A new fluorescent sensor for Cd2+ and its application in living cells imaging

Article abstract of DOI:10.1016/j.tetlet.2015.02.010

A novel, fluorescent sensor based on 2,7-dichlorofluorescein with aromatized diethylene triamine as ionophore was developed. Under physiological pH conditions, the sensor exhibited about 70-fold enhancement in fluorescence emission upon binding with Cd²⁺. It demonstrates that the sensor can be used to detect Cd²⁺ in aqueous solution and it was successfully applied to image Cd²⁺ in living cells.

Full text of DOI:10.1016/j.tetlet.2015.02.010

Tetrahedron Letters 56 (2015) 1322–1327
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
A new fluorescent sensor for Cd2+ and its application in living cells
imaging
a,
Xiao-yan Liu a, Da-ying Liu a, Jing Qi a, Zhi-gang Cui b, He-xi Chang b, Hua-rui He b, , Guang-ming Yang
⇑
⇑
a Department of Chemistry, Nankai University, Tianjin, China
b Heowns Biochem Technologies LLC, Tianjin, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel, fluorescent sensor based on 2,7-dichlorofluorescein with aromatized diethylene triamine as ion-
ophore was developed. Under physiological pH conditions, the sensor exhibited about 70-fold enhance-
ment in fluorescence emission upon binding with Cd2+. It demonstrates that the sensor can be used to
detect Cd2+ in aqueous solution and it was successfully applied to image Cd2+ in living cells.
Ó 2015 Elsevier Ltd. All rights reserved.
Received 11 November 2014
Revised 24 January 2015
Accepted 2 February 2015
Available online 7 February 2015
Keywords:
Fluorescence sensor
Ionophore
Cadmium
Cell imaging
Introduction
binding selectivity and sensitivity of Cd2+ against Zn2+ in aque-
ous solution.
Cadmium, an essential element in the earth, is widely used in
agriculture and industry.1 These sources of cadmium cause exces-
sive exposure to human society.2 Whereas Cd2+ is also known for
its toxicity, which can induce serious health and environmental
problem, such as renal dysfunction, Calcium metabolism disorders,
prostate cancer, etc.1–3 Thus, there is a great need for methods of
detecting and monitoring Cd2+ levels in living cells and
environment.4
Compared with other analytical methods, such as atomic absorp-
tion5 and ICP (inductively coupled plasma) atomic emission spectros-
copy,6 fluorescent chemosensors have many advantages such as high
sensitivity and simplicity, real-time monitoring, which has been con-
sidered one of the most useful and powerful tools for detecting and
monitoring cations, anions, and neutral molecules for many years.7
Although a number of fluorescent sensors for Cd2+ have been
reported up to now,8 most of them suffered from the interference
of Zn2+ because they are located in the same group of the Periodic
Table and resulted in many similarities of chemical properties.9
Hence, we present the design and synthesis of a novel, 2,7-
dichlorofluorescein-based sensor with aromatized diethylene-
triamine as ionophore. The sensor possesses relatively high
The selection of 2,7-dichlorofluorescein as fluorophore was
based on the following considerations: 2,7-dichlorofluorescein as
the most important derivatives of fluorescein has been widely
employed as signalling handles for molecular imaging and chemo-
sensing applications.10 One advantage of 2,7-dichlorofluorescein is
that its pKa is 4.69, which is much lower than that of fluorescein
(6.44). Hence, the fluorescence and absorption properties of 2,7-
dichlorofluorescein are less sensitive to the solvent media in the
physiological pH range.11
The selection of aromatized diethylenetriamine as ionophore
was driven by several design criteria: (a) diethylenetriamine is
one of the most useful skeletons and has been found significant
applications in a variety of involving metal complexes;12 (b) aro-
matization can greatly reduce the pKa of diethylenetriamine, lead
to its less sensitive to the value of pH at physiological conditions;13
(c) to enhance metal binding selectivity, a series of substituents
including diethylsulfane, 6-methylpicolinic acid, acetic acid, were
introduced at the NH and NH2 groups of aromatized diethylenetri-
amine. Among all the available ionophores tested, the one with
acetic acid as the substituent was found to be the best sensor can-
didate for Cd2+. In fact, the ionophore is the aromatized version of
diethylenetriamine-pentaacetic acid (ADTPA), which is also one of
the most important chelators for metal ions.14
⇑
Corresponding authors. Tel.: +86 22 83717408; fax: +86 22 83717748 (H.-r.H.);
Scheme 1 displays the synthetic route of Sensor 1, the detailed
procedures and characterization of the novel sensor are described
in the Supporting information.
tel.: +86 22 23501230; fax: +86 22 2350 8545 (G.-m.Y.).
E-mail addresses: huarui.he@heowns.com (H.-r. He), yanggm@nankai.edu.cn
(G.-m. Yang).
http://dx.doi.org/10.1016/j.tetlet.2015.02.010
0040-4039/Ó 2015 Elsevier Ltd. All rights reserved.

X.-y. Liu et al. / Tetrahedron Letters 56 (2015) 1322–1327
1323
NH2
NO2
NH2
NO2
NH2
NO2
Br
H2,Pd/C
H
H
N
Br
N
N
Br
N
H
N
H
H
3
1
4
2
O
O
O
N
ONa
N
O
ONa
O
O
O
NaO
Br
N
O
NaOH
O
O
N
O
O
ONa
N
N
O
O
F
M
D
5
,
ionophore
l3
C
O
P
Cl
OH
O
N
O
O
N
ONa
N
O
N
O
O
O
ONa
O
O
O
N
O
O
NaO
O
O
O
OH
DDQ
O
O
N
O
O
ONa
O
N
O
NaOH
N
N
N
O
O
N
O
O
O
O
N
O
OH
Cl
Cl
Cl
NaO
Cl
O
Cl
HO
Cl
O
CHO
HO
OH
OH
O
O
6
7
8
Sensor 1
Scheme 1. Synthesis of Sensor 1.
Fluorescent properties of Sensor 1
Results and discussion
UV–vis properties of ionophore
In order to further illustrate the binding properties, fluores-
cence responses of Sensor 1 (25 M, kex = 470 nm) to various metal
l
ions in the physiological condition (20 mM HEPES buffer, pH = 7.4)
at room temperature were also carried out. As shown in Figure 2a
and b, Sensor 1 showed a weak fluorescence in aqueous solution in
the absence of metal ions. Upon the addition of 5.0 equiv of metal
To obtain the binding properties, the ionophore was also syn-
thesized. Figure shows the UV–vis spectra of ionophore
(77
M) in the presence of different concentrations of Cd2+ in
20 mM HEPES buffer at pH 7.4. As shown in Figure 1a, upon
increasing the amount of Cd2+, the absorbance spectra peak at
249 nm decreased gradually. A comparison of the A249 nm ratio of
the absorbance before and after the addition of Cd2+ exhibited that
1
l
ions (125 l ,
M), including Na+, K+, Ca2+, Mg2+, Zn2+, Cu2+, Cd2+, Fe2+
Fe3+, Hg2+, Mn2+, Ni2+, Cr3+, Co2+, Pb2+, Sr2+ to Sensor 1, It was found
that Cd2+ and Hg2+ caused the remarkable fluorescence enhance-
ment, with Cd2+ about 70ꢀfold enhancement and Hg2+ about
100ꢀfold enhancement. By contrast, nearly no or very slight fluo-
rescence intensity changes of Sensor 1 were observed in the
presence of others metal ions under the identical conditions.
there was about 4.7 fold decrease in the range of 0–120
(Fig. 1b) because of the electron density of the aniline ring changes
corresponded to the concentration of Cd2+ 15
The results provided
that the ionophore can form effective complexation with Cd2+
lM
.
.
Figure 1. (a) Change in the absorption spectra of ionophore (77
before and after the addition of Cd2+
l lM. (b) A comparison of the I249 nm ratio of the absorbance
M) upon titration of Cd2+ in the range of 0 to 120
.

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X.-y. Liu et al. / Tetrahedron Letters 56 (2015) 1322–1327
Figure 2. (a) Fluorescence responses of Sensor 1 (25
l
M, kex = 470 nm) in HEPES buffer solutions (20 mM, pH = 7.4) to various metal ions(125 l ,
M), for example, Na+, K+, Ca2+
Mg2+, Zn2+, Cu2+, Cd2+, Fe2+, Fe3+, Hg2+, Mn2+, Ni2+, Cr3+, Co2+, Pb2+, Sr2+. (b) Represent the relative fluorescence intensity of Sensor 1 (F/F0) in the presence of various metal ions.
(c) Fluorescence responses at 530 nm of Sensor 1 (25
concentrations of NaCl.
l
M, kex = 470 nm) towards Cd2+ and Hg2+ (125
l
M) in HEPES buffer solutions (20 mM, pH = 7.4) containing of different
n
Fmax½Mꢂ þ FminKd
Surprisingly, nearly no fluorescence enhancement was observed
with Zn2+, which is one of the major interfering ions for almost
all Cd2+ sensors. Thus this sensor possesses a high selectivity
against Zn2+. The results provided that Sensor 1 can identify
Cd2+ and Hg2+ over others metal ions in HEPES buffer solution.
F ¼
n
Kd þ ½Mꢂ
where F denotes the observed fluorescence. Fmax is the maximum fluo-
rescence for the Sensor 1—Cd2+ complex at a saturation concentration
of Cd2+, while Fmin is the fluorescence for free Sensor 1, and n is the num-
ber of Cd2+ bound per Sensor. Job’s plots indicted that the stoichiometry
between Sensor 1 and Cd2+ is 2:1 (Fig. 5a), which means the value of n is
0.5. As shown in Figure 5b, the dissociation constant Kd between Sensor
1 and Cd2+ was determined from intercept of the linear plot, which was
calculated to be 4.08 ꢃ 10ꢁ6 M. These results demonstrate that the Sen-
sor 1 possesses high affinity with Cd2+ in aqueous solution.
To improve the selectivity of Sensor 1 towards Cd2+ and Hg2+
,
we found that a large amount of Clꢁ can successfully mask the
response of Hg2+, to a certain degree. As depicted in Figure 2c,
when 150 mM NaCl, which is close to the concentration of the bio-
logical system, was added into the aqueous solution of Sensor 1
(25 lM) in 20 mM HEPES buffer at pH 7.4, the fluorescence
enhancement of Sensor 1 towards Hg2+ at 530 nm decreased from
100ꢀfold to 10ꢀfold, nearly 90% of fluorescence response was
eliminated. While the fluorescence enhancement of Sensor 1
towards Cd2+ only decreased from 70ꢀfold to 60ꢀfold, in other
words, about 90% of fluorescence response remain. Addition of
600 mM NaCl further suppressed the fluorescence enhancement
down to 2 fold, about 2% of response left. However, considering
the response of Cd2+ under the identical condition, the interference
of Hg2+ is almost negligible.
The fluorescence titration of Sensor 1 (25 lM, kex = 470 nm) in
different concentrations of Cd2+ solution containing of 600 mM NaCl
were also carried out. As shown in Figure 4a and b, the fluorescence
intensity at 530 nm increased gradually and reached saturation
with the addition of 30 l
M Cd2+. A plot of relative fluorescence
intensity (F/F0) clearly illustrated that there was about 50 fold
increase compared to Sensor 1 without Cd2+, where F and F0 repre-
sent the fluorescence intensity in the presence and absence of Cd2+
.
However, under the identical conditions, nearly no fluorescence
enhancement of Sensor 1 towards Hg2+ was observed. The result
explicitly indicates that the Sensor 1 has excellent selectivity for
Cd2+ over Hg2+ in the aqueous solution of NaCl.
The fluorescence spectra of Sensor 1 (25 lM) taken in the
course of titration with Cd2+ in 20 mM HEPES buffer at pH 7.4 were
shown in Figure 3a and b. As expected, with the increase concen-
tration of Cd2+ into Sensor 1 resulted in obvious fluorescence
enhancement and fluorescence emission intensity at 530 nm
became saturated (about 70ꢀfold) when Cd2+ reached 20
l
M.
Fluorescent detection of Cd2+ in living cells
Therefore, on the basis of fluorescence titration data, the dissocia-
tion constant of Sensor 1 with Cd2+ was calculated by using the fol-
lowing fitting equation.16
To further demonstrate the practical application of Sensor 1 in
biological field, fluorescence imaging experiments were conducted

X.-y. Liu et al. / Tetrahedron Letters 56 (2015) 1322–1327
1325
Figure 3. (a) Fluorescence spectra of Sensor 1 (25
l
M, kex = 470 nm) in HEPES buffer solutions (20 mM, pH = 7.4) upon the titration of Cd2+ in the range of 0–20
lM. (b) The
relative fluorescence intensity at 530 nm of Sensor 1 in the presence of various concentrations of Cd2+ (0–20
lM).
Figure 4. (a) Fluorescence spectra of Sensor 1 (25
35
M). (b) Comparison of fluorescence intensity at 530 nm of Sensor 1 versus increasing Cd2+ and Hg2+ concentrations in HEPES buffer solution (20 mM, pH = 7.4) containing
of 600 mM NaCl.
l
M, kex = 470 nm) in HEPES buffer solution (20 mM, pH = 7.4) containing of 600 mM NaCl upon the titration of Cd2+ (0–
l
Figure 5. (a) Job’s plot for Sensor 1 (from 2:1 complex) in 20 mM HEPES buffer solution at pH 7.4. The total concentration of Sensor 1 and Cd2+ is 5
(fluorescence intensity at 530 nm) of Sensor 1 with Cd2+
lM. (b) Linear plot
.
with HeLa cells and the images were taken with Olympus IX81
inverted research microscope. As revealed in Figure 6, first the cells
buffer saline (PBS), the cells were further incubated with Cd2+
(5 M) in the same medium for another 1 h, the strong green
l
were only treated with 10
l
M of Sensor 1 for 18 h at 37 °C, no fluo-
fluorescence emission was shown. Bright image transmission and
rescence was observed. After three times washing with phosphate
fluorescence images overlaid perfectly, which confirm that the

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X.-y. Liu et al. / Tetrahedron Letters 56 (2015) 1322–1327
Figure 6. Fluorescence field image and their corresponding merge field. (a): HeLa cells were incubated with Sensor 1 (10
1 for 18 h, washed three times, and then further incubated with 5
M Cd2+ for 1 h.
lM) for 18 h. (b): HeLa cells incubated with Sensor
l
fluorescence signal results from the intracellular region.17 All of
these facts implied that Sensor 1 has good membrane permeability
and can be used as a sensor for detecting Cd2+ in living cells.
010. These data include MOL files and InChiKeys of the most
important compounds described in this article.
References and notes
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.tetlet.2015.02.

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