Using Bioluminescence Turn-On to Detect Cysteine in Vitro and in Vivo

Article abstract of DOI:10.1021/acs.analchem.8b00682

Cysteine (Cys) is an essential amino acid and plays important roles in many biological processes. Bioluminescence (BL) is advantageous in sensitivity but BL probes that were intentionally developed for the selective detection of Cys were rarely reported. Herein, employing a fast conjugate addition between Cys and acrylic ester, we synthesized a caged BL probe acrylic ester luciferin (1) and used it to selectively detect Cys in vitro and image Cys in living cells and in tumor sites. We envision that, in the future, probe 1 might be used for evaluating the Cys roles in more biological processes.

Full text of DOI:10.1021/acs.analchem.8b00682

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Using Bioluminescence Turn-On to Detect Cysteine in Vitro and in Vivo
Miaomiao Zhang, Lin Wang, Yangyang Zhao, Fuqiang Wang, Jindao Wu, and Gaolin Liang
Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b00682 • Publication Date (Web): 02 Apr 2018
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Using Bioluminescence Turn-On to Detect Cysteine in Vitro and in
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†
‡
‡,
§
§
Miaomiao Zhang, Lin Wang, Yangyang Zhao, Fuqiang Wang, Jindao Wu, * and Gaolin
Liang†,*
†CAS Key Laboratory of Soft Matter Chemistry, Department of Chemistry, University of Science and Technology
of China, 96 Jinzhai Road, Hefei, Anhui 230026, China
‡Key Laboratory of Living Donor Liver Transplantation of Ministry of Public Health, Department of Liver
Transplantation Center of The First Affiliated Hospital, Nanjing Medical University, 140 Hanzhong Road, Nanjing,
Jiangsu 210029, China
‡School of Life Sciences, University of Science and Technology of China, Huangshan Road, Hefei, Anhui 230027,
China
ABSTRACT: Cysteine (Cys) is an essential amino acid and plays important roles in many biological processes.
Bioluminescence (BL) is advantageous in sensitivity but BL probes that were intentionally developed for the selective
detection of Cys were rarely reported. Herein, employing a fast conjugate addition between Cys and acrylic ester, we
synthesized a caged BL probe acrylic ester luciferin (1) and used it to selectively detect Cys in vitro and image Cys in
living cells and in tumor sites . We envision that, in the future, probe 1 might be used for evaluating the Cys roles in
more biological processes.
Cysteine (Cys) is an essential amino acid to living
subjects and involved in several important biological
processes including protein synthesis, detoxification, and
metabolism.1 Elevated levels of Cys were reported to
associate with neurotoxicity2 and cardiovascular
diseases,3 and Cys deficiency may lead to a lot of
pathological symptoms such as edema, liver damage,
skin lesions, and weakness.4 Consequently, precise
assessment of Cys levels in human bodies may aid the
early diagnoses of some diseases. So far, a lot of
analytical methods have been reported for sensing Cys
including high performance liquid chromatography
(HPLC),5 capillary electrophoresis,6 voltammetry,7 mass
spectrometry (MS),8 colorimetric,9 and fluorometry.10-12
Among them, fluorescence is widely used due to its high
sensitivity, low limit of detection (LOD), and simple
implementation. However, fluorescence always suffers
from photo bleaching of the fluorophores, interference
by the auto-fluorescence from the detection samples, etc.
In contrast to fluorescence, bioluminescence (BL) has
higher signal-to-noise ratio because it does not need
light excitation.13-14 Moreover, due to their biological
sources, BL probes always have relatively lower
cytotoxicity than fluorescence probes. To date, various
BL probes have been synthesized for sensing (or imaging)
of a series of important biomakers.15-19 However, BL
probes that were intentionally developed for the selective
detection of Cys were rarely reported.
fluorescence sensors, all of which share an acryloyl
appendage, have been reported for Cys detection
operating via a Michael addition and an intramolecular
cyclization between Cys and the appendage.21-24
Inspired by above mentioned studies, herein we
synthesized a silent BL probe acrylic ester luciferin (1),
whose hydroxyl group of D-luciferin motif being caged by
acrylic ester, for selective detection of Cys in vitro and
sensing Cys inside cells and tumors. As outlined in
Scheme 1, a conjugate addition of Cys to the acrylate of 1
occurs to yield the thioether intermediate, which
instantly undergoes an intramolecular cyclization to
yield a stable seven-member heterocyclic compound and
D-luciferin. In the presence of adenosine triphosphate
(ATP), O2, and Mg2+, -luciferin will be converted by the
D
enzyme firefly luciferase (fLuc) to yield decarboxylated
luciferin, accompanied by a photon emission.25
It is well known that the condensation reaction
between acrylate and Cys can be used for the preparation
of substituted 1,4-thiazepines.20 Recently, many
Scheme 1. Proposed mechanism of probe 1 for cysteine
sensing.
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Figure 1. (a) Bioluminescence spectra of 25 μM probe 1 with (red) or without (black) 100 μM Cys in phosphate buffered
saline (10 mM, pH 7.4) containing 0.1 mg/mL fLuc and 10 mM MgCl2. (b) The linear response of 25 μM probe 1 towards Cys in
the range of 0-25 μM. (c) Selectivity of 1 (25 μM) towards Cys (25 μM), HCys (25 μM), GSH (25 μM) or other 19 natural amino
acids (each at 250 μM), respectively. The error bar represents the standard deviation of three independent experiments. The
relative (B-B0) (%) indicates the ratio of the bioluminescence intensity of probe 1 with the analyte to that of probe 1 with Cys.
We firstly synthesized and characterized probe 1
(Scheme S1 and Figures S1-S3). Next, we studied its
chemical response to Cys. After adding 4-fold Cys to the
solution of 1 and incubated the mixture for 30 minutes at
37 °C, we observed the UV absorption peak 300 nm of 1
(GSH)) or 250 μM of other 19 natural amino acids (Val,
Tyr, Trp, Ser, Pro, Phe, Met, Lys, Leu, Arg, Ile, His, Gly,
Gln, Glu, Asp, Asn, Ala, Thr), respectively. As shown in
Figure 1c, compared with B - B0 value induced by other
selected species in the presence of fLuc, 25 μM Cys
showed significantly BL enhancement after addition to
25 µM 1 (23-182 folds of those induced by the other
tested species). HPLC analysis indicated that some
amino acids (e.g., Gln) could also react with probe 1 to
yield tiny amount of D-luciferin to generate BL (Figure
S9). This result suggested that probe 1 has excellent
selectivity toward Cys over other intracellular small
biological molecules. Interference tests indicated that the
bioluminescence intensity of probe 1 (25 μM) with Cys
(25 μM) was almost not affected in the presence of above
analytes, suggesting our probe 1 could be applied for
sensing Cys in real samples (Figure S10).
red shifted to 330 nm which is the typical UV peak of -
D
luciferin (Figure S4), indicating the removal of the
acryloyl appendage from 1 to yield D-luciferin. Then, BL
response of 1 toward Cys was preliminarily investigated.
As shown in Figure 1a, after addition of 100 μM Cys into
the solution of 25 μM 1 in phosphate buffered saline (10
mM PBS, pH 7.4, with 10 mM MgCl2) and incubation at
37 °C for 30 min, addition of 1 mM ATP and 0.1 mg/mL
fLuc into the mixture immediately induced the increase
of the BL intensity significantly . For the control group
without Cys addition, BL signal was barely observed
under the same condition. This result further validated
our hypothesis that probe 1 could react with Cys to yield
free D-luciferin to generate BL. Under this condition,
about 98.4% of probe 1 was calculated to convert to
-
D
luciferin using HPLC analysis (Figure S5). Kinetic study
of the reaction between Cys and 1 indicated that this
reaction had a second-order rate constant of 60.2 M-1·S-1
(Figure S6).
After confirming the rapid response of probe 1 to Cys,
we performed its sensitivity studies. After incubation
with different concentrations of Cys for 30 min at 37 °C,
probe 1 exhibited a different increase of BL intensity in
the range of 0-100 μM (Figure S7) and a good linear
relationship (Y = 6.489X + 3.886, R2 = 0.997) between
the BL intensity enhancement (i.e., B - B0) of 25 µM
probe 1 and Cys concentration in the region of 0-25 µM
(Figure 1b and Figure S8). A LOD 88.0 nM of probe 1
toward Cys was obtained based on the general 3σ
method (3σ/s).26 which is comparable to the methods
that have been reported (Table S1).
It is known to all that the selectivity of a probe to its
target analyte among potential competitive species is
very important. To further evaluate its feasibility for
biomedical applications, we incubated 25 μM 1 with 25
μM biothiols (Cys, homocysteine (Hcy), glutathione
Figure 2. BL images of fLuc-transfected MDA-MB-231
cells in different times that pre-incubated with 2 mM MBA
for 30 min followed by incubation with 25 μM Cys for 30
min and then with 25 μM 1 (top row), pre-incubated with 2
mM MBA for 30 min followed by 25 μM 1 (middle row),
incubated with 25 μM 1 (bottom row) acquired at 0, 10, 20,
50, 80, 110, 140, and 170 min at 37 °C in culture medium
without serum.
After verifying the good property of 1 for Cys detection
in vitro, we prepared to apply probe 1 for BL imaging of
Cys in living cells. Before that, we evaluated the
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cytotoxicity of 1 on MDA-MB-231 cells using 3-(4,5-
dimethylthiazol-2-yl) 2,5 diphenyltetrazplium bromide
(MTT) assay. More than 95% of the cells survived in 100
µM 1 up to 8 h (Figure S11), suggesting that probe 1 at a
concentration below 100 µM was safe for living cell
imaging. We thus chose probe 1 at 25 µM for the below
cell imaging experiment. As we know, cancer cells are
characterized with a high concentration of intracellular
GSH (~mM).27 Therefore, interference from intracellular
GSH should be studied before applying 1 for sensing Cys
inside cells. Through kinetic study, the second-order rate
constant of the reaction between probe 1 and GSH was
measured to be 14.7 M-1·S-1 (Figure S12), which was
much smaller than that of 1 toward Cys. In addition, only
a very small proportion of probe 1 was transformed into
D-luciferin after the reaction with GSH (Figure S13). This
demonstrated that probe 1 had good selectivity for
imaging Cys over GSH. Indeed, as shown in figure S14,
although the BL from 1 generated by 250 µM GSH was
lower than that by 25 µM Cys, BL by 1 mM GSH was
much higher. Therefore, we blocked the thiol activity
with maleimidobutyric acid (MBA) before applying the
cells for BL imaging of Cys with probe 1. MDA-MB-231
cells that transfected with fLuc were pre-incubated with
2 mM MBA for 30 min to consume all of the free thiols
within the cells and washed with PBS buffer for three
times. For Cys detection, after co-incubation with 25 μM
Cys for 30 min, the cells were washed with PBS buffer for
three times and then put in a 96-well plate at 5 × 105
cells/well. After addition of 25 μM 1, in virtue of a small
animal imaging system, we gained real-time recording of
BL signals that generated from the cells. As shown in
Figure 2, after the addition of probe 1, BL signal from the
cells in the top row was the brightest. Quantification of
the BL intensity indicated that the BL signal gradually
increased to its plateau (9.88 × 104 of total flux, Figure
S15) at around 50 min, followed by a slow decrease. For
the control cells whose thiol activity was blocked by 2
mM thiol-reactive MBA for 30 min but without Cys
addition, very weak BL signal (5.01 × 104 of total flux at
50 min, Figure S15) was observed after 30 min
incubation with 25 μM 1 (middle row in Figure 2),
suggesting above strong BL signal was induced by the
added Cys. For the cells without any treatment, weak BL
signal (5.15 × 104 of total flux at 50 min, Figure S15) was
also observed after 25 μM 1 addition (bottom row in
Figure 2). Since Hcy is hardly detectable in healthy
mammalian cell, thus the weak BL should be ascribed to
the products of 1 that reacts with intracellular Cys and
GSH. In addition, cellular uptake tests of 1 indicated that
the probe gradually entered MDA-MB-231 cells in 2 h
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Figure 3. BL imaging of nude mice that xenografted with
fLuc-transfected MDA-MB-231 tumors at different times
after i.p. injection of MBA at 0.5 mmol/kg followed by i.v.
injection of Cys at 1.0 mmol/kg 30 min later and i.v.
injection of 1 at 12.5 μmol/kg 30 min later (top row), i.p.
injection of MBA at 0.5 mmol/kg followed by i.v. injection
of 1 at 1.0 mmol/kg 30 min later (middle row), i.v. injection
of 1 at 1.0 mmol/kg (bottom row) in PBS at 15, 20, 25, 30,
and 40 min.
We then applied probe 1 for BL imaging of Cys in
tumors in living mice. A fLuc-transfected MDA-MB-231
tumour was xenografted in the right thigh of each nude
mouse and the mice were randomly divided into three
groups. One group of mice were intraperitoneally (i.p.)
injected with 0.5 mmol/kg MBA followed by intravenous
(i.v.) injection of Cys at 1.0 mmol/kg and finally i.v.
injection of probe 1 at 12.5 μmol/kg and used as the
experimental group. The remaining two groups were
used as control groups. In detail, one group of mice were
injected with MBA followed by the injection of 1 and
another group of mice were injected with 1 only. In
virtue of a small animal imaging system, we gained the
real-time recording of BL signal that generated from the
tumours in the nude mice. As shown in the top row of
Figure 3, during the observation time (i.e., 15-40 min),
the BL signal from the experimental group gradually
increased to reach its peak at 25 min (5.73 × 106 of total
flux, Figure S17) and slowly decreased thereafter,
and rapidly reacted with Cys to yield -luciferin (Figure
D
S16). These results indicated that probe 1 is applicable
for BL imaging of Cys in living cells.
suggesting that our probe 1 reacted with Cys to yield -
D
luciferin to generate BL in the tumors. For the control
group whose thiol activity was blocked by injected MBA
but without Cys injection, almost no bioluminescent
signal (1.53 × 105 of total flux at 25 min, Figure S17) was
observed after probe 1 injection (middle row in Figure 3),
indicating above BL signal in the experimental group
was induced by the injected Cys. Interestingly, for the
control group that only injected with probe 1, weak BL
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signal was also observed (7.20 × 105 of total flux at 25
min, Figure S17, bottom row in Figure 3). This indicated
that the intracellular Cys and GSH in tumors reacted
with probe 1 to yield D-luciferin to generate the BL. These
results suggested that probe 1 was applicable for imaging
Cys in tumours in living mice.
In summary, we developed a silent bioluminescent
probe 1 to selectively sense Cys in vitro and image Cys in
vivo. The high selectivity of probe 1 for Cys detection lies
in the fast conjugate addition of Cys to the acrylate motif
of 1 and subsequent intramolecular cyclization to release
D-luciferin to generate BL in the presence of fLuc. We
successfully applied probe 1 to selectively detect Cys in
the 0-25 μM linear concentration range with a LOD of 88
nM in vitro. Moreover, we also evaluated the capability
of probe 1 for sensing Cys in living cells and tumours. We
envision that, in the future, probe 1 might be applied to
evaluate the Cys roles in more biological processes.
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ASSOCIATED CONTENT
Supporting Information
General methods; Syntheses and characterizations of 1;
Scheme S1; Figure S1-S17; Table S1-S3. This material is
available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*E-mail: wujindao@njmu.edu.cn (J. Wu).
*E-mail: gliang@ustc.edu.cn (G. Liang).
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
This work was supported by the Ministry of Science and
Technology of China (2016YFA0400904), the National
Natural Science Foundation of China (Grants 21725505 and
21675145), Major program of Development Foundation of
Hefei Center for Physical Science and Technology
(2016FXZY006), and Jiangsu Youth Medical Talents
(QNRC2016580).
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