A "turn-on" fluorescent probe for the detection of Cu2+ in living cells based on a signaling mechanism of NN isomerization

Article abstract of DOI:10.1039/c5nj03649f

A novel fluorescent probe A based on coumarin associated with a NN double bond was conveniently designed and synthesized for Cu²⁺, and both rapid colorimetric and fluorescence turn-on detection can be achieved, showing a visible color change from pink to yellow and a significant fluorescence enhancement, respectively. The probe exhibits high selectivity toward Cu²⁺ over other metal ions and excellent sensitivity with a detection limit of 20 nM, and it can be monitored in a wide range of pH (3-10) in aqueous systems. Besides, the probe has been applied in living cells.

Full text of DOI:10.1039/c5nj03649f

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DOI: 10.1039/C5NJ03649F
Journal Name
ARTICLE
2+
A “turn-on” fluorescent probe for detection of Cu in living cells
based on signaling mechanism of N=N isomerization
a
abc
a
a
*ad
Shuo Cao, Qiaoying Jin, Lin Geng, Lingyun Mu and Shouliang Dong
Received 00th January 20xx,
Accepted 00th January 20xx
A novel fluorescent probe A based on coumarin associated with a N=N double bond was conveniently designed and
2+
DOI: 10.1039/x0xx00000x
synthesized for Cu , and both rapid colorimetric and fluorescence turn-on detection can be achieved, showing a visible color
change from pink to yellow and a significant fluorescence enhancement, respectively. The probe exhibits high selectivity
toward Cu2+ over other mental ions and excellent sensitivity with a detection limit of 20 nM, and it can be monitored at a
wide range of pH (3 - 10) in aqueous system. Besides, the probe has been applied in living cells.
www.rsc.org/
sensitivity and high interference by other closely related metal
ions still remain in those reported strategies. Therefore,
development of fluorescent probes specifically toward
detecting Cu in aqueous solution and under physiological
relevant conditions is still of great necessity.
Introduction
Selective detection of metal ions has attracted considerable
attention in recent years. Among the various approaches to
detecting ions, fluorescent probes are regarded as the most
powerful tools due to their selective, sensitive, non-destructive,
2+
1
Nowadays, several signaling mechanisms have been widely
used in chemosensors, such as photo-induced electron/ energy
2
-4
and real-time monitoring qualities. Actually, in the past few
years, great efforts have been devoted to the development of
2
5-27
28-30
transfer,
fluorescence resonance energy transfer.
fluorescence signaling mechanisms, isomerization
the fact that C=N isomerization signaling mechanism has
intramolecular charge transfer
and
31-33
Among the various
2
+
fluorescent probes for Cu due to its indispensable roles in
environmental and biological processes ranging from bacteria
3
4-36
based on
5-7
to mammals. As a transition metal, copper is the third most
abundant essential trace element in the body, and usually
37-42
43-
already been utilized in sensors,
while N=N isomerization
45
with better quenching effect is still unexplored precisely
8
presents at a low level in cells and tissues. If excessively
draws our special attention. Meanwhile, coumarin is a popular
fluorescent dye with widespread applications particularly as an
ideal fluorophore in the designing of chemosensors due to its
superior photophysical and spectroscopic properties, such as
long absorption and emission wavelengths, high fluorescence
accumulated to the extent that the cellular homeostasis is
broken, copper can cause many seriously adverse
neurodegenerative damages, such as Alzheimer’s disease and
9-11
2+
Wilson’s disease.
Owing to its paramagnetic nature, Cu is
1
2-14
known as an efficient fluorescence quencher,
and early-
reported Cu probes generally underwent fluorescence
46, 47
quantum yields and good stability.
2+
Actually, a number of fluorescent probes working on
coumarin for detection of metal ions have been reported based
2
+ 15-19
quenching upon binding of Cu .
Fluorescence turn-off
probes may exhibit false positive results, while fluorescence
turn-on probes possess more excellent advantages.20 In fact,
recent years have witnessed a growing number of fluorescence
48-51
on isomerization,
while most of them exploited the
inhibition of C=N isomerization by metal ions’ binding-induced
conformational restriction rather than formation of covalent
bridging of C=N bond, which is more stable. Meanwhile, rare
2
+ 21-24
enhancement probes for Cu .
Unfortunately, in most cases,
the fluorescence increase is fairly weak while the background
signal is strong. Besides, the poor water solubility often limits
the application of the probes in living tissues. Moreover, the low
2+
fluorescent probes for Cu based on N=N isomerization have
ever emerged. Therefore, in this paper, we put forward a novel
2+
strategy of suppression of N=N isomerization by Cu -catalyzed
cyclization from 2-aminoazobenzene to triazole to devise a
2+
turn-on fluorescent probe (A) for Cu (Scheme 1). The new
a.Institute of Biochemistry and Molecular Biology, School of Life Sciences, Lanzhou
University, 222 Tianshui South Road, Lanzhou 730000 China.
E-mail: dongsl@lzu.edu.cn; Fax: (+) 86 931 8912428
b.
c.
d.
nd
Laboratory of Medical Genetics, The 2 Hospital of Lanzhou University, Lanzhou,
30030 China
Key Laboratory of Digestive System Tumors of Gansu Province, Lanzhou 730030
China
7
Key Laboratory of Preclinical Study for New Drugs of Gansu Province, 222
Tianshui South Road, Lanzhou 730000, China
†
Electronic Supplementary Information (ESI) available: Detailed synthetic
1
13
procedures, characterization and
compounds, and additional cell images. See DOI: 10.1039/x0xx00000x
H
NMR, C NMR and HRMS spectra of
Scheme 1 The structure of probe
mechanism of Cu detection
A
and proposed sensing
2+
This journal is © The Royal Society of Chemistry 20xx
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ARTICLE
Journal Name
A was synthesized on the basis of the route spectrophotometer. Fluorescence spectra wereViteawkAertnicleoOnnlinae
coumarin derivative
shown in Scheme 2, containing an unbridged N=N bond which PerkinElmer LS-55 spectrofluorometer. DFlOuIo: 1r0e.s1c0e39n/cCe5NimJ0a36g4in9gF
can reversibly isomerize between trans and cis configurations at was performed on a Nikon ECLIPSE TE2000-S inverted
the cost of consuming energy of the molecule, resulting in no fluorescence microscope.
2
+
fluorescent emission. Whereas upon being exposed to Cu ,
N=N isomerization was suppressed by selective catalytic Synthesis of probe A
5
2, 53
cyclization to triazole,
therefore probe A displayed a
In refluxing n-butanol, 4-diethylamino salicyldehyde was
coupled with 1.2 equiv. of ethyl nitroacetate via a one-pot
remarkable green florescence enhancement along with a visible
color change from pink to yellow in an aqueous acetonitrile
solvent. And it was proved to be an efficient probe selectively
for Cu over other metal ions at nanomolar level. As a key facet,
it can be successfully utilized in detecting Cu in living cells.
reaction for 24 h to afford the product
Subsequently, was reduced to amino product
presence of stannous chloride at 85 C for 4 h. And the final
compound (63% yield) was obtained by a one-pot reaction
which involved two continuous steps, diazotization and
3
in 73% yield.
3
4
in the
2+

2+
A
coupling, and be sure that the reaction proceeded under 0

C all
1
along. The structure of
C NMR. HRMS: Mr calculated for C19
A
was confirmed by HRMS, H NMR, and
13
H
21
N
5
O
2
, 351.17, found
1.23 (t, J = 6.9
+
m/z 352.17 [M + H] . 1H NMR (CDCl
3
, 300 MHz):

Hz, 6 H), 3.43 (q, J = 7.2 Hz, 4 H), 5.89 (d, J = 2.4 Hz, 1 H), 6.16
dd, J = 6.3 Hz, 1 H), 6.55 (d, J = 2.4 Hz, 1 H), 6.61 (dd, J = 6.3 Hz,
H), 7.38 (d, J = 8.7 Hz, 1 H), 7.60 (d, J = 8.7Hz, 1 H), 7.92 (s, 1
(
1
1
3
3
H), without four active hydrogens showed on it. C NMR (CDCl ,
7
1
4
5 MHz):  12.27, 12.36, 14.03, 54.86, 59.71, 96.21, 96.50,
06.11, 108.19, 109.57, 122.62, 130.45, 130.57, 132.97, 146.37,
6.56, 149.87, 153.03, 155.11, 159.87.
Scheme 2 Synthetic route to probe
A
Solution preparation
Probe was dissolved in acetonitrile to make a 100 μM stock
A
solution, which was further diluted to 10 μM (in spectroscopic
measurements) and 5 μM (in cellular imaging) in a mixture of
phosphate buffer saline (PBS, 0.01 M, pH 7.4) and acetonitrile
(9: 1, v/ v) for subsequent measurements. Stock solutions of
Cu and other ions were prepared in PBS (0.01 M, pH 7.4) and
diluted to the required concentrations for following
To the best of our knowledge, this is the first fluorescence
2+
turn-on probe based on coumarin for Cu detection by bridging
N=N bond covalently to block its isomerization, which is
2+
triggered by Cu -catalyzed cyclization to trizole. The
for Cu2+ sensing was compared with
performance of probe
other representative turn-on probes for Cu reported recently
2+
A
2+
2
4, 54-59
experiments.
UV-Vis
absorption
and
fluorescence
based on various mechanisms
in Table S1. As shown in it,
measurements were carried out at room temperature.
the proposed probe exhibits excellent analytical performance
and therefore is a potent turn-on fluorescent probe for Cu2+
monitoring. And it also opens up a new path to applying N=N
isomerization to designing fluorescence turn-on probes.
Cell culture and imaging
HepG2 cells were cultured in Dulbecco’s Modified Eagle’s
Medium (DMEM) supplemented with 10% fetal bovine serum
(
FBS) and antibiotics (100 units/ mL penicillin, 100 μg/ mL
streptomycin) in a humidified atmosphere of CO / air (5: 95%)
at 37 C. Prior to imaging experiments, HepG2 cells were seeded
in 12-well plates and cultured for 24 h.
Experimental
Materials
2

All reagents in the syntheses were purchased from J & K (Beijing,
China) and used without purification unless otherwise. Solvents
were dried by standard methods prior to use. Human liver
carcinoma cell line HepG2 was purchased from Type Culture
Collection of the Chinese Academy of Sciences (Shanghai,
China).
For labeling, the medium was removed and cells were rinsed
three times with PBS. Then HepG2 cells were incubated with
probe
at 37
HepG2 cells were preloaded with probe
of PBS and acetonitrile (9: 1, v/ v) at 37
A (5 μM) in a mixture of PBS and acetonitrile (9: 1, v/ v)
2+

C for 30 min as control. For Cu imaging, another set of
A
(5 μM) in a mixture

C for 30 min, rinsed
three times with PBS and further treated with different
concentrations of Cu (5 μM, 20 μM and 50 μM) in PBS at 37 C
for additional 30 min. Cells rinsed three times with PBS and
bathed in it, imaging was carried out. Images were acquired
using an inverted fluorescence microscope and fluorescence
imaging was conducted in green channel.
Instruments
2+
1H NMR and 13C NMR spectra were recorded on a Varian
spectrometer (300 MHz and 75 MHz, respectively). HRMS were
recorded on a Bruker maXis 4G mass spectrometer. All pH
measurements were made on a Model PHS-3C pH meter. UV-
Vis absorption spectra were measured on a TU-1901
2
| J. Name., 2012, 00, 1-3
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Fig. 1 UV-Vis absorption spectra (a) and fluoresceVniecweAertmicleisOsniolinne
0. 9/C 36 9F
Results and discussion
spectra (b) of probe
A
(10 μM) and
B
obt
D
a
O
inI:
e
1
d
f
1
r
0
o
3
m
A
5N(1J0
0 μM)
4
Probe
in a yield of 63% (Scheme 1). Compound
the reduction of compound
A
was achieved by diazo-couple reaction from compound
2+
reacting with Cu (100 μM) in a mixture of PBS (0.01 M, pH 7.4)
4
4
was obtained via and CH CN (9: 1, v/ v). Excitation wavelength was set at 420 nm
3
3
, which was prepared from for (b). Insets: images of
A and B prepared using the same
coupling 4-diethylamino salicyldehyde with ethyl nitroacetate. experimental conditions under room light (a) and under a UV
1
13
lamp at 365 nm (b).
All compounds were characterized by HRMS, H NMR, and
C
NMR.
Spectroscopic properties of probe A and its response to Cu2+
Fluorescence titration and sensitivity
and its response to Cu2+
were investigated in a mixture of phosphate buffer saline (PBS,
.01 M, pH 7.4) and acetonitrile (9: 1, v/ v) at room temperature.
Both the UV-Vis absorption and fluorescence spectra are
depicted in Fig. 1. It was found that the maximum absorption
(10μM) shifted from 421 nm to 538 nm
COO) (100μM) (Fig. 1a), and the
fluorescence of the solution increased dramatically with the was estimated to be 0.02 μM, which is satisfied for Cu2+
From the fluorescence titration experiments (Fig. 2a), we can
Spectroscopic properties of probe
A
2+
see on addition of increasing concentrations of Cu (0 – 80 μM)
to the solution of probe (10 μM), fluorescence emission at λ =
10 nm gradually arose, and fluorescence intensity ratio (I/ I
at λ = 510 nm also presented a gradual increase as can be found
A
0
5
0
)
2+
from the inset. The equivalent of Cu (10 μM) was sufficient to
wavelength of probe
A
complete the reaction, displaying the splendid sensitivity of
with the addition of Cu(CH
3
2
2+
probe
A for Cu . Furthermore, the detection limit of probe A
maximum emission wavelength exhibiting a red-shift from 497 detection in drinking water within the US EPA limit (~ 20 μM).
nm to 510 nm due to the formation of the new compound As can be seen in Fig. 2b, there was a linear correlation between
fluorescence intensity ratio (I/ I
concentration ranging from 0.02 μM to 5 μM, which made the
quantitative determination of Cu at a low level in aqueous
solution feasible.
) at λ = 510 nm and Cu2+
0
trizole
B (confirmed by HRMS in Fig. S9, Fig. 1b). Actually, an
obvious color change from pink to yellow (Fig. 1a, inset) and
fluorescence emission from none to green (Fig. 1b, inset) of the
solution can be easily observed by the naked eye.
2+
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Fig. 2 (a) Fluorescence emission spectra of probe
Journal Name
(10 μM) presence of Cu (50 μM) in a mixture of PBS (0.01 M, pH 7.4)
2+
A
View Article Online
2+
upon addition of increasing concentrations of Cu (0 – 80 μM) and CH
in a mixture of PBS (0.01 M, pH 7.4) and CH CN (9: 1, v/ v). Inset: NaOH and HCl aqueous solution. λex = 420 nm.
the plot of fluorescence intensity ratio (I/ I ) at λ = 510 nm vs.
Cu concentration. (b) The linear correlation between
3
CN (9: 1, v/ v). Different pH valu
D
e
O
s
I: 1
w
0
e
.1
r
0
e
39
a
/C
d
5
ju
N
s
J
t
0
e
3
d
649F
b
y
3
0
2+
2+
fluorescence intensity ratio (I/ I
0
) at λ = 510 nm and Cu
Selectivity of probe A toward Cu2+
concentration (0.02 – 5 μM). λex = 420 nm.
One of the most indispensable qualities of a fluorescent probe
is the high selectivity toward the specific analyte over other
relevant species. To evaluate the selectivity of probe
for Cu (30 μM), a variety of metal ions (30 μM) including Ag ,
A
(10 μM)
Kinetics and pH effects
2+
+
In addition, we evaluated the time course of fluorescent
3
+
2+
2+
2+
2+
3+
+
+
2+
2+
+
+
2+
Al , Ca , Cd , Co , Fe , Fe , K , Li , Mg , Mn , Na , Ni , Pb
2
+
response of probe
a, the reaction of
completed within 5 min. Therefore, probe
A (10 μM) to Cu (50 μM). As shown in Fig.
A
2+
2+
and Zn were examined under the same condition with Cu . As
2+
3
and Cu went rapidly, which can be
absolutely could be
2+
shown in Fig. 4, only addition of Cu resulted in a dramatic
fluorescence enhancement of the solution of probe , while
other metal ions did not lead to obvious fluorescence. After all,
A
2+
A
applied in real-time determination of Cu in environmental and
biological conditions. For practical application, we also
2+
evaluated the appropriate pH condition for probe
base titration experiments, probe (10 μM) reacting with Cu
50 μM) to produce
presenting almost equivalent fluorescence emission (Fig. 3b),
suggesting that probe could operate at a wide pH range. In
other words, we successfully provided an ideal florescent probe
A
. In acid- we induced Na
2
S equivalent to Cu to the above fluorescent
2+
2+
A
solution to remove Cu by precipitating Cu
2
S, and fluorescence
(
B remained unaffected at pH 3 - 10 with intensity was still unchanged, reflecting that the reaction
preferred catalysis rather than chelation. Furthermore, the
A
2+
performance of probe
A
in Cu detection in the presence of
other competitive species was also tested. The competition
experiments (Fig. 4) showed that these metal ions displayed no
evident interference in the fluorescence increase caused by
2+
for Cu detection under approximate physiological conditions.
2+
Cu . All the above results indicate that the selectivity of probe
2+
A
for Cu in aqueous system is remarkably high.
Fig. 4 Fluorescence responses of probe
of metal ions (30 μM) respectively in a mixture of PBS (0.01 M,
pH 7.4) and CH CN (9: 1, v/ v) at λem = 510 nm in the absence
grey bars) and presence (black bars) of Cu (30 μM). The
second bar represents fluorescence intensity of the reaction
A (10 μM) toward kinds
3
2+
(
2+
system of probe
2
A and Cu on subsequent addition of Na S (30
μM). λex = 420 nm.
Cell imaging
Due to the outstanding spectroscopic and chemical properties
of probe , we further investigated its biological application in
living cells. As is known to all, Cu accumulates in liver,
A
2+
Fig. 3 Time-dependent (a) and pH-dependent (b) changes of
fluorescence intensity at λ = 510 nm of probe
A
(10 μM) in the therefore, we chose human liver carcinoma cell line HepG2 as
the target. HepG2 cells were first incubated with only probe
A
4
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Journal Name ARTICLE
9
1
1
1
1
1
1
1
1
1
1
2
2
2
E. Madsen and J. D. Gitlin, Annu. Rev. Neurosci., 2V0ie0w7A,rt3ic0le,O3n1li7ne-
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0 S. S. Percival, G. P. Kauwell, E. Bowser and M. Wagner, J. Am.
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(
1
5 μM) in a mixture of PBS (0.01 M, pH 7.4) and acetonitrile (9:
, v/ v) for 30 min, which led to barely intracellular florescence
as determined by inverted fluorescent microscope (Fig. 5b).
Then the cells preloaded with probe (5 μM) were further
3
DOI: 10.1039/C5NJ03649F
A
1 E. Gaggelli, H. Kozlowski, D. Valensin and G. Valensin, Chem.
Rev., 2006, 106, 1995-2044.
2+
incubated with Cu (50 μM) in PBS for 30 min, whereupon a
significant increase of fluorescence inside the cells was
observed (Fig. 5c). Bright-field measurements confirmed that
the cells were viable throughout the experiments (Fig. 5a).
Bioimaging of lower concentrations of Cu (5 μM and 20 μM)
was also realized (Fig. S11). These results indicate that probe
is low cytotoxic, cell membrane permeable and capable of
2 H. Mu, R. Gong, Q. Ma, Y. Sun and E. Fu, Tetrahedron Lett.,
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007, 48, 5525-5529.
3 A. W. Varnes, R. B. Dodson and E. Wehry, J. Am. Chem. Soc.,
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2+
A
3
5 Y. Zheng, K. M. Gattás-Asfura, V. Konka and R. M. Leblanc,
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2+
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Lett., 2008, 10, 5115-5118.
2+
Fig. 5 Fluorescence imaging of probe
A
with Cu in HepG2 cells.
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543-2555.
1 G.-K. Li, Z.-X. Xu, C.-F. Chen and Z.-T. Huang, Chem. Commun.,
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(
a) Bright-field image of HepG2 cells preloaded with probe A (5
2
μM) in a mixture of PBS (0.01 M, pH 7.4) and acetonitrile (9: 1,
v/ v) for 30 min and then incubated with Cu2+ (50 μM) in PBS for
2
008, 1774-1776.
additional 30 min at 37
channel of HepG2 cells treated with

C. (b) Fluorescence image in green
(5 μM) for 30 min at 37
2 X. Qi, E. J. Jun, L. Xu, S.-J. Kim, J. S. Joong Hong, Y. J. Yoon and
A
J. Yoon, J. Org. Chem., 2006, 71, 2881-2884.

C. (c) Fluorescence image in green channel of HepG2 cells 23 Y. Zhou, F. Wang, Y. Kim, S.-J. Kim and J. Yoon, Org. Lett., 2009,
preloaded with probe
A
(5 μM) for 30 min and then incubated
with Cu (50 μM) for additional 30 min. Scale bar represents
00 μm.
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2+
24 G. He, X. Zhao, X. Zhang, H. Fan, S. Wu, H. Li, C. He and C. Duan,
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In summary, we have designed and synthesized a novel
coumarin-based fluorescence turn-on probe
signaling mechanism of N=N isomerization, which can be
utilized in aqueous solution at a wide pH range. The probe
features a rapid response to Cu2+ with an evident fluorescence
enhancement owing to the formation of the new compound
triazole. Remarkably, probe
selectivity for Cu over other metal ions. Significantly, the
intracellular Cu imaging capacity in HepG2 cells further
demonstrates that probe is a valuable tool in living systems.
2
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2
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Page 7 of 7
New Journal of Chemistry
View Article Online
DOI: 10.1039/C5NJ03649F
Graphical abstract
A “turn-on” probe A based on coumarin with mechanism of N=N isomerization was
2
+
developed for detecting Cu in living cells.