectra Max Paradigm
Multi-Mode Detection Platform. In vivo imaging was performed
using a CRI Maestro small animal in vivo imaging system.
Synthesis
(E)-2-(3-(3-Chloro-4-hydroxystyryl)-5,5-dimethylcyclohex-2-en-
limits its application in the living system. This encourages us to 1-ylidene)malononitrile (DCM-COH). To a solution of compound
develop new NIR fluorescent carriers with better biocompatibility 1 (1.0 g, 5.37 mmol) and piperidine (0.15 mL) in acetonitrile
for in vivo molecular imaging.
(40 mL) was added 3-chloro-4-hydroxybenzaldehyde (925 mg,
In this study, a new near-infrared emission fluorophore 5.91 mmol), the mixture was further stirred at 80 1C for 6 h. After
DCM-COH was developed. It did not need a high proportion the reaction, the precipitate was filtered, washed, and recrystal-
of DMSO or other organic solvents, but exhibited long emission lized with ethanol to obtained DCM-COH as a red crystal (1.2 g,
1
wavelength and a large Stokes shift under physiological conditions. 68.8%). H NMR (500 MHz, CDCl3) d 7.51 (s, 1H), 7.35 (d, J =
In addition, it has excellent chemical and optical properties, 7.3 Hz, 1H), 7.04 (d, J = 8.4 Hz, 1H), 6.93 (d, J = 16.1 Hz, 1H), 6.85
such as simple preparation (step synthesis), suitable pKa (7.60), (d, J = 16.1 Hz, 1H), 6.82 (s, 1H), 5.75 (s, 1H), 2.60 (s, 2H), 2.44
deep tissue penetration, near-infrared emission in a physiological (s, 2H), 1.08 (s, 6H). 13C NMR (101 MHz, CDCl3) d 189.59 (s),
environment and so on, which showed a broad application 169.20 (s), 156.54 (s), 153.60 (s), 152.58 (s), 135.20 (s), 130.75 (d, J =
prospect. As a proof of concept, it has been successfully applied 14.8 Hz), 129.66 (s), 128.29 (s), 127.99 (d, J = 17.8 Hz), 123.45 (s),
121.10 (s), 120.76 (s), 116.79 (d, J = 14.1 Hz), 113.51 (s), 112.73 (s),
78.66 (s), 42.99 (s), 39.21 (s), 32.05 (s), 28.04 (s). (ESI-MS) m/z calcd
for C19H17ClN2O [M À H]+, 323.0957; found 323.0955.
(E)-2-(3-(4-Hydroxystyryl)-5,5-dimethylcyclohex-2-en-1-ylidene)-
malononitrile (DCI-OH). To a solution of compound 1 (1.0 g,
5.37 mmol) and piperidine (0.15 mL) in acetonitrile (40 mL) was
added 4-hydroxybenzaldehyde (656 mg, 5.37 mmol), the mixture
was further stirred at 80 1C for 6 h. After the reaction, the
precipitate was filtered, washed, and recrystallized with ethanol
to obtained DCI-OH as a red crystal (1.2 g, 69.8%). 1H NMR
(500 MHz, DMSO) d 9.97 (s, 1H), 7.55 (d, J = 8.5 Hz, 2H), 7.21
(d, J = 8.5 Hz, 2H), 6.83–6.75 (m, 3H), 2.59 (s, 2H), 2.53 (s, 2H),
1.01 (s, 6H). 13C NMR (101 MHz, CDCl3) d 191.17 (s), 169.64 (s),
157.51 (s), 154.61 (s), 137.05 (s), 132.57 (s), 129.55 (s), 128.68 (s),
127.12 (s), 122.86 (s), 116.17 (d, J = 14.7 Hz), 113.84 (s), 113.08 (s),
43.16 (s), 39.36 (s), 32.18 (s), 28.17 (s).
(E)-2-Chloro-4-(2-(3-(dicyanomethylene)-5,5-dimethylcyclohex-
c1-en-1-yl)vinyl)phenyl acrylate (CYS-1). To a solution of DCM-
COH (0.5 g, 1.54 mmol) and DIPEA (0.58 mL) in dry dichloro-
methane (10 mL) was added acryloyl chloride (374 mL, 4.62 mmol)
in 10 mL of dry dichloromethane. Under an air atmosphere,
the reaction mixture was stirred at room temperature for 3 h.
Fig. 2 Structures of reported fluorophores with an optically tunable
hydroxyl group.
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The resulting organic layer was washed with water and dried over A549 cells were treated with CYS-1 (10 mM) after incubation
Na2SO4. After dichloromethane removal and further purification with cysteine (20 mM) and then NEM (1 mM). The images of the
with silica gel column chromatography (petroleum ether/ethyl cells were taken under a laser confocal microscope.
acetate), the desired compound CYS-1 was afforded as a yellow
1
In vivo imaging of cysteine in living mice
solid (0.51 g, 87.5%). H NMR (500 MHz, DMSO) d 8.00 (d, J =
1.6 Hz, 1H), 7.71 (dd, J = 8.4, 1.6 Hz, 1H), 7.52 (d, J = 16.2 Hz, 1H),
7.42 (d, J = 8.4 Hz, 1H), 7.28 (d, J = 16.2 Hz, 1H), 6.93 (s, 1H), 6.61
(d, J = 17.3 Hz, 1H), 6.47 (dd, J = 17.2, 10.4 Hz, 1H), 6.24 (d, J =
10.5 Hz, 1H), 2.63 (s, 2H), 2.53 (s, 2H), 1.02 (s, 6H). 13C NMR
(126 MHz, DMSO) d 170.28 (s), 163.13 (s), 155.18 (s), 146.64 (s),
135.89 (s), 134.84 (d, J = 9.7 Hz), 131.23 (s), 128.57 (s), 127.96 (s),
126.72 (s), 126.52 (s), 124.61 (s), 123.61 (s), 113.70 (s), 112.90 (s),
77.18 (s), 42.24 (s), 38.11 (s), 31.68 (s), 27.44 (s). (ESI-MS) m/z
calcd for C22H19ClN2O2 [M À H]À, 377.1062; found 377.1061.
All animal procedures were performed in accordance with the
requirements of the Animal Ethics Committee of Zhengzhou
University, and the guidelines of the National Institutes of
Health. All animal experiments were approved by the Animal
Ethics Committee of Zhengzhou University.
For in vivo imaging, the mice were divided into two groups.
(a) Fluorescence imaging of cysteine in living mouse after
intraperitoneal injection of PBS (100 mL) as blank; (b) fluores-
cence imaging of cysteine in living mouse after injection of
CYS-1 (200 mM, 50 mL) followed by treatment with cysteine
(200 mM, 50 mL) for 25 min. Then, the in vivo imaging was
obtained by using a CRI Maestro small animal in vivo imaging
system.
General procedure to monitor cysteine in vitro
All the spectrum measurements were performed in PBS buffer
(10 mM, pH 7.4) at 37 1C. The stock solution of probe CYS-1
(5 mM) was prepared in DMSO. Various analytes (Gly, Leu,
KH2PO4, Na2SO3, NaHCO3, MgSO4, KAl(SO4)2, KCl, NaAC, KI,
NaIO4, CaCl2, NaCl, GSH, Hcy, and cysteine) were prepared in
ultrapure water. The calculated amount of probe in the test
solution was prepared at a final concentration of 10 mM.
Cysteine and other analytes were also dissolved in test solution
with an appropriate concentration. The fluorescence emission
spectra were collected (lex = 490 nm, lem = 655 nm).
Results and discussion
Fluorophore structure optimization and performance study
Dicyanoisophorone-based systems with attractive attention
have been widely used in bioimaging due to their many
advantages including simple synthesis, fine water solubility, a
large Stokes shift, excellent biocompatibility and so on. As a
dicyanoisophorone-based fluorescent dye, DCI-OH has been
widely used as a fluorophore to design a variety of fluorescent
probes; its excitation and emission wavelengths are 433 nm and
570 nm in PBS solution respectively. Because near-infrared red
emissions with higher penetrability can avoid auto-fluorescence
interference from indigenous biomolecules compared with
the shorter wavelength, many analytical chemists usually force
DCI-OH to dissociate a proton for NIR emission through the
addition of more dimethyl sulphoxide or other organic solvent
in detecting systems, which may cause enzyme degeneration to
damage organisms in living systems. This encouraged us to
modify its structure to develop a new NIR fluorophore with
better biocompatibility for molecular bioimaging in vivo. The
key issue is that the high pKa value of DCI-OH leads to its low
proton dissociation under physiological conditions and limits
its precise bioimaging application in vivo.16,30 Nevertheless,
electron-withdrawing substitution at the ortho position of
hydroxyl on the fluorophore skeleton can reduce the pKa value
of the molecular dye. As expected, new chloro-substituted DCM-
COH (pKa = 7.60) exhibits a better optical performance under
mimetic physiological conditions compared with DCI-OH
(pKa = 9.02) (Fig. 3). Taken together, DCM-COH is more suitable
than DCI-OH for developing activated in vivo bioimaging fluor-
escent probes.
Activation mechanism
ESI-MS was used to verify the activation mechanism. In detail,
CYS-1 (50 mM) was incubated with cysteine (50 mM) for 5 min in PBS
solution, then extracted with dichloromethane and concentrated.
The concentrated solution was analyzed by ESI-MS.
Cell culture and cytotoxicity assay
A549 cells were provided by Cell Bank of Shanghai Institute of
Biochemistry and Cell Biology, Chinese Academy of Sciences
(Shanghai, China), and were grown in DMEM medium supple-
mented with 10% fetal bovine serum (FBS) at 37 1C in a 5%
CO2/95% air incubator. For MTT assays, cells were initially
seeded into a 96-well plate at a density of 6 Â 103 cells per well
and incubated for 24 h, and then media were replaced with
200 mL DMEM media with different concentrations (0, 5.0, 10,
20, 30, and 40 mM) of the probe. After incubation for 24 h
0.02 mL of the MTT (0.5 mg mLÀ1) were added to each well
followed by culture for another 4 h. After that, the media were
abandoned and the formed formazan was dissolved in 150 mL
DMSO in each well. Then, the absorbance values were mea-
sured at 570 nm with a reference wavelength of 630 nm using
an ELISA reader (Spectra Max Plus 384, Molecular Devices,
Sunnyvale, CA) and the cell viability rate was calculated.
Imaging of cysteine in living cells
Design and synthesis of CYS-1
Cells were seeded into a 96-well plate and cultured in DMEM
medium supplemented with 10% FBS in an atmosphere of 5% Cysteine participates in a variety of life activities, and its
CO2 at 37 1C. After 24 h, the cells were treated with cysteine (20 mM), abnormal change can lead to serious diseases including acquired
then incubated with 10 mM probe or without probe as control. immunodeficiency syndrome, liver damage and skin damage.31,32
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Scheme 1 Synthesis of DCM-COH and probe CYS-1.
implied its favorable water solubility. The excitation and emission
spectra of DCM-COH (10 mM) were measured in PBS solution, the
maximum excitation and the maximum emission was at 490 nm
and 655 nm respectively (Fig. 5A), the NIR emission of DCM-COH
with an obviously large Stokes shift (165 nm) was suitable for
imaging analysis due to the efficiently decreased measurement
error caused by the overlapped excitation light and scattered
light. The time-dependent fluorescence response of CYS-1 toward
cysteine was tested. As shown in Fig. 5B, the fluorescence intensity
at 655 nm increased rapidly with time, and reached the maximum
value in approximately 25 min. The significantly increasing
maximum fluorescence intensity at 655 nm suggested that the
cleavage reaction initiated by cysteine resulted in the release of
free DCM-COH, indicating that the probe can be used as a
fluorescent sensor sensitive to cysteine. Subsequently, with the
increase of cysteine concentration, the fluorescence intensity at
655 nm increased gradually. The continuous increase of cysteine
resulted in a significant enhancement of fluorescence intensity
at 655 nm (Fig. 5C). In the concentration range of 0–150 mM,
the fluorescence signal intensity has a linear relationship with
cysteine (R2 = 0.9993, Fig. 5D). The detection limit of the probe
(3s/slope method) was as low as 173 nM.
Fig. 3 (A) Structure modification of fluorophore DCI-OH. (B) Fluores-
cence intensity at the maximum emission wavelength of DCI-OH and
DCM-COH vs. pH.
Up to now, many fluorescent probes have been developed for the
detection of cysteine, but there is still difficulty in monitoring the
in vivo cysteine concentration due to their insufficient sensitivity
and their short emission wavelengths (Table S1, ESI†).4 Though
some NIR fluorescent probes with long excitation wavelengths
(4650 nm) were reported, the relatively short Stokes shift or high
proportion of organic solvent in test systems limited further
biological applications.2,33–38 Development of fluorescent probes
with a large Stokes shift which can penetrate deeper tissue under
physiological conditions for cysteine tracking is still urgent.
As a proof-of-concept, DCM-COH was used as an NIR fluoro-
phore for developing a new probe CYS-1 to detect biomarker
cysteine for the first time. Herein, we chose the acryl ester as a
selective reaction trigger for cysteine to realize fluorescence ‘‘off–on’’.
After interaction of CYS-1 with cysteine, the free DCM-COH was
released, yielding a remarkable fluorescence signal (Fig. 4).
CYS-1 was synthesized within two steps as shown in Scheme 1,
DCM-COH or DCI-OH was obtained by reaction compound 1
with 3-chloro-4-hydroxybenzaldehyde or 4-hydroxybenzaldehyde.
Compound DCM-COH in dry dichloromethane was added to acryl
chloride to obtain CYS-1. The spectra of 1H NMR and 13C NMR are
shown in Fig. S3–S9 in the ESI.†
Spectral response of CYS-1 towards cysteine
Firstly, the spectral character of this probe was initially inves-
tigated in the buffer solution (PBS, 10 mM, pH = 7.4), which
Fig. 5 (A) Excitation and emission spectra of DCM-COH (10mM);
(B) fluorescence response of 10 mM CYS-1 after incubation with Cys during
time in PBS buffer (10 mM, pH 7.4); and (C) fluorescence response of 10 mM
CYS-1 after incubation with Cys (0–150 mM) in PBS buffer (10 mM, pH 7.4).
(D) Linear relationship of fluorescence response of 10 mM CYS-1 with a
function of the concentrations of Cys from 0 to 150 mM at 655 nm in PBS
(lex = 490 nm, lem = 655 nm).
Fig. 4 Design principle of molecular probe CYS-1 for cysteine detection
based on DCM-COH.
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The detection mechanism of CYS-1 towards cysteine
ionization of cysteine, promoted the thiolysis reaction to
restore the fluorophore. The results showed that the probe
has obvious advantage and practical value for the application of
bio-imaging in living systems. In order to adapt to the physio-
logical conditions of the cellular system, a pH of 7.4 was selected
as the experimental condition.
As aforementioned, the cleavage of acryl ester by the cysteine-
reactive reaction could release the electro-donating OH gene-
rating DCM-COH. The activation mechanism was verified by
electrospray ionization mass spectrometry (ESI-MS). CYS-1 was
incubated with cysteine in PBS solution and extracted by
dichloromethane and concentrated. The concentrated solution
was analyzed by ESI-MS. As shown in Fig. S3–S5 (ESI†), the peak
at m/z 174.0231 [M À H]À corresponds to a seven-membered
cyclized by-product, the peak at m/z 323.0931 [M À H]À corre-
sponds to DCM-COH and the peak at m/z 377.1059 [M À H]À
corresponds to CYS-1, indicating that the addition of CYS-1 in
the presence of cysteine did produce DCM-COH.
The cysteine detection by CYS-1 in bottled water samples
Next, the selectivity of CYS-1 was tested by determining the
concentration of Cys in water samples through a recovery test.
The fluorescence signal at 655 nm was recorded by adding
different Cys to the water sample. As shown in Table 1, CYS-1
determined the concentration of Cys in the water samples, with
a good recovery ranging from 98.4% to 101.6%. The results
indicated that CYS-1 could be used for the quantitative detec-
tion of Cys in water samples.
Selectivity of cysteine
In order to further verify the specificity of the probe for the
fluorescence sensing of cysteine, the fluorescence signal
changes of the probe to various analytes (including Gly, Leu,
KH2PO4, Na2SO3, NaHCO3, MgSO4, KAl(SO4)2, KCl, NaAC, KI,
NaIO4, CaCl2, NaCl, GSH, Hcy, and cysteine) were recorded.
As shown in Fig. 6, the addition of other analytes made the
probe exhibit negligible fluorescence response. In conclusion,
probe CYS-1 possessed excellent specificity under physiological
conditions.
Cytotoxicity and cell imaging of CYS-1
The excellent performance of the probe in vitro detecting
system enabled us to further explore its ability to track
cysteine in living cells. Before cellular application, the bio-
compatibility of the probe on A549 cells was detected by the
MTT method. After incubation with a high concentration of
40 mM CYS-1 for 24 h, the survival rate of A549 cells was close
to 90%, indicating that the cytotoxicity of the probe on cells
was very small (Fig. S2, ESI†). We further used the probe to
evaluate the intracellular cysteine change in A549 cells. The
results in Fig. 7 show that when the probe (10 mM) was added,
a fluorescence signal can be observed indicating that A549
tumor cells contain a certain amount of cysteine. When the
cells were pretreated with cysteine (20 mM) and incubated with
the probe (10 mM), an obvious near-infrared fluorescence
signal of living cells was observed, which indicated that
the probe had good cell permeability and potential to detect
cysteine in living cancer cells.
The detection character of CYS-1 under different pH
conditions
In addition, we studied the fluorescence character of the probe
at different pH values to study its application in biological
imaging (Fig. S1, ESI†). No obvious emission changes at 655 nm
were observed in the pH range of 6–9, indicating that the probe
could remain stable under acidic and alkaline conditions.
As expected, in the presence of cysteine, the emission increased
distinctly in the pH range from 6–9, especially under the
alkaline conditions. The alkaline solution increased the
In vivo imaging of CYS-1 towards cysteine
For accurate disease diagnostics, in vivo imaging is a powerful
tool particularly for suspicious lesions with high spatio-
temporal precision. We examined the applicability of the probe
in the in vivo detection of the endogenous cysteine. Mice were
divided into two groups. In the first group, the mice were
injected subcutaneously with PBS (100 mL) for 25 min as a
control. In the second group, the mice were subcutaneously
injected with CYS-1 (200 mM, 50 mL) followed by injection with
cysteine (200 mM, 50 mL) for 25 min. The in vivo imaging was
captured on a CRI Maestro small animal in vivo imaging system.
As shown in Fig. 8, almost no fluorescence was observed in the
first group. However, a significant NIR fluorescence enhancement
Table 1 Results of the recovery test for applicability evaluation in bottled
purified water
Fig. 6 Fluorescence intensity changes of CYS-1 upon exposure to different
types of analytes. Fluorescence intensity at 655 nm of CYS-1 (10 mM) upon
interaction with other analytes (40 mM) in PBS buffer (10 mM, pH 7.4).
Columns a–q: Gly, Leu, KH2PO4, Na2SO3, NaHCO3, MgSO4, KAl(SO4)2, KCl,
NaAC, KI, NaIO4, CaCl2, NaCl, GSH, Hcy, and cysteine.
Sample no.
Cys spiked (mM)
Recovered (mM)
Recovery (%)
1
2
50
100
49.0 Æ 0.75
100.2 Æ 3.7
98.4
101.6
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Conclusions
In conclusion, this study aimed to modify a dicyanoisophorone-
based fluorophore platform with an optically hydroxyl tuneable
group for better biocompatibility for molecular bioimaging
in vivo, chloro-substituted dicyanoisophorone-based DCM-COH
(lex/em = 490/655 nm) with long emission wavelength and large
Stokes shift was constructed and synthesized as a new fluoro-
phore with NIR emission under physiological conditions.
In addition, it has excellent chemical and optical properties,
such as simple preparation (one-step synthesis), suitable pKa
(7.60), a NIR emission for deeper tissue penetration in a physio-
logical environment, and a large Stokes shift (165 nm) which
shows a wide range of potential application prospects. As a
proof-of-concept, DCM-COH has been successfully applied to
the design of a probe (CYS-1) for detecting cysteine. CYS-1
possessed a large Stokes shift (165 nm), low toxicity, high
sensitivity (detection limit of B173 nM), good cell membrane
permeability, and selectivity for the rapid detection of cysteine.
More importantly, the NIR emission fluorescence of the probe
had fast response and low background interference, which made
it possible to detect cysteine in vivo, indicating that the probe
could be applied as a promising tool for detecting cysteine
in complex bio-systems and diagnosing cysteine-associated
diseases in clinical trials. We hope that the platform of this
excellent and easy-to-use NIR fluorophore (DCM-COH) with an
optically tunable hydroxyl group may be exploited for the capable
tracking of various analytes in biological chemistry and preclinical
applications.
Fig. 7 Fluorescence imaging of cysteine in A549 cells by probe CYS-1 in
cancer cells. Fluorescence images of A549 cells treated without CYS-1;
fluorescence images of A549 cells treated with CYS-1 (10 mM); and
fluorescence images of A549 cells treated with CYS-1 (10 mM) after being
incubated with cysteine (20 mM). Fluorescence images of A549 cells
treated with CYS-1 (10 mM) after being incubated with NEM (1 mM) and
cysteine (20 mM). The images were taken under a laser confocal micro-
scope. Scale bar: 50 mm.
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
This research work was supported by the National Natural
Science Foundation of China (Grant No. 81903770) and the
Joint Construction Project of Medical Science and Technology
Research Plan of Henan Province, LHGJ20190266.
Notes and references
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