A fast, highly selective and sensitive dansyl-based fluorescent sensor for copper (II) ions and its imaging application in living cells

Article abstract of DOI:10.1016/j.tetlet.2017.01.090

Since the copper ions (Cu²⁺) play a fatal role in many foundational physiological processes, it is important to develop a simple, highly sensitive and selective sensor for Cu²⁺detection in living systems. Herein, an intramolecular charge transfer (ICT) and dansyl-based fluorescent chemosensor 1 was designed, synthesized and characterized for the sensitive and selective quantification of Cu²⁺. It exhibited remarkable fluorescence quenching upon addition of Cu²⁺over other selected metal ions, attributed to the complex formation between 1 and Cu²⁺with the association constant 6.7?×?10⁵?M^?1. The sensor 1 showed a fast and linear response towards Cu²⁺in the concentration range from 0 to 12.5?×?10^?6?mol?L^?1with the detection limit of 2.5?×?10^?7?mol?L^?1. This detection could be carried out in a wide pH range of 5.0–14. Furthermore, sensor 1 can be used for detecting Cu²⁺in living cells.

Full text of DOI:10.1016/j.tetlet.2017.01.090

Accepted Manuscript
A fast, highly selective and sensitive dansyl-based fluorescent sensor for copper
(II) ions and its imaging application in living cells
Ming Zhou, Xiaobo Wang, Kunzhu Huang, Yuzhao Huang, Shou Hu, Wenbin
Zeng
PII:
S0040-4039(17)30134-X
DOI:
http://dx.doi.org/10.1016/j.tetlet.2017.01.090
TETL 48593
Reference:
Tetrahedron Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
22 November 2016
20 January 2017
25 January 2017
Please cite this article as: Zhou, M., Wang, X., Huang, K., Huang, Y., Hu, S., Zeng, W., A fast, highly selective and
sensitive dansyl-based fluorescent sensor for copper (II) ions and its imaging application in living cells, Tetrahedron
Letters (2017), doi: http://dx.doi.org/10.1016/j.tetlet.2017.01.090
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An fast, highly selective and sensitive dansyl-based fluorescent s nsor for copper (II) ions and its
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imaging application in living cells
Ming Zhou,a,b Xiaobo Wang,a,c Kunzhu Huang,a,d, Yuzhao Huangb, Shou Hub, Wenbin Zenga,*

1
Tetrahedron Letters
A fast, highly selective and sensitive dansyl-based fluorescent sensor for copper (II) ions and its
imaging application in living cells
Ming Zhou,a,b Xiaobo Wang,a,c Kunzhu Huang,a,d, Yuzhao Huangb, Shou Hub, Wenbin Zenga,*
a Xiangya School of Pharmaceutical Sciences, Central South University, 172 Tongzipo Road, Changsha, 410013, P. R. China.
b Xiangya Hospital, Central South University, Changsha 410078, P. R. China.
c Department of Nuclear Medicine, Henan Provincial People's Hospital and People's Hospital of Zhengzhou University, Henan, 450003, P. R. China.
d School of Pharmaceutical Sciences, Changsha Medical University, Changsha, 410219, P.R.China,
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
Since the copper ions (Cu2+) play a fatal role in many foundational physiological processes, it is
important to develop a simple, highly sensitive and selective sensor for Cu2+ detection in living
systems. Herein, an intramolecular charge transfer (ICT) and dansyl-based fluorescent
chemosensor 1 was designed, synthesized and characterized for the sensitive and selective
quantification of Cu2+. It exhibited remarkable fluorescence quenching upon addition of Cu2+
over other selected metal ions, attributed to the complex formation between 1 and Cu2+ with the
association constant 6.7×105 M-1. The sensor 1 showed a fast and linear response towards Cu2+
in the concentration range from 0 to 12.5 × 10-6 molꢀL−1 with the detection limit of 2.5×
10-7ꢀmolꢀL−1. This detection could be carried out in a wide pH range of 5.0–14. Furthermore,
sensor 1 can be used for detecting Cu2+ in living cells.
Available online
Keywords:
Fluorescent sensor
Copper (II) ions
Dansyl
2009 Elsevier Ltd. All rights reserved.
to the highly sensitive, rapid and non-destructive in vivo
characteristics.16-17 Unfortunately, they still show deficiency in
practical application, such as cross-sensitivity toward other metal
ions, a narrow pH range, a low and slow fluorescence response in
aqueous media, cytotoxicity.18-19 In this regard, there still is a great
need for the design and synthesis of new fluorescent chemosensors
with improved properties.
Introduction
Abundant attention recently has been attracted by the development
of fluorescent chemo-sensors for the detection of metal ions such as
Cu2+, Fe3+, Hg2+, Pb2+, and Zn2+ in the environment or biological
system.1-3 Copper, as the third most abundant essential trace element
in human body, plays a fatal role in many foundational physiological
processes.4 For example, copper ions act as a cofactor for electron
transport, or as a catalyst in redox reactions in many proteins.5 On
the other hand, an excess of copper ions in living systems can lead to
the production of reactive oxygen species (ROS), which can degrade
biomacromolecules such as lipids, nucleic acids, and proteins. This
process is the common pathological basis of many serious diseases,
such as Alzheimer’s disease,6-7 Indian childhood cirrhosis (ICC),8
Parkinson’s disease,9-10 Menkes11 and Wilson diseases.12 In view of
the critical role of copper ion in the biological systems, it is in
demand and of considerable significance to exploit a rapid, efficient
and convenient strategy for Cu2+ detection in living systems.
Herein, an intramolecular charge transfer (ICT) and dansyl-based
fluorescent chemosensor
1 for the sensitive and selective
quantification of Cu2+ was designed, synthesized and characterized.
It exhibited remarkable fluorescence quenching upon addition of
Cu2+ over other selected metal ions, which was attributed to the
complex formation between 1 and Cu2+ with the association constant
6.7×105 M-1. Sensor 1 showed a fast and linear response towards
Cu2+ in the concentration range from 0 to 12.5 × 10-6 molꢀL−1 with
the detection limit of 2.5× 10-7 molꢀL−1. The whole process could be
carried out in a wide pH range of 5.0-14.0 and was not disturbed by
other metal ions. Furthermore, the capability of sensor 1 for
detecting Cu2+ in living cells was demonstrated. This suggests that 1
could be used for practical detection of Cu2+ in living systems.
Currently, several methods such as liquid-liquid extraction
(LLE)-flame atomic absorption spectrometry (FAAS),13 inductively
coupled plasma mass spectroscopy (ICP-MS),14 and inductively
coupled plasma atomic emission spectrometry (ICP-AES)15, have
been proposed for detection of copper ions at trace quantity levels in
Result and Discussion
As shown in Scheme 1, the synthesis of sensor 1 started with
readily available starting materials pyridine-2,6-dicarboxylic acid (2)
and 5-(dimethylamino)naphthalene-1-sulfonyl chloride (3), and
ended up with reaction of nucleophilic substitution of
2,6-bis(bromomethyl)pyridine (6) and,N-dimethyl-5-(piperazin-1-
ylsulfonyl)naphthalen-1-amine (7) in 89% yield.20,21 This route is a
five-step synthetic procedure with a 49% overall yield. These
chemical structures were fully characterized and confirmed by
various samples. However, these approaches have
a
few
disadvantages including relatively high cost, tediousness,
time-consuming, post-mortem sample preprocessing and destruction
of cells or tissues, which constrain their wide application, in many
native biological studies. Therefore, considerable attentions have
been focused on the design of fluorescent chemosensors for Cu2+ due

2
1H-NMR, HRMS, 13C-NMR and MS (Figure S1-S3, Supporting
Information).
=349.05X+445.68, where X denotes the equiv. of Cu2+. Thus 1
can be a sensitive fluorescent sensor for the quantitative detection of
Cu2+ in aqueous solution. The association constant of sensor 1 and
Cu2+ was also calculated with a value of 6.7×105 M-1 and with the
detection limit of 2.5× 10-7ꢀmolꢀL−1.
Scheme 1. Synthetic route of sensor 1.
Most frequently, 4-hydroxyerhylpiperazine-1-propanesulfonic
acid (HEPPS) buffe`r system was employed to maintain probe
solutions in physiological pH window. We firstly investigated the
effect of HEPPS buffer on the fluorescence characteristics of sensor
1 in diverse concentration of HEPPS. As shown in Figure S4, the
fluorescence intensity was observed and indicated that sensor 1
would work in physiological conditions. In addition, the optimal
concentration of HEPPS for sensor solution is 10 mmol/L, and that is
applied in spectroscopic measurement.
To evaluate the selectivity of sensor 1, the effect of other typical
interfering metal ions, on the fluorescence behavior of sensor 1 in
HEPPS buffer solution was investigated (Figure 1 and S6). The
results revealed slight fluorescence change upon addition of 4
equiv of various interfering metal ions, such as Na+, Ag+, K+, Pb2+,
Hg2+, Cr3+, Gd3+, Ca2+, Zn2+, Fe2+, Fe3+, Mg2+, Co2+ and Ni2+.
However, upon addition of Cu2+, a drastic exhaustive fluorescence
quenching was observed at the peak of 540 nm, which indicated that
sensor 1 could selectively recognize Cu2+ in aqueous condition.
Compared with the blank solution of sensor 1, the fluorescence
intensity at the peak of 540 nm after addition of Cu2+ was decreased
by 40-fold, which further demonstrated that sensor 1 has a high
selectivity for Cu2+ over other typical interfering metal ions.
Figure 2. The saturation curve between the relative fluorescence intensity and the
equiv. of Cu2+. Insert: The linear correlation between fluorescence intensity and
Cu2+ concentration (λex/λem= 330/540 nm, 25 μM sensor 1, 10 mM HEPPS buffer,
pH=6.8).
To verify whether sensor 1 is suitable for detecting Cu2+ in
physiological environment, we evaluated the effect of pH on
fluorescence intensity. As shown in Figure 3, almost no changes in
fluorescence intensity of sensor 1 was observed ranging from pH 5 to
14. However, when pH value is lower than 5, the fluorescence was
quenched，which is most likely caused by protonation of dansyl
core.22 The results demonstrated that sensor 1 has the capability to
detect Cu2+ in the physiological pH window.
Figure 3. Effect of pH on the fluorescence intensity of sensor 1
Figure 1. Fluorescence intensity of sensor 1 (25 μM) in the presence of 4 equiv. of
various metal ions (λex/λem= 330/540 nm, 25 μM sensor 1, 10 mM HEPPS buffer,
pH=6.8).
An important property of chemosensors is their high selectivity
toward the analyte over the other competitive metal ions. Therefore,
the competition experiments were also conducted when various
metal ions including Cu+, Co+, K+, Na+ Cr3+, Fe2+, Gd3+, Hg2+, Mg2+,
Na+, Ni+, Pb2+, Zn2+, Fe3+ and Cu2+ co-existed in the system. As
presented in Figure 4, the fluorescent intensity is hardly affected in
response to other metal ions. But when added with Cu2+, the
fluorescence exhibited almost sharp quenching, which suggested that
sensor 1 has an excellent anti-interference ability over other metal
ions when used to detect Cu2+.
To get further insight into the ability of sensor 1 to detect Cu2+,
fluorescence titration experiment was performed (Figure S6,
Supporting Information). As shown in Figure 2, with incremental
addition of Cu2+ (0-40 equiv.), the fluorescence intensity of sensor 1
at 540 nm decreases sharply and finally saturates at about 2 equiv. of
Cu2+. There is a good linear correlation (R2= 0.9997) between the
fluorescence intensity and concentration of Cu2+ in the range from 0
to 12.5×10-6 M. The linear regression equation was
F

3
Figure 6. Reversibility of Cu2+ binding to the probe upon addition of EDTA or S2-.
Black line: only sensor 1; Red line: fluorescence quenching upon addition of 1
equiv of Cu2+; Blue line: increase in fluorescence intensity after addition of 1 equiv
of S2-. Green line: increase in fluorescence intensity after addition of 1 equiv. of
EDTA (λex/λem= 330/540 nm).
Figure 4. The fluorescence responses of sensor 1 in the present of various metal
ions. Red bars represent the addition of 4 equiv. metal ions to a solution of the
probe. Black bars represent the subsequent addition of 4 equiv. Cu2+ to the
solution. (λex/λem= 330/540 nm, 25 mM sensor 1,100 mM metal ions).
As shown in Figure 5, the saturation curve of fluorescence was
determined in nearly 5 seconds, which indicates the response of
sensor 1 with Cu2+ is fast. Besides, the incremental addition of Cu2+
promoted a negligible effect on the response. All these results clearly
proved that it is a preponderant property of probe 1 for Cu2+
detection, indicating that this probe enables to detect Cu2+ in real
time.
To determine the binding stoichiometry of senser 1-Cu2+
complexes, Job’s plot experiments were performed. A series of
solution containing certain Cu2+ and sensor 1 were prepared, in
which their total concentration are constants. Fluorescence intensity
of those mixtures of Cu2+ and probe 1 in varying molar ratios (1:9,
2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2 and 9:1) were measured. As recorded
in Figure 7, the maximum fluorescence emission at 540 nm was
observed when copper ion was close to 0.5 fractions, which proved
the 1:1 complex formation between 1 and Cu2+.
Figure 5. Reaction-time profiles of sensor 1 in the absence (Black) or presence of
Cu2+ [0.5, 1, 2, 3 equiv]. (λex/λem= 330/540 nm, 25 mM sensor 1,100 mM metal
ions)
Figure 7. Job plot of the complexion between the sensor 1 and Cu2+ (λex/λem
=
Furthermore, the reversibility of the binding between Cu2+ and
sensor 1 was performed. We chose ethylene diamine tetraacetic acid
(EDTA) and S2- as the metal chelating agents. When added EDTA or
S2- to the mixture of sensor 1 and Cu2+ in HEPPS buffer, the
fluorescence was restored, which suggested that the binding of
sensor 1 to Cu2+ was a reversible process in the presence of EDTA or
S2- (Figure 6). In addition, the fluorescence intensity was invariant
when addition 3 equiv. of EDTA or S2-, and EDTA has a greater
impact on fluorescence recovery (Figure S7 and S8, Supporting
information).
330/540 nm, 25 μM sensor 1, 10 mM HEPPS buffer, pH=6.8). The total molar
concentration of 1 and Cu2+ is 25 μM.
Fluorescence quantum yields we detected using quinine as a
standard with a known F value of 0.546 in 0.1 M H2SO4. The sensor
1 and quinine were excited at the same wavelength (330 nm),
maintaining nearly equal absorbance (0.05). The fluorescent
quantum yield was 12% in the absence of Cu2+ which was calculated
as follows:
Φs/Φf=(As/Af)×(Abss/Absf)×(ηs2/ηf2)
where Φs and Φf are the fluorescence quantum yields of quinine and
the sensor 1 respectively; As (1192) and Af (262) are the emission
areas of quinine and the sensor 1, respectively; Abss and Absf are the

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absorbance of quinine and the sensor 1 at the wavelength (340nm) of
excitation; ηs and ηf are the refractive indices of the standard and
reference, respectively.
The ability of sensor 1 to detect Cu2+ within living cells was also
evaluated by fluorescence microscopy. HeLa cells were incubated
with 10 μM sensor 1 for 20 mins at 37ºC and then washed with
phosphate buffer saline (PBS) to remove the remaining sensor. As
shown in Figure 8, a bright fluorescence was observed. However, the
fluorescence changed significantly after addition of CuCl2 for 10 min
(10 equiv.) at 37ºC, which indicated that sensor 1 could easily
penetrate the cell membrane and detect Cu2+ within living cells.
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Figure 8. Fluorescence imaging of sesnor 1 in HeLa cells; (a) Black and white
field image of HeLa cells; (b) Sensor 1(10 μM) was incubated for 20 min; (c)
Bright field image of HeLa cells shown in panel (d); (d) HeLa cells were pretreated
with sensor 1 for 10 min, then were incubated with Cu2+(10 equiv.).
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Conclusion
In summary, we have developed an ICT and dansyl-based
fluorescent sensor 1, specific for Cu2+. Results showed that sensor 1
has excellent selectivity and high sensitivity for Cu2+ over other
common metal ions and can be used to quantitatively detect Cu2+
with a fast response in a wide pH range from 5 to 14. The sensor 1
displayed a dramatic fluorescence quenching selectively for Cu2+
over other metal ions. More importantly, application in bioimaging
was illustrated and the living cell imaging experiment further
demonstrated the excellent cell-membrane permeability and the
potential capability of sensor 1 for detecting Cu2+ in living cells.
Acknowledgments
This work was supported by National Natural Science
Foundation of China (81271634, 81671756 and 81601554).
Supplementary Material
Electronic Supplementary Information (ESI) available: Copies
of fluorescence spectra and NMR spectra. This material is
available free of charge via the Internet.
References and notes
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Hosseinzadeh, R.; Nemati, M.; Mohadjerani, M. Beilstein. J. Org.
Chem. 2016, 12, 1749-1757.
Saeed, M. A.; Powell, D. R. Hossain, M. A. Tetrahedron Lett.
2010, 51, 4904-4907.

5
Highlights
A novel fluorescent chemosensor 1 for the
detection of copper (II) ion was developed.
1 exhibited remarkable fluorescence quench
for the sensitive and selective quantification of
Cu2+.
1 showed an ultrafast and linear response
towards Cu2+ in wide range and can be used for
detecting Cu2+ in living cells.