A novel and effective benzo[: D] thiazole-based fluorescent probe with dual recognition factors for highly sensitive and selective imaging of cysteine in vitro and in vivo

Article abstract of DOI:10.1039/c9nj03202a

Cysteine (Cys) plays a key role in all life forms. In the present study, a novel and effective fluorescent probe, 2-(benzo[d]thiazol-2-yl)-1,4-phenylene diacrylate (BT-AC), was developed for selectively sensing Cys over other analytes. BT-AC alone essenti

Full text of DOI:10.1039/c9nj03202a

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A novel and effective benzo[d]thiazole-based fluorescent probe
with dual recognition factors for highly sensitive and selective
imaging of cysteine in vitro and in vivo
Received 00th January 20xx,
Accepted 00th January 20xx
‡a
‡a
a
a
a
a
a
DOI: 10.1039/x0xx00000x
Meiqing Zhu, Xiaoqin Wu, Linfeng Sang, Fugang Fan, Lijun Wang, Xiangwei Wu, Rimao Hua,
a*
b
Yi Wang, Qing X. Li
Cysteine (Cys) plays a key role in all life forms. In the present study, a novel and effective fluorescent probe, 2-
(benzo[d]thiazol-2-yl)-1,4-phenylene diacrylate (BT-AC) was developed for selectively sensing Cys over other analytes. BT-
AC alone essentially emits no fluorescence, whereas it achieves a 4725-fold enhancement with a large Stokes shift (135
nm) in the presence of Cys. Moreover, the limit of detection the fluorescent assay was as low as 32.6 nM of Cys. The
fluorescence response of BT-AC to calf serum provided strong evidence of the practical applicability of this probe. This
probe
was
successfully
used
for
imaging
of
Cys
in
HepG2
cells
and
zebrafish.
detection of thiols, fluorescence analysis and colorimetry are
currently the most convenient and fast. In particular,
fluorescence analysis has the advantages of simple operation
Introduction
As components of most proteins and large molecules,
3
3
and low detection limits. Since Sippel
used N-(4-(7-
1-8
thiols play vital roles in biophysiological activities
Intracellular thiols mainly include cysteine (Cys), homocysteine
Hcy) and glutathione (GSH), maintaining the dynamic redox
.
diethylamino-4-methylcoumarin-3-yl) phenyl) maleimide as a
probe for detecting small-molecule thiols in 1981, substantial
breakthroughs have been made in the design, synthesis and
application of small-molecule thiol probes. Fluorescent probes
for the detection of thiols are evolving rapidly due to the
(
equilibrium of organisms by establishing a balance between
the reduced and oxidized states. Cys is not only a precursor of
GSH, acetyl-CoA and taurine, but also a provider of sulfur
3
4-36
ability of these probes to monitor analytes in cells
.
1, 9-11
ligands to the sulfur-iron complexes in cells
. High
To date, many fluorescent probes for thiol detection have
been developed with different mechanisms of reactions, such
as aldehyde cyclization, Michael addition, and cleavage of
concentrations of Hcy may cause cardiovascular disease and
12-15
Alzheimer’s disease
. GSH is the most abundant non-
protein mercapto amino acid in cells, playing a key role in the
3
7-39
disulfide and sulfonamide bonds
. However, most of these
3, 11, 16-20
maintenance of the physiological functions of cells
.
probes cannot reliably distinguish Cys from Hcy and GSH due
Given the importance of thiols in physiological activities and
the strong association between these molecules and various
diseases, research on small-molecule thiol detection in
biological samples has received much attention and interest
4
0-44
to their similar structures and reactivity
. Compared with
traditional single-recognition-mode probe molecules, probes
with dual-recognition groups achieve efficient detection of
target analytes via a tandem or synergistic dual molecular
recognition reaction, thus significantly improving the
21-28
from scientists
.
Classic methods for the detection of small-molecule thiols
include electrochemical detection and high-performance liquid
chromatography, which can detect only the total amount of
thiols present and require large amounts of sample and long
4
5-
selectivity of the probes and achieving optimum recognition
4
8
.
In addition, these probes provide new ideas and methods
4
9-56
for the design of optical probes
. Therefore, the
development of sensitive and selective probes with dual-
recognition groups for detecting Cys has great scientific
significance.
29-32
detection times
. Therefore, the development of a
convenient and inexpensive method for qualitative and
quantitative analysis of thiols and for real-time examination of
biological samples is important. Among the many methods for
In the present study, a novel dual-site fluorescent probe
2
-(benzo[d]thiazol-2-yl)-1,4-phenylene diacrylate (BT-AC)
based on 2-(benzo[d]thiazol-2-yl) benzene-1,4-diol (BT-OH)
was synthesized. The application potential of the probe was
also studied. The bisacryloyl groups of BT-AC are used not only
as recognition groups but also as an electrophile. The main
characteristic of BT-AC is that the products of the first step
reaction can usually continue to react with Cys in the next
a. Key Laboratory of Agri-food Safety of Anhui Province, School of Resources and
Environment, Anhui Agricultural University, Hefei 230036, China
Department of Molecular Biosciences and Bioengineering, University of Hawaii at
Manoa, 1955 East-West Road, Honolulu, HI 96822, USA
† Electronic supplementary information (ESI) available. See DOI:
b.
‡
These authors contributed equally to this work.
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step, and both reaction mechanisms of the two steps are 1H), 6.92 – 6.88 (m, 1H), 6.84 (m, 1H). HRMS (ESI, m/z)
View Article Online
DOI: 10.1039/C9NJ03202A
9 2
H NO S + H] : 244.0432, found: 244.0429.
+
similar. BT-AC makes optimal use of the difference in structure calculated for [C13
and chemical properties between Cys and other thiols. BT-AC 2-(Benzo[d]thiazol-2-yl)-1,4-phenylene diacrylate (BT-AC).
can only undergo complete tandem reaction with Cys in a BT-OH (0.245 g, 1 mmol) and ET N (0.202 g, 2 mmol) were
certain period of time, while it reacts with Hcy through mixed in an eggplant-shaped flask containing 20 ml of
incomplete tandem reaction and cannot react with GSH. anhydrous dichloromethane, and the temperature of the
Therefore, BT-AC is capable of specifically detecting Cys over mixture was lowered to 0 °C. Then, acryloyl chloride (0.272 g,
other thiols. Moreover, BT-AC exhibits good cell permeability 3.0 mmol) was slowly added to the mixture through a constant
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and has been successfully used here for fluorescence imaging pressure dropping funnel under N atmosphere. After stirring
of Cys in live cells and zebrafish.
for 1 hour, the temperature of the mixture was raised to room
temperature, and the mixture was stirred at room
temperature until the reaction was complete (the reaction was
monitored using thin layer chromatography). The mixture was
Experimental
Reagents and apparatus
washed three times with saturated aqueous NaHCO
mL) and dried over anhydrous MgSO . Finally, the solvent was
evaporated under reduced pressure and the crude product
3
(3×10
4
2
,5-Dihydroxybenzaldehyde, 2-aminobenzenethiol and
were purchased from Energy Chemical Co. Ltd.
Shanghai, China). All amino acids were purchased from Sigma-
Aldrich (St. Louis, MO, USA). N-Ethylmaleimide (NEM, thiols
scavenger), NaHS, Fe(NO ·9H O, glucose, KNO
Mg(NO ·6H O, NaNO , H , Cu(NO ·3H O, Ca(NO
and Zn(NO ·6H O were acquired from J&K (Beijing, China). All
2 5
NaS O
(
was purified on silica gel column. (Vethyl acetate/Vpetroleum ether
=
1
/4, v/v) to obtain 2-(benzo[d]thiazol-2-yl)-1,4-phenylene
1
diacrylate (303 mg, 85% yield). H NMR (600 MHz, DMSO-d
6
):
3
)
3
2
3
,
δ 8.19 – 8.12 (m, 2H), 8.05 (d, J = 8.1 Hz, 1H), 7.58 – 7.54 (m,
H), 7.55 – 7.45 (m, 3H), 6.66 (dd, J = 17.3, 1.5 Hz, 1H), 6.64 –
.56 (m, 2H), 6.45 (dd, J = 17.3, 10.4 Hz, 1H), 6.30 (dd, J = 10.0,
.4 Hz, 1H), 6.20 (dd, J = 10.3, 1.2 Hz, 1H). C NMR (151 MHz,
): δ 164.53, 164.40, 160.95, 152.35, 148.53, 145.72,
35.47, 135.36, 134.56, 127.87, 127.85, 127.34, 126.88,
3
)
2
2
3
2
O
2
)
3 2
2
3 2 2
) ·4H O,
1
6
1
3
)
2
2
reagents were used without further purification unless
otherwise specified. Zebrafish were obtained from Shanghai
FishBio Co.,Ltd (Shanghai, China).
Nuclear magnetic resonance (NMR) spectra were recored
on Agilent 600MHz DD2（DirectDrive2）spectrometer (Santa
Clara, CA, USA) at for HNMR (600 MHz) and C NMR (150
MHz), and tetramethylsilane (TMS) were used as internal
standards for calibration purposes. All fluorescence and UV-vis
absorption spectra were collected on Agilent Cary Eclipse
fluorescence spectrophotometer (Santa Clara, CA, USA) and
Shimadzu UV-1800 spectrophotometer (Shimadzu, Kyoto,
Japan) respectively. Agilent Accurate-Mass-Q-TOF MS 6520
mass spectrometer (with an electrospray ionization source
1
3
DMSO-d
6
1
1
4
26.43, 125.98, 125.85, 123.51, 122.68, 122.63, 40.49, 40.37,
0.23, 40.17, 40.09, 39.95, 39.81, 39.68, 39.54. HRMS (ESI,
1
13
+
m/z) calculated for [C19
52.0640.
Fluorescence quantum yield
The fluorescence quantum yield of BT-AC was calculated
using quinine sulfate solution containing 1% H SO as a
standard. The calculation formula was as follows 58-62:
H
13NO
4
S + H] : 352.0644, found:
3
2
4
2
2
ϕX = ϕs(FX
FS)(AS AX)
(
ȠX ȠS
)
(
(
(
ESI)) were used for High-resolution mass spectrometry
HRMS) analyses. Shanghai REX pH-25 digital display pH meter
Shanghai, China) was used for pH values. Zebrafish and HepG2 represents the quantum yield of BT-AC, F represents the
integral area of the fluorescence emission spectrum, A is the
microscope in epifluorescence mode equipped different filters absorbance of BT-AC, and η is the refractive index of the
where “X” and “S” represent the sample BT-AC and the
reference substance quinine sulfate, respectively, Φ
cells imaging were acquired on a Nikon inverted Ti-S
for excitation and emission, respectively (Tokyo, Japan).
Synthesis and characterization
solvent.
Preparation of Fluorescence and UV-vis spectroscopy
2
-(Benzo[d]thiazol-2-yl)benzene-1,4-diol (BT-OH). 2-
The measurement conditions for all the fluorescence
spectroscopy experiments were as follows: E = 335 nm; slit width,
x
Aminothiophenol (500 mg, 3.99 mmol) and 2,5-
dihydroxybenzaldehyde (580 mg, 4.2 mmol) were added to the
eggplant bottle containing 20 ml DMF and stirred to dissolve
10 nm; emission wavelength range, 350-650 nm. The detection
conditions of ultraviolet-visible spectra are as follows: scanning
step, 1.0 nm; scanning wavelength range, 200-500 nm. The BT-AC
stock solution (10 mM) was prepared in analytical-grade DMSO. All
analytes solutions were dissolved in deionized water (100 mL) to
2 2 5
them. Then Na S O (0.70 g, 4.20 mmol) was added with
continuous stirring. The reaction mixture was heated to reflux
until the reaction was complete (The reaction was monitored
by TLC). Next, the mixture was then cooled to room
temperature and washed with deionized water (50 mL). The
solid precipitate was collected on a filter and recrystallized
-3
obtain 5×10 M aqueous solutions.
Cytotoxicity
4
First, HepG2 cells (Density of 4 × 10 cells per well) were
seeded in 96 well plates. Then, a series of concentration gradients
10, 20, 30, 40 μM) were set to incubate HepG2 cells, and untreated
HepG2 cells were used as a control. After 24 hours of BT-AC
from methanol to obtain the pure product 2-(benzo[d]thiazol-
1
2
-yl)benzene-1,4-diol (702 mg, 72% yield). H NMR (600 MHz,
(
DMSO-d
6
): δ 10.85 (s, 1H), 9.16 (s, 1H), 8.11 (dd, J = 7.1, 2.6 Hz,
1
H), 8.01 (dd, J = 7.6, 3.1 Hz, 1H), 7.64 – 7.47 (m, 2H), 7.42 (m,
treatment, the cells were washed three times with PBS buffer
2
| J. Name., 2012, 00, 1-3
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solution to remove BT-AC. The cell viability of HepG2 cells was then
analyzed using the CCK-8 assay and the MTT assay, respectively.
Absorbance was measured at 450 nm (CCK-8 assay) and 490 nm
demonstrate that the probe BT-AC exhibited no fluorescence at 470
View Article Online
DOI: 10.1039/C9NJ03202A
nm. Upon addition of Cys to the buffer solution containing BT-AC,
the fluorescence intensity exhibited an approximately 4725-fold
enhancement. Although the fluorescence intensity increased after
addition of Hcy, the fold increase was negligible compared to the
(
MTT assay) using a microtiter plate reader (ELx808, BioTek, USA).
Cell imaging
HepG2 cells were incubated in DMEM-H containing 10% calf
serum under humidified conditions at 37 °C and with 5% carbon
dioxide. First, the cells were plated in a 24-well plate and placed in
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an incubator for 12 h. Then, HepG2 cells were cultured in a 95% CO
incubator at 37 °C with a configured BT-AC, N-ethylmaleimide
NEM) or thiols solutions, and imaging experiments were performed
2
(
under an inverted fluorescence microscope (λex = 375 nm).
Untreated HepG2 cells were used as a blank control.
Imaging of zebrafish
The purchased zebrafish eggs were incubated in a 1000 ml
beaker containing E3 embryo culture medium, and the beaker was
placed in a constant temperature incubator (temperature setting
was 28 °C). After the zebrafish hatch, replace the E3 medium once a
day for three days. Normally growing three-day-old zebrafish were
selected and incubated with BT-AC and N-ethylmaleimide (NEM) in
an incubator at 28 °C. After washing three times with E3 medium,
imaging experiments were performed on zebrafish under
fluorescence microscope (λex = 375 nm). Untreated zebrafish served
as a blank control. All animal procedures were performed in
accordance with the Guidelines for Care and Use of Laboratory
Animals of Anhui Agricultural University and experiments were
approved by the Animal Ethics Committee of Anhui Agricultural
University.
Scheme 1 (a) Synthetic route of BT-AC and (b) fluorescence “turn on” mechanism of
BT-AC in the presence of Cys.
fold increase observed after addition of Cys. Moreover, the addition
of amino acids and analytes other than Cys and Hcy caused hardly
any changes in the fluorescence signal. As shown in Figure 1a
inset), compared with the other three groups of experiments, the
solution of BT-AC + Cys exhibited strong blue fluorescence under a
65-nm excitation ultraviolet lamp. The results showed that BT-AC
(
3
could specifically recognize Cys over other analytes. The UV-vis
spectra of BT-AC show absorption peaks at approximately 310 nm
Results and discussion
Synthesis and characterization of BT-AC
Considering the characteristics of easy synthesis, low
background fluorescence, high selectivity and sensitivity and low
cytotoxicity, a fluorescent probe was designed and synthesized by
combining 2-(benzo[d]thiazol-2-yl)benzene-1,4-diol as a
fluorophore with bisacryloyl groups as the recognition moieties of
Cys. As expected, BT-AC exhibited no fluorescence after
modification by bisacryloyl groups. The electron-poor acrylate
group is readily cyclized and cleaved by the Michael addition
reaction in the presence of a thiol, releasing a fluorophore, which
results in enhanced fluorescence to achieve the desired results. The
stepwise reaction of the thiols with the double recognition group
results in a different cyclization cleavage process, enabling BT-AC to
specifically recognize Cys. The synthetic process of BT-AC is as
described in Scheme 1a. BT-AC was characterized by H NMR, 13C
NMR and HRMS (Figures S1-5, ESI†). Optimization of detection
conditions with BT-AC is shown in Figures S6-8 (ESI†).
Figure 1 Fluorescence spectrum (a) and UV-vis spectrum (b) of the probe BT-AC (10 μM)
to various analytes (200 μM). The inset shows a photograph of the BT-AC solutions in
the presence and absence of thiols.
in buffer solution (pH 7.4, v/v, 7/3, λexꢀ=ꢀ335ꢀnm).
The UV-vis spectra of BT-AC changed significantly after
treatment with Cys and Hcy. The maximum absorption peak for BT-
AC exhibited a redshift of approximately 25 nm (Figure 1b). The
absorption spectra from the UV titration experiments showed that
the absorption intensity of BT-AC increased at 335 nm and
disappeared at 310 nm as the concentration of Cys/Hcy increased.
The above results indicated that only Cys and Hcy can react with BT-
AC, respectively. In addition, we obtained pseudo first-order rate
constants for the reaction of BT-AC with thiols by time-dependent
curve (Fig. S6B, ESI†). The constants of BT-AC and Cys/Hcy were
calculated (from the linear curve shown in Figure. S6b, ESI†) to be
1
Examination of the selectivity for Cys and resistance to
interference of BT-AC
To verify the selectivity of BT-AC for Cys, we studied the
reactions between BT-AC and various analytes, including several
familiar amino acids (Cys, Thr, Hcy, Lys, GSH, Asp, Arg, Pro, Try, Tyr,
+
-
+
-
2+
2+
2+
2+
His, Glu, Gly), different ions (K , NO
3
, Na , HS , Mg , Cu , Zn , Ca
-4
-5 -1
3
+
8.04 × 10 and 8.13 × 10 S , respectively. K is approximately 10
and Fe ), H
2
O
2
and glucose. The results shown in Figure. 1a clearly
Cys
This journal is © The Royal Society of Chemistry 20xx
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times the value of KHcy. The results show that BT-AC can recognize
Cys more quickly than Hcy.
AC probe solution exhibited strong absorption at 310 nm and
View Article Online
DOI: 10.1039/C9NJ03202A
almost no fluorescence at 470 nm (Figure 3a, b) (Ф = 0.001, using
In order to verify the interference of various analytes with the
selective recognition of Cys by BT-AC, various analytes that might
interfere with the specific recognition of Cys by BT-AC were
excluded. As shown in Figure 2, the results showed that the above
analytes did not hinder the selective recognition of Cys by BT-AC.
quinine sulfate as a standard). However, after the addition of Cys,
the absorption intensity of the BT-AC solution at 310 nm gradually
decreased with time and gradually increased with the absorption
peak at 335 nm, accompanied by the isoelectric point occurring at
approximately 330 nm. Interestingly, with constant addition of Cys,
the BT-AC solution completed a transition from no fluorescence to
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“
turn-on” fluorescence at 470 nm, and the change in the
fluorescence signal caused the solution to exhibit intense blue
fluorescence (ФBT-AC = 0.37, using quinine sulfate as a standard). In
the presence of equal amounts of Cys, the fluorescence intensity
and absorbance enhancement trend of BT-AC showed a good linear
relationship with the concentrations of Cys (Figure 3c-d). After
addition of 30 equivalents of Cys, the fluorescence of BT-AC
increased approximately 4725-fold. The limit of detection of Cys
was determined to be 32.9 nM by the unified detection limit
calculation formula (CDL = 3δ/S, where δ is the standard deviation
of the blank sample, and S is the slope). Compared to previously
reported thiol probes (Table S2, ESI†), BT-AC exhibits such
advantages as low detection limits and large Stokes shifts (135 nm).
These results indicate that BT-AC can selectively and quantitatively
detect Cys. Importantly, the large Stokes shift of the BT-AC-Cys
system increased the signal-to-noise ratio in imaging applications,
leading to the distribution of Cys in cells and zebrafish being highly
distinguishable during observation.
Sensing mechanism of BT-AC
Figure 2 Ratiometric response (I470nm/I387nm) of BT-AC (10 μM) in the presence and
absence Cys (200 μM) with various analytes (200 μM) in DMSO-HEPES buffer
solution (7:3, v/v, pH 7.4, λex = 335 nm, slit: 10.0 nm). Data are the mean ± ꢀSE
The mechanism of the reaction between BT-AC and Cys was
1
confirmed by H NMR and LC-HRMS. BT-AC and Cys were mixed in a
DMSO-d solution and allowed to react to completion (CBT-AC/CCys
/2), followed by filtration. The filtrate was subjected to H NMR
(bars) (nꢀ=ꢀ3).
6
=
Quantitative detection of Cys
1
1
analysis. As shown in Figure. 4, the peak for the H atom on the
bisacryloyl ester group of BT-AC was located at 6.20-6.66 ppm.
After completion of the reaction of BT-AC with Cys, the peaks for
the hydrogen atoms of the diacrylate group disappeared, which was
accompanied by the emergence of new peaks (Figure. 4). These
results indicate that the BT-AC undergoes a Cys-triggered
substitution-cyclization reaction to release a fluorescent group,
The responsiveness of BT-AC (10 μM) to Cys was investigated
in DMSO/HEPES solutions (pH 7.4, v/v, 7/3) at ambient
temperature. Under the above-mentioned optimum conditions, UV- which enhances the fluorescence signal.
1
Figure 4 H NMR spectrum of BT-AC before and after reaction with Cys.
Figure 3 (a) Fluorescence and (b) UV-via titration spectra of BT-AC (10 μM) vs Cys
(0~30 equiv.) in a DMSO-HEPES buffer solution (7:3, v/v, λexꢀ=ꢀ335ꢀnm). Plot of (c)
fluorescence and (d) UV-vis ratiometric response as a function of Cys equivalents.
Data are the mean ± ꢀSE (bars) (nꢀ=ꢀ3).
The reaction mixture of the probe BT-AC and Cys was also
examined by LC-HRMS (Figure S9, ESI†), and the conditions used are
listed in Table S1 (ESI†). Observation of the addition of Cys to BT-AC
produced two different emission peaks at 387 and 470ꢁnm (Figure
1), excited-state intramolecular proton transfer (ESIPT) process
vis and fluorescence titration experiments were performed. The BT-
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might occur between protons on phenolic hydroxyl group and
Practical applications of BT-AC
View Article Online
DOI: 10.1039/C9NJ03202A
nitrogen atoms on benzothiazole group. As the reaction between
BT-AC and Cys exhibits the ESIPT process from the beginning, we
conclude that Cys is first conjugated with the acrylate group near
the N atom to form thioether (BT-AC-Cys calculated [H ]: 473.0836,
found m/z: 473.0836). The BT-AC-Cys thioether system is cleaved at
Several potential applications of BT-AC were also investigated.
As BT-OH exhibits bright fluorescence in the solid state (Figure S13,
ESI†), a test strip experiment was carried out for convenient
detection of Cys. Therefore, neutral filter paper is first used to make
test strips with the same size. The paper was then wetted in BT-AC
solution (10 mM in DCM) and dried in the oven to prepare test
strips for convenient detection of Cys. A change in color was
+
the ester bond position and cyclizes to form a seven-membered ring
and releases a fluorescent group (calculated [H ]:
+
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98.0532/176.0376, found m/z: 298.0533/176.0390, respectively).
Cys then reacts with another acryloyl group on BT-AC in the same
manner as above (calculated [H ]: 419.0730, found m/z: 419.0731).
Finally, the thioether formed by Cys with another acryloyl group is
cleaved and cyclized to form BT-AC (calculated [H ]:
+
+
4
19.0730/176.0376, found m/z: 419.0731/176.0376, respectively).
Compared with the reaction of BT-AC and Hcy, the activation
entropy required for the seven-membered ring reaction of BT-AC
and Cys is low; it is easier for BT-AC to react with Cys than with Hcy,
leading to specific recognition of Cys (Scheme 1b).
Density functional theory (DFT) calculations
Figure 6 (a) Relative fluorescence intensity of calf serum (%) with BT-AC (10ꢀμM) in
aqueous HEPES buffer (pHꢀ7.4, DMSO content < 0.2%), λexꢀ=335ꢀnm; (b) correlation
curve between relative fluorescence intensity and calf serum content. Data are the
mean ± ꢀSE (bars) (nꢀ=ꢀ3).
To better understand the fluorescence mechanism of BT-AC
and Cys. First, the spatial distribution and orbital energies of the
HOMO and LUMO of BT-OH, BT-OH-AC and BT-AC were optimized
according to the B3LYP method in the Gaussian 09 program (Figure
S10-12, ESI†). Figure 5 shows that LUMO is mainly distributed on
the fluorophore group near the bisacryloyl ester group on the BT-
AC, while HOMO is uniformly distributed on the fluorophore. When
the acryloyl group interacts with Cys, the HOMO energy of BT-AC is
lower than that of BT-OH, which causes the intramolecular transfer
of electrons on BT-AC to occur again, leading to the transition of the
upper electrons on the fluorescent group back to the ground state
and producing fluorescence. This result indicates the bisacryloyl-
modified BT-AC undergoes electron transfer from the receptor to
the fluorescent group, which hinders radiation relaxation and thus
quenches the fluorescence of BT-AC. After Cys reacted with one of
the acryloyl groups, BT-OH-AC was released, which led to the
transfer of HOMO to the fluorescent group. Further reaction of Cys
with BT-OH-AC led to cleavage of the second acryloyl group,
allowing HOMO to be completely transferred to the fluorescent
group. Thus, 2-(benzo[d]thiazol-2-yl) benzene-1,4-diol was released
completely, which led to fluorescence enhancement. The DFT
analysis is consistent with the above-mentioned experimental
results and confirms the mechanism proposed previously.
promptly and clearly observed with Cys when various analyte
solutions were dripped onto the test strip with a straw. The strips
were visualized under a portable UV lamp with excitation at 365
nm. In comparison, the other analytes did not induce any change in
color. This result indicates that the BT-AC test strip is easy to
prepare and can specifically detect Cys.
In addition, we investigated the effect in fluorescence response of
foetal calf serum by BT-AC in HEPES buffer (pH 7.4) at ambient
temperature. As shown in Figure 6, the fluorescence intensity of BT-
AC changed as the concentration of calf serum increased.
Interestingly, the calf serum content exhibited a close linear
2
relationship with the fluorescence intensity at 470 nm (R = 0.9967).
Therefore, BT-AC can be used to detect foetal calf serum levels in
practical applications.
Cell imaging
The effects of BT-AC on HepG2 cells were studied by two kinds
of cytotoxicity assays (CCK-8 and MTT) prior to cell imaging
experiments. As shown in Figure 7, as the concentration of BT-AC
Figure 7 Cell viability values (%) estimated by a CCK-8 and MTT assay versus
incubation concentrations of BT-AC. HepG2 cells were cultured in the presence
of 0-40 μM BT-AC. Data are mean  SE (bars) (n = 3).
Figure 5 HOMO and LUMO of BT-OH, BT-OH-AC and BT-AC, respectively.
This journal is © The Royal Society of Chemistry 20xx
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increased, the survival rate of HepG2 cells did not change
significantly. Although the concentration of BT-AC increased to 40
μM, more than 85% of HepG2 cells survived. The experimental
results show that BT-AC is less toxic to cells and not affect the
HepG2 cells experimental results.
Thus, we used BT-AC to image Cys in HepG2 cells. HepG2 cells
were pretreated with N-ethyl maleimide (NEM, thiols scavenger)
prior to incubation with BT-AC. Subsequently, HepG2 cells were
incubated with exogenous thiols. As shown in Figure 8, the
Conclusions
View Article Online
DOI: 10.1039/C9NJ03202A
The fluorescent probe BT-AC with dual-recognition groups
was developed by taking full advantage of the structural
characteristics of different thiols (Cys, Hcy and GSH). The
probe BT-AC is highly selective towards Cys, and its sensitivity
for Cys is negligibly affected by other amino acids and metal
ions. HRMS and NMR were used to confirm the reaction
mechanism, and DFT was used to verify the results. BT-AC has
been utilized in test strips and calf serum to detect Cys. In
addition, the probe has the advantages of low cytotoxicity, a
large Stokes shift (135 nm) and a low limit of detection of Cys
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(
32.6 nM). BT-AC has been also successfully applied in the
imaging of live HepG2 cells and zebrafish. The application of
dual-recognition groups can provide a reference for further
structural optimization of fluorescence probes to improve
selectivity and sensitivity.
Conflicts of interest
No conflicts of interest.
Acknowledgements
Figure 8 Fluorescence microscope images (λex = 375 nm) of BT-AC in living HepG2
cells to identify Cys. HepG2 cells were incubated with BT-AC (20 μM) for 20 min (a);
HepG2 cells pretreated with NEM (1 mM) for 1 h and then incubated with BT-AC (20
μM) for 20 min (b); the last three groups of HepG2 cells pretreated with NEM (1.0
mM) for 1 h, then incubated with GSH (c), Hcy (d), and Cys (e) (200 μM) for 20 min,
respectively, and finally incubated with BT-AC (20 mM) for 20 min. From top to
bottom: Dark field (UV light), Bright field, Merge. Scale bar: 20 µm.
This work was financially supported in part by the National
Natural Science Foundation of China (NSFC) (no. 31601657)
and the National Undergraduate Innovation and
Entrepreneurship Training Program (No. 201810364088).
fluorescence intensity of HepG2 cells treated with Cys was
significantly enhanced, while the fluorescence intensity of HepG2
cells added with Hcy and GSH was not changed. The results show
that BT-AC can realize the visualization of endogenous Cys in HepG2
cells.
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