Detection of Intracellular Selenol-Containing Molecules Using a Fluorescent Probe with Near-Zero Background Signal

Article abstract of DOI:10.1021/acs.analchem.6b01545

Selenocysteine (Sec), encoded as the 21st amino acid, is the predominant chemical form of selenium that is closely related to various human diseases. Thus, it is of high importance to identify novel probes for sensitive and selective recognition of Sec and Sec-containing proteins. Although a few probes have been reported to detect artificially introduced selenols in cells or tissues, none of them has been shown to be sensitive enough to detect endogenous selenols. We report the characterization and application of a new fluorogenic molecular probe for the detection of intracellular selenols. This probe exhibits near-zero background fluorescence but produces remarkable fluorescence enhancement upon reacting with selenols in a fast chemical reaction. It is highly specific and sensitive for intracellular selenium-containing molecules such as Sec and selenoproteins. When combined with flow cytometry, this probe is able to detect endogenous selenols in various human cancer cells. It is also able to image endogenous selenol-containing molecules in zebrafish under a fluorescence microscope. These results demonstrate that this molecular probe can function as a useful molecular tool for intracellular selenol sensing, which is valuable in the clinical diagnosis for human diseases associated with Sec-deficiency or overdose.

Full text of DOI:10.1021/acs.analchem.6b01545

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Article
Detection of Intracellular Selenol-Containing Molecules Using
A Fluorescent Probe with Near-Zero Background Signal
Qi Sun, Shu-Hou Yang, Lei Wu, Qing-Jian Dong, Wen-Chao Yang, and Guang-Fu Yang
Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b01545 • Publication Date (Web): 10 May 2016
Downloaded from http://pubs.acs.org on May 13, 2016
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Detection of Intracellular Selenol-Containing Molecules Using A
Fluorescent Probe with Near-Zero Background Signal
a †
a †
a
b
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Qi Sun, Shu-Hou Yang, Lei Wu, Qing-Jian Dong, Wen-Chao Yang, * Guang-Fu Yang *
a
Key Laboratory of Pesticide & Chemical Biology of Ministry of Education, College of
Chemistry, Central China Normal University, Wuhan 430079, P.R. China;
b
Department of Nuclear Medicine, Tongji Hospital, Tongji Medical College, Huazhong University
of Science and Technology. Wuhan 430030, P.R. China.
—
————————
†
*
These two authors made equal contributions.
Wen-Chao Yang, e-mail: tomyang@mail.ccnu.edu.cn; Tel: 86-27-67867706; Fax:
8
6-27-67867141; Guang-Fu Yang, e-mail: gfyang@mail.ccnu.edu.cn; Tel: 86-27-67867800;
Fax: 86-27-67867141.
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ABSTRACT
Selenocysteine (Sec), encoded as the 21st amino acid, is the predominant chemical
form of selenium that is closely related to various human diseases. Thus, it is of high
importance to identify novel probes for sensitive and selective recognition of Sec and
Sec-containing proteins. Although a few probes were reported to detect artificially
introduced selenols in cells or tissues, none of them has been shown to be sensitive
enough to detect endogenous selenols. We report the characterization and application
of a new fluorogenic molecular probe for the detection of intracellular selenols. This
probe exhibits near-zero background fluorescence but produces remarkable
fluorescence enhancement upon reacting with selenols in a fast chemical reaction. It is
highly specific and sensitive for intracellular selenium-containing molecules such as
Sec and selenoproteins. When combined with flow cytometry, this probe is able to
detect endogenous selenols in various human cancer cells. It is also able to image
endogenous selenol-containing molecules in zebrafish under fluorescence microscope.
These results demonstrate that this molecular probe can function as a useful molecular
tool for intracellular selenol sensing, which is valuable in the clinical diagnosis for
human diseases associated with Sec-deficiency or overdose.
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Keywords: Selenocysteine, fluorescent probes, benzoquinolizine coumarin, cellular
imaging, sensors
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INTRODUCTION
Selenium, discovered in 1817, is a micronutrient known to be important to human
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health. The year 2017 was announced to be the selenium year in recognition of the
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significance of selenium for 200 years. Intracellular selenium mainly takes the form
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of selenocysteine (Sec),
which is an essential building block for selenoproteins
10-16
(
SePs).
SePs have been implicated in various cellular functions and linked to
several human diseases, such as cancers, cardiovascular diseases, neurodegenerative
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, 17-21
diseases and inflammations.
Due to the importance of biological selenols, there
is a growing interest in developing highly selective fluorescent molecular probes that
can facilitate intracellular selenol-containing molecule detection and creation of
molecular tools to diagnose human diseases associated with selenium deficiency and
2
2-25
overdose.
However, designing selenol-selective probes represents a significant
challenge mostly due to the interference of biological thiols that share similar
chemical properties to selenols but are much more abundant in cells and other
biological samples. To date, only a few fluorescent probes have been designed to
2
2, 24, 26
selectively detect biological selenols.
Although these probes are able to detect
artificially introduced selenols in cells or tissues (thus at significantly elevated levels),
none of them has been shown to be sensitive enough to detect endogenous selenols,
an important issue that we sought to solve in the current study.
a
The selenol pK value in Sec (∼5.8) is lower than thiophenol (∼6.5) and biological
thiols (∼8.3), which means Sec is more likely to exist as deprotonated state. So, the
thiophenol probe may also react with Sec in acidic conditions due to the same
mechanism of nucleophilic attack. We have previously reported a thiophenol probe,
2
7
denoted 1, that is only weakly fluorescent (quantum yield Φ = 0.0050). Probe 1
indeed reacted with selenol, such as Sec, resulting in the release of the free
fluorophore, 7-N, N-diethylaminocoumarin (2). Because the quantum yield (Φ) of 2 is
0
.14, this reaction leads to the generation of a strong fluorescence. Probe 1 shows
excellent selectivity and sensitivity for Sec, with a limit of detection (LOD) is ∼62
2
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nM. It is sensitive enough for quantifying the serum concentration of selenium in
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healthy adults (>500 nM), but not sufficient for accurate selenium determination in
the selenium-deficient patients with Keshan disease or Kashin-Beck disease (~100
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nM).
Therefore, further modification of probe 1 is necessary in order to further
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improve its detection sensitivity.
Recently, we hypothesized that the blockage of internal twisting of the nitrogen
atom at 7-position on coumarin ring of probe 1 can significantly enhance the quantum
yield of the freed fluorophore, and then discovered a new thiophenol probe with
improved the sensitivity through the strategy of blocking the internal twisting of the
3
0
nitrogen atom at 7-position on coumarin ring (denoted 3 in Scheme 1). Inspired by
the good detection capability against selenol with thiophenol probe 1, the newly
developed probe 3 was applied for selenol-containing molecule detection in this study.
After comprehensive characterization, the new probe shows much improved
properties for selenol-containing molecules: (1) exhibits first-order rate constant (k) of
-1
2.15 min , the best among all reported Sec probes to date; (2) about 250-fold
fluorescence intensity enhancement in pure aqueous solution; and (3) sufficient LOD
(~18 nM) for selenium determination in selenium-deficient patients. More importantly,
probe 3 is able to detect intracellular selenols with flow cytometry, and discriminate
the cancer cells from normal cells. To the best of our knowledge, this is the first report
that cancer cells showed higher intracellular level of selenol-containing molecules
compared to normal cells. In addition, the probe successfully imaged the endogenous
selenol-containing molecule and exogenous Sec in zebrafish, demonstrating its
potential for the biological function study of SePs, and the clinical diagnosis for the
selenium-related diseases.
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Scheme 1. The mechanism of Sec detection with molecular probe 3.
O N
2
O
H
NH2
N S
O
NO2
Sec
aqueous
solution
N
O
O
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N
O
O
3
4
No fluorescence
Stronge fluorescence
EXPERIMENTAL SECTION
Organic Synthesis
Unless otherwise noted, all chemical reagents were commercially available and
treated with standard methods before use. Silica gel column chromatography (CC):
silica gel (200-300 mesh); Qingdao Makall Group Co., Ltd (Qingdao; China).
1
Solvents were dried in a routine way and redistilled. H NMR was recorded in
6 3
DMSO-d or CDCl on a Varian Mercury 400 MHz and 600 MHz spectrometer, while
1
3
the C NMR spectra were recorded at 100 MHz and 150 MHz spectrometer. The
resonances (δ) are given in ppm relative to tetramethylsilane (TMS). The following
abbreviations were used to designate chemical shift mutiplicities: s = singlet, d =
doublet, t = triplet, m = multiplet, br = broad. High resolution Mass Spectra (HRMS)
were acquired in positive mode on a WATERS MALDI SYNAPT G2 HDMS (MA,
USA). Melting points were taken on a Buchi B-545 melting point apparatus and
uncorrected.
Synthesis of Probe 3
The major intermediates (M and 4) and the final product (probe 3) were
31-33
synthesized according to reported methods.
(
10-nitro-2,3,6,7-tetrahydro-1H,5H,11H-pyrano[2,3-f]pyrido[3,2,1-ij]q
1
uinolin-11-one) (M): purple solid, yield: 77%, H NMR (400 MHz, CDCl
3
): δ
8
4
.55 (s, 1H), 6.98 (s, 1H), 3.41 (q,
H).
J = 5.3 Hz, 4H), 2.76-2.86 (m, 4H), 1.99 (s,
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(10-amino-2,3,6,7-tetrahydro-1H,5H,11H-pyrano[2,3-f]pyrido[3,2,1-ij]
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quinolin-11-one) (4): yellow solid, yield: 61%, H NMR (400 MHz, DMSO-d6):
δ
6.79 (s, 1H), 6.62 (s, 1H), 4.97 (s, 2H), 3.11-3.14 (m, 4H), 2.67-2.74 (m, 4H),
+
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1.85-1.91 (m, 4H). HRMS calcd for [M ]: 256.1206. Found: 256.1211.
(2,4-dinitro-N-(11-oxo-2,3,6,7-tetrahydro-1H,5H,11H-pyrano[2,3-f]py
rido[3,2,1-ij]quinolin-10-yl)benzenesulfonamide) (probe 3): black solid,
o
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yield: 86 %. m.p.: 206-207 C. H NMR (600 MHz, CDCl
1H), 8.41 (d, = 6 Hz, 1H), 8.13 (d, = 8.4 Hz, 1H), 7.78 (d,
6.94 (s, 1H), 3.25-3.30 (m, 4H), 2.77 (q, = 8.0 Hz, 4H), 1.92-1.98 (m, 4H). C
NMR (100 MHz, DMSO- ): 164.32, 154.90, 154.65, 152.34, 150.94, 145.58,
3
):
δ
8.76 (d,
J = 1.8 Hz,
J
J
J
= 3.6 Hz, 2H),
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J
d
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δ
142.95, 137.05, 131.84, 130.54, 124.59, 123.66, 118.07, 111.83, 110.16, 59.78,
+
54.22, 53.69, 31.72, 25.68, 24.65. HRMS calcd for [M+H] : 487.0918. Found:
18 4 8
487.0914. Anal. Calcd for C21H N O S: C, 51.85; H, 3.73; N, 11.52; S, 6.59;
Found: C, 51.82; H, 3.72; N, 11.53; S, 6.56.
Sec Sensing and Selectivity Evaluation
The sensing reaction of probe 3 (10 μM) with certain amount of Sec was carried
out in 100 mM phosphate buffer at desired pH environment at 30 ˚C. The Sec was
2
freshly prepared by the preincubation of equal molar of (Sec) with DTT in the same
phosphate buffer. The fluorescence intensity of the above system was monitored at the
wavelength of 535 nm (λex = 380 nm).
To study the interference that may be caused by small molecules or ions, probe 3
(10 μM) was mixed with various analytes, such as amino acids (Phe, Arg, His, Tyr,
2
+
2+
+
+
Ala, Trp, Lys and Met), metal ions (Ca , Mg , Na and K ) and biological thiols
(Cys, Hcy and GSH ), respectively. After 30 min incubation, the fluorescence
intensity of each sample was measured at the wavelength of 535 nm (λex = 380 nm).
Similar selectivity study were performed against protein and enzymes (bovine serum
albumin (BSA), human elastase, acetylcholinesterase (AChE), butyrylcholinesterase
(BChE) and trypsin, and their final concentrations were around 0.01 mg/mL, while
the concentration of recombinant human thioredoxinredutase (hTR) is about 0.01
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mg/mL. The fold of fluorescence increase (F/F
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) was normalized to the base
fluorescence intensity of probe 3 for the above studies.
The recombinant hTR was generated by overexpression of hTR-pGEX plasmid
that was purchased form Protech Company (Wuhan, China). The mutants U498C and
U498S were obtained by site-directed mutagenesis.
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Determination of Quantum Yield
According to the reported method, the quantum yields for probe 3 and its
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fluorophore (compound 4) were determined with rhodamine B as reference.
More specifically, using rhodamine B (Φ = 0.88, ethanol) as reference, compounds 3
and 4 were firstly diluted to suitable concentration by PBS (0.1 M, pH 7.0) buffer to
make their absorption less than 0.05. Then, their absorbance and integral areas were
studied at this concentration. Finally, the quantum yields of these two compounds
were calculated by Abbe’s refractometer and the following equation.
Fs ⋅ Ac
φs =
φc
(1)
Fc ⋅ A
s
Determination of Second Order Reaction Rate Constant
For a second order reaction, A and B are reactants, while the C and D are
products.
k
A + B ⎯⎯⎯ ⎯→ C + D
If the starting concentrations of compound A and B are equal, such as a, the
reaction rate can be calculated by the following equation. K is the second order
reaction rate, X means the concentrations of products, such as C and D.
dx
2
=
K ×(a − x)
(2)
dt
Then the equation (3) was converted from equation (2).
1
= K ×t
(3)
a − x
For our system, the fluorescence intensity F and concentration of products are in
the linear relationship at the early stage. So the concentration of the product can be
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calculated by the equation (4)
Ft − F0
F∞ − Ft
x =
× a
(4)
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In the above equation, F
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represent the start fluorescence intensity of reaction
systems at t = 0. F represent the total fluorescence intensity of reaction systems
∞
when the reaction had been complete response. F means the fluorescence intensity of
t
reaction systems after incubation time t. The fluorescence intensity of reaction
systems was monitored at the wavelength of 535 nm (λ = 380 nm).
ex
The equation (4) was substituted into equation (3) to yield the equation (5).Based
on this equation, the second order reaction rate K could be calculated.
Ft − F0
=
K ×a × t
(5)
F∞ − F
t
Living Cell Imaging and Cytotoxicity Study
H460 cells were cultured in DMEM supplemented with 10% (V/V) fetal
bovine serum, 1% (V/V) penicillin/streptomycin and maintained in the atmosphere of
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5% CO at 37 °C. The cells were seeded on 6-well plates at 1×10 cells per well in 2
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mL culture medium and incubated overnight in 5% CO at 37 °C. Then both (Sec)2
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and DTT (20 μM) were added into the cells and incubated for different times (0h, 6 h
and 12 h). After washing the cells with PBS three times to move the remaining (Sec)2
and DTT，the cells were further incubated with probe 3 (20 μM) for 20 min at 37 °C.
After washing the cells with PBS three times, the treated cells were imaged by
inverted fluorescence microscopy (Olympus IX71, Japan). In order to induce
endogenous Sec, the H460 cells were treated with sodium selenite (20 μM) for
different times (0 h, 6 h and 12 h). And then the cells were incubated with the probe 3
(20 μM) for 20 min at 37 °C. After the cells were washed three times to remove the
remaining probe 3, the fluorescence images were acquired with inverted fluorescence
microscopy. In the mean time, the control group just incubated with the probe 3 (20
μM) for different times (0h, 6 h and 12 h) at 37 °C and then imaged by the
microscopy.
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To study the cytotoxicity, HEK293 cells were seeded at 1×10 cells per well in
96-well plates and incubated for 24 h before treatment, followed by exposure to
different concentrations (0, 1, 5, 10, 20, 50 μM) of probe 3, Sec, Na SeO and
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compound 4 for additional 24 h. To determine the cell viability, the colorimetric assay
with 3-(4, 5-dimethylthiazol-2-yl)-3, 5-diphenytetrazolium bromide (MTT) was
applied for evaluating the metabolic activity. Cell samples were incubated with MTT
solution (containing 5 mg/mL MTT reagent in PBS) for 4 h, then DMSO was added to
dissolve the formazan crystals after removal of the MTT solution. The absorbance
was measured at 540 nm with a microplate reader (SpectraMax M5, Molecular
Devices). Each experiment was performed at least three times. The cytotoxic effect
was evaluated accordingly.
Flow Cytometry Analysis
Flow cytometry (C6, BD Biosciences) was employed to determined endogenous
selenol with developed probe. Cancer and normal cell cultures were prepared for
sample analysis. After removing media and rinsing with PBS buffer, the cells were
treated with trypsin (0.25 %). The digested cells were centrifugated and further
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suspended at a density of 2×10 /mL with PBS in 1.5 mL eppendorf tubes. Following
by treat with a range of concentrations (0, 0.1 μM, 1 μM, 10 μM, 100 μM) of probe 3
at room temperature for 10 min. the fluorescence signal were determined in FL1-A
detector by flow cytometry. Analysis was conducted for a minimum of 10000
counting events and median values of histograms were used to quantify the Sec
recognition with probe 3.
In Vivo Imaging of Zebrafish
To explore the potential of probe 3 as an in vivo imaging tool, we applied it to
detection in living zebrafish. The 3-7 days zebrafishes post-fertilization were
purchased from Eze-Rinka Company (Nanjing, China). The zebrafishes were cultured
in 5 mL embryo medium supplemented with 1-phenyl-2-thiourea (PTU) in 6-well
plates for 24 h at 30 °C. Then the embryo zebrafishes were washed three times to
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remove the remaining (Sec)
2
and DTT, and further incubated with probe 3 (10 μM) for
24 h at 30 °C. After removing the embryo medium and washing the zebrafish with
PBS for three times, the fluorescence images were acquired with stereo microscopy
(Olympus SZX16, Japan). In order to detect endogenous selenol contents, the
zebrafish was directly treated with embryo medium containing probe 3 (10 μM) for
24 h. After washing the zebrafish for three times, the zebrafishes were imaged by
stereo microscopy. As the negative control, the zebrafishes without any treatment
were imaged directly.
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0
RESULTS AND DISCUSSION
Probe Synthesis
The new probe 3 was prepared in three steps and the detailed synthetic route is
shown in Scheme 2, and the structures of probe 3 and intermediate molecules were
fully characterized (see Figure S1-S6). The water solubility of probe 3 was
investigated prior to the fluorescence property characterization. It revealed that and
the maximum absorbance in 0.1 M phosphate buffer (pH 7.4) linearly increased with
increasing the concentration of probe 3 (0-200 μΜ, see Figure S7), indicating the
good water solubility of the newly synthesized probe. Thus probe 3 is readily soluble
in phosphate buffer system, and there is no need for co-solvent for biological
selenol-containing molecule detection.
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Scheme 2 The synthetic route of probe 3.
n-BuOH
acetic acid
CHO
OH
NO2
O
O
+
O N
2
piperidine
reflux
N
O
N
O
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M
SnCl2
HCl
r.t.
O N
2
O
H
pyridine
DCM
r.t.
NH2
N S
O
NO2
N
O
O
N
O
O
2
,4-dinitrobenzenesulfonyl
chloride,
4
3
Fluorescence, pH dependence and Sensitivity of Probe 3
Based on the primary characterization, probe 3 exhibits several very desirable
properties in pure aqueous solution. First, the new probe has near zero fluorescence
background (Φ = 0.0014). Second, molecule 4, the fluorophore of probe 3, has an
increased quantum yield (0.30; in comparison, Φ of 2 is 0.14) and a notable red shift
(
λ = 380 nm and λ = 535 nm; Figure S8). Third, probe 3 exhibits first-order rate
ex
em
-
1
constant (k) of 2.15 min when reacting with ten times higher concentration of Sec at
o
3
0 C (Figure 2a), the best for all reported Sec probes to date (for comparison, k is
-
1
o
-1
o
24, 26
~
1.34 min for Sel-green at 30 C and ~0.16 min for HD-Sec at 25 C).
To
2
2
compare with the reported BESTthio probe with second-order rate constant of 740
-1. -1
o
M s at 37 C, the second-order rate constant of probe 3 reacting with Sec was
o
measured at 30 C (Figure 3). The second-order rate constant was calculated to be
-1. -1
o
~
700 M s by equation 5, which should be even higher at 37 C. In addition, The
Sec-reactivity of probe 3 has a bell-shaped pH-dependence (Figure S9), with the
maximal value at pH 6.0. Based on this observation, pH 6.0 was chosen for the
remaining experiments in this study. From the concentration titration experiment
shown in Figure S10 and Figure 2b, Sec can produce up to 250-fold fluorescence
enhancement. On the basis of the calibration curve of response of probe 3 to Sec in
aqueous solution, its LOD for Sec is further determined to be about 18 nM (Figure
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3
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6
S11).
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9
0
Figure 1. (a) Time dependence for the fluorescence spectra (λex = 380 nm) of probe 3
10 μΜ) in the presence of Sec (50 μΜ) in 0.1 M phosphate buffer (pH 7.0) at 30 °C.
(
Inset: photo of samples illuminated with 365 nm UV light with and without the
addition of Sec (50 μΜ). (b) The change of the relative fluorescence intensity (F/F ) at
0
535 nm of the probe 3 (10 μΜ) with increasing concentrations of Sec.
a
400
320
b
0.5
0.4
0.3
0.2
0.1
2
1
40
60
8
0
0
0.0
0
200 400 600 800 1000
0
30 60 90 120 150
t
(s)
t
(s)
Figure 2. (a) Time dependence for the fluorescence spectra (λem = 515 nm) of probe 3
(10 μΜ) in the presence of Sec (10 μΜ) in 0.1 M phosphate buffer (pH 7.0) at 30 °C.
(b) The plot of the reaction progress ((F -F )/(F -F )) vs the reaction time. F is the
t
0
∞
t
0
initial fluorescence intensity of reaction system, F is the fluorescence intensity of
t
reaction system at the corresponding time, F is the total fluorescence intensity when
∞
100% substrate converted, and t is incubation time in second.
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It is noteworthy that the turn-on fluorescence sensing mechanism of probe 3 upon
the recognition of Sec was validated by HRMS and HLC analysis. As the reference,
+
Figure 3a shows the HRMS peak of the probe [3+H ] (calcd. for 487.0918) before the
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addition of Sec. When monitoring the reaction system of probe 3 and Sec that are
solved in dd-water containing 0.1% (V/V) DMSO (the final concentration are 20 μM),
the HRMS peak of compound 4 (calcd. for 256.1206) was also found in Figure 3b. To
further validate this reaction mechanism, the reaction of probe 3 in the absence and
presence of Sec was monitored by HPLC, with fluorophore (4) as reference (Fig. S12).
All of them were solved in CH CN, the mobile phase (ACN:H O) is 60%: 40%, and
3
2
the flow rate is 0.3 mL/min. It shows that the signal peak of reaction system between
probe 3 and Sec correlates well with the signal peaks of the fluorophore (4). These
results indicate that probe 3 actually reacted with Sec and gave out 4 as a product.
4
a
1.4x10
1.2x10
b
487.09122
256.12116
4
4
4
3
4
4
3
3
3
3
2.0x10
1.5x10
1.0x10
1.0x10
8.0x10
6.0x10
4.0x10
5.0x10
2.0x10
0.0
0
.0
5
0
5
0
5
0
5
0
7
8
8
9
9
0
4
4
4
4
4
5
5
m/z
m/z
Figure 3. HPLC-MS spectrum of pure probe 3 (a), and the reaction system between
+
probe 3 and Sec. Molecular ion mass peak of (a) [3+H ] (calcd. for 487.0918), (b)
+
[
4] (calcd. for 256.1206).
Probe Selectivity
We tested the selectivity profile of probe 3. The data presented in Figure 4a
shows that this molecular probe is highly selective for selenols (represented by Sec)
over competing amino acids (Phe, Arg, His, Tyr, Trp, Ala, Lys and Met), metal ions
2+
2+
+
+
(
Ca , Mg , Na and K ) and biothiols (Cys, Hcy and GSH ). It displayed 120-fold
fluorescence enhancement upon treatment with Sec, indicating that probe 3 is
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particularly selective toward Sec. We also tested the reactivity of probe 3 with SePs.
We chose human thioredoxin redutase (hTR), a well-characterized human SeP for this
examination. It is known that hTR contains a single Sec residue locating at the C
terminal. We prepared and tested the recombinant version of hTR (1 µg/mL), as well
as two hTR mutants, U498C and U498S, in which Sec was substituted with cysteine
and serine, respectively. In addition, we also tested five control proteins (10 µg/mL) –
bovine serum albumin (BSA), human elastase, acetylcholinesterase (AChE),
butyrylcholinesterase (BChE) and trypsin –that do not contain Sec but commonly
exist in serum. As shown in Figure 4b, only wild type hTR (wt hTR) was able to
produce significant fluorescence enhancement, indicating our molecular probe is able
to selectively detect SePs. In addition, the second-order reaction rate constant for the
0
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3
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7
8
9
0
-1. -1
reaction between probe 3 and hTR was measured to be about 1200 M s . Thus the
reaction between probe 3 and hTR was only slightly faster than that of reaction
-1. -1
between probe 3 and Sec (700 M s ) at 30 °C, which indicates that the probe 3 could
not discriminate the hTR and Sec.
a
140
b
100
80
60
40
20
0
1
20
00
80
1
60
40
20
0
Figure 4. (a) The selectivity profile of probe 3 against metal ions, amino acids,
biological thiols and Sec. F/F represents the relative fluorescence response. The
0
probe concentration was 10 µM, the Sec concentration was 100 µM and other
analytes were added at 1000 µM. (b) The selectivity profile of probe 3 against hTR (1
µg/mL) and proteins (10 µg/mL) that do not have Sec residue. F/F represents the
0
relative fluorescence response. The probe concentration was 10 µM.
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Cytotoxity of Probe 3 and Its Application in Cell Imaging
We next carried out an imaging study using probe 3 against human lung cancer
cells (A549). We first studied the response of probe 3 to the exogenous Sec in cells at
different time points. After treated with Sec newly prepared by equimolar complex of
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(Sec) and DTT to the cultured cells, a bright and strong green fluorescence could be
2
observed after 6 h and the green fluorescence became stronger after 12 h, while no
significant fluorescence signal in control cells all the time (Figure 5a and Figure 5c).
We also examined the cells treated with sodium selenium (Na SeO ), a classical
2
3
precursor of Sec biosynthesis. It was expected that the cells treated with Na SeO3
2
would produce increased level of reactive selenol species. This was confirmed by the
data presented in Figure 5b, and the green fluorescence also increased with the
extension of the incubation time. These results show that probe 3 is capable of
detecting purposely elevated Sec in mammalian cells. In addition, the results obtained
from MTT assay indicated that, probe 3 and its fluorophore did not cause significant
cytotoxicity against HEK293 cells, whereas the representative selenocompounds, Sec
and Na SeO , caused severe cytotoxicity (see Figure 6).
2
3
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Figure 5. Fluorescence imaging in A549 cells at different incubation times (0 h, 6 h
and 12 h). The cells were untreated (a), with 20 μΜ Na SeO (b), and treated with 20
2
3
μΜ (Sec) and 20 μΜ DTT(c), and the bright-field images (top panel of each time
2
point), fluorescence images (bottom panel of each time point) were acquired.
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b 120
a
120
00
3
4
(
Sec)
2
100
1
Na SeO
2 3
80
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40
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20
0
20
0
0
10
20
30
40
50
0
10
20
30
40
)
50
Concentraton (μΜ
)
Concentration (μΜ
Figure 6. Cytotoxicity of representative selenocompounds (a) and new synthesized
compounds (b) against HEK293 cells. The cell viability was determined by the MTT
assay. Data represent average values of three independent experiments.
Endogenous Cellular Selenol/Sec Determination
We then examined whether probe 3 was useful for detecting endogenous selenols
in single cells using flow cytometry. Probe 3 was tested at four different
concentrations (0.1, 1, 10 and 100 μM) with A549, a lung cancer cell. The results
depicted in Figure 7a (as well as Figure S13) and the resultant plot of the fluorescence
intensity versus the concentration of probe 3 was shown in Figure 7b. Moreover, the
time dependent profile of the fluorescence for A549 cells was investigated and the
corresponding results were showed in Figure 7c and 7d. Notably, those data
demonstrate that probe 3 can indeed detect intracellular selenols in individual cells,
reaching the highest signal at 10 μM of probe and a cell-probe incubation time of 10
minutes.
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Figure 7. (a) The flow cytometric analysis of A549 cells exposed to different
concentrations of probe 3 (I, 0 μΜ; II, 0.1 μΜ; III, 1 μΜ; IV, 10 μΜ; V, 100 μΜ) after
1
0 min incubation. (b) Plot of the fluorescence signal versus the probe concentration
in flow cytometry. (c) The flow cytometric analysis of A549 cells exposed to probe 3
10 μΜ) after different time of incubation. (d)Plot of the fluorescence signal versus
(
the incubation time in flow cytometry.
The above flow cytometry-based method was further used to measure the relative
selenol concentrations by probe 3 in both normal cells (HEK293 and COS) and other
three cancer cells (A549, H460, and HeLa). Interestingly, the data presented in Figure
8
revealed that selenol levels in the chosen cancer cells are noticeably higher than that
of normal cells, a novel observation that has never been described before.
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200
0
HEK293 COS HeLa H460 A549
Figure 8. The flow cytometric analysis of normal cells (HEK293 and COS) and
cancer cells with probe 3 (10 μΜ).
In Vivo Selenol/Sec Imaging of Zebrafish with Probe 3
We also investigated the suitability of probe 3 as in vivo imaging agent. Zebrafish
were chosen for this investigation mainly for experimental simplicity (it can be
3
6, 37
conveniently imaged under fluorescence microscopy).
Zebrafish were divided
into three groups: the control group (group a in Figure 9; no probe 3 added), intrinsic
selenol test group (group b in Figure 9, treated with probe 3 alone), and chemically
enhanced selenol test group (group c in Figure 9, treated with probe 3 as well as
equimolar complex of (Sec) and DTT to produce enhanced level of selenols). No
2
significant fluorescence was observed in the control group. However, probe 3 was
able to detect both intracellular selenol-containing molecules in the intrinsic selenol
group b (strong fluorescence was observed in the head) and chemically enhanced
selenol group c (strong fluorescence was seen in both the head and the venter). These
results demonstrate that probe 3 can perform sensitive Sec imaging in the living
organisms.
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Figure 9. Confocal imaging of selenols in zebrafish embryos, 30 min after incubation
of probe 3: (a) Control group (no probe), (b) Probe group (only 10 μΜ probe 3 was
added), (c) Sample group (10 μΜ probe 3, 20 μΜ (Sec) and 20 μΜ DTT were
2
added).
CONCLUSION
In conclusion, we have successfully identified a new molecular probe for
selenol-containing molecule recognition. This probe exhibits several very desirable
properties, including near-zero background fluorescence, large fluorescence
enhancement upon reacting with Sec (~250-fold), a low LOD (18 nM), and a large
-1
Sec-reacting rate constant (~2.15 min ). Furthermore, probe 3 can be used to carry
out sensitive intracellular selenol-containing molecule detection using live cells and
living organisms. We expect this molecular probe can find useful applications both in
the study of biological function related to SePs and in the clinical diagnosis for human
diseases associated with Se-deficiency or overdose.
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AUTHOR INFORMATION
†
These authors contributed equally.
Corresponding authors
*
8
*
8
Wen-Chao Yang, e-mail: tomyang@mail.ccnu.edu.cn; Tel: 86-27-67867706; Fax:
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6-27-67867141;
Guang-Fu Yang, e-mail: gfyang@mail.ccnu.edu.cn; Tel: 86-27-67867800; Fax:
6-27-67867141.
ACKNOWLEDEMENTS
We are grateful for the financial support from the National Natural Science
Foundation of China (Grant No. 21332004 and 21372094).
SUPPORTING INFORMATION
The NMR, LC-MS and HPLC spectra for the newly synthesized compounds, and
some figures for probe 3 characterization. This material is available free of charge via
the Internet at http://pubs.acs.org.
REFERENCES
(1)
(2)
(3)
(4)
(5)
(6)
(7)
(8)
(9)
Foster, L. H.; Sumar, S. Crit. Rev. Food Sci. Nutr. 1997, 37, 211-228.
Rayman, M. P. Lancet 2000, 356, 233-241.
Chen, J.; Berry, M. J. J. Neurochem. 2003, 86, 1-12.
Thomson, C. D. Eur. J. Clin. Nutr. 2004, 58, 391-402.
Duntas, L. H.; Benvenga, S. Endocrine 2015, 48, 756-775.
Du, X.; Wang, C.; Liu, Q. Curr. Top. Med. Chem. 2016, 16, 835-848.
Ogawa-Wong, A. N.; Berry, M. J.; Seale, L. A. Nutrients 2016, 8, 80-99.
Weekley, C. M.; Harris, H. H. Chem. Soc. Rev. 2013, 42, 8870-8894.
Hatfield, D. L.; Tsuji, P. A.; Carlson, B. A.; Gladyshev, V. N. Trends Biochem.
Sci. 2014, 39, 112-120.
(10) Arthur, J. R. Cell Mol. Life Sci. 2000, 57, 1825-1835.
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