Toward a highly sensitive and selective indole-rhodamine-based light-up probe for Hg2+ and its application in living cells

Article abstract of DOI:10.1016/j.tetlet.2017.06.024

We herein designed and synthesized a light-up fluorescent probe L1 for Hg²⁺ species, which is based on indole derivative and Rhodamine fluorophore. The new probe can show a linear response to Hg²⁺ with high sensitivity and selectivity. As the Hg²⁺ concentration changed from 0 to 450?μM, the fluorescence intensity of L1 at 575?nm changed from 50 to 6181 (～120-fold). The detection limit of the probe was 5.0?×?10^?8?M. Besides, we have successfully applied L1 to monitor Hg²⁺ species in living MCF-7 cells by way of fluorescence imaging.

Full text of DOI:10.1016/j.tetlet.2017.06.024

Accepted Manuscript
Toward a Highly Sensitive and Selective Indole-Rhodamine-Based Light-up
Probe for Hg2+ and Its Application in Living Cells
Yao Tang, Guo-Fang Jiang
PII:
S0040-4039(17)30746-3
DOI:
http://dx.doi.org/10.1016/j.tetlet.2017.06.024
TETL 49016
Reference:
Tetrahedron Letters
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Revised Date:
Accepted Date:
2 April 2017
6 June 2017
9 June 2017
Please cite this article as: Tang, Y., Jiang, G-F., Toward a Highly Sensitive and Selective Indole-Rhodamine-Based
Light-up Probe for Hg2+ and Its Application in Living Cells, Tetrahedron Letters (2017), doi: http://dx.doi.org/
10.1016/j.tetlet.2017.06.024
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Tetrahedron Letters
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Toward a Highly Sensitive and Selective Indole-Rhodamine-Based Light-up
Probe for Hg2+ and Its Application in Living Cells
Yao Tang, Guo-Fang Jiang*
State Key Lab of Chemo/Biosensing and Chemometrics,College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, P. R. China
*Corresponding author. Tel.: +86-731-88821488; fax: +86-731-88713642; e-mail: gfjiang@hnu.edu.cn
ARTICLE INFO
ABSTRACT
Article history:
Received
We herein designed and synthesized a light-up fluorescent probe L1 for Hg2+ species, which is
based on indole derivative and Rhodamine fluorophore. The new probe can show a linear
response to Hg2+ with high sensitivity and selectivity. As the Hg2+ concentration changed from 0
to 450 µM, the fluorescence intensity of L1 at 575 nm changed from 50 to 6181 (∼120-fold).
The detection limit of the probe was 5.0 × 10-8 M. Besides, we have successfully applied L1 to
monitor Hg2+ species in living MCF-7 cells by way of fluorescence imaging.
Received in revised form
Accepted
Available online
Keywords:
Fluorescent probe
Hg2+
2016 Elsevier Ltd. All rights reserved.
Sensitivity
Selectivity
Cell imaging
In the past decade, researches on sensors or probes have
attracted a lot of interests owing to their high selectivity for the
detection of toxic metal ions, and these metal ions were one of
the main factors affecting the environmental and physical
health.[1-4] Among these various toxic metal ions, ionic mercury
has attracted growing research interests because of its high
toxicity and easily absorbed and accumulated in living
organisms.[5] High exposures to Hg2+ may cause gastrointestinal
injury and serious diseases such as kidney dysfunction, prenatal
brain damage and disorders of the central nervous system. In
addition, it is difficult to be excluded in a short time when the
ionic mercury into the human body. Thus, sensitive and
quantitative detection of Hg2+ is highly necessary for
environment and biomedicine.[6]
Scheme 1. Synthesis and chemical structures of probes L1-L3.
So far, a large number of fluorescent chemosensors for Hg2+
with high selectivity and relatively simple operation have been
reported.[1, 6-23] Most of probes are based on sulfur donor centers
via utilizing the strong binding between mercury and sulfur.
Although applications of these probes for real samples have
increased, they still suffer from several problems such as easy to
oxidation of these functional groups by oxidants and non-
recycling, undesired air-oxidation of amines and especially
sulfides in long-term storage.[24-27] On the other hand, some other
probes which detect Hg2+ through the coordination of multiple
nitrogen atoms have resolved the above issues. However, these
probes subject to selectivity problems due to the strong
interference of Cu2+, Pb2+, Fe3+, Mg2+ and Ag+.[6, 26-33] Therefore,
it is necessary to develop a highly selective fluorescent probe for
the detection and quantification of Hg2+ ions.
derivatives are widely used in bioimaging and fluorescent
sensors.[34-37] Herein, we have prepared a small library with three
light-up fluorescent probes (L1-L3) for Hg2+ species by way of
coupling the carbohydrazone block with Rhodamine B (Scheme
1). Screening investigations[38] shown that among the three
probes, L1 displayed high sensitivity and selectivity towards
Hg2+ species with the best linear response. Furthermore, we have
successfully applied L1 to detect Hg2+ ions in living cells.
As probes L1-L3 were prepared, we firstly examined the
fluorescence changes of L1-L3 in DMF-phosphate buffer
solution (PBS, 25 mM, pH 7.4, containing 40% DMF) upon
addition of Hg2+ and other metal ions (Cu+, Cd2+, Cu2+, Li+, Ni2+,
Zn2+, Ca2+, Cr3+, Pb2+, Fe3+, Al3+, Na+, Mg2+, K+, Mn2+, Co2+ and
Ag+ ions). As shown in Figure 1 and Figure S1-2, all the three
probes showed significant fluorescence turn-on response at 575
nm when treated with Hg2+, which was possibly due to the Hg2+
facilitated ring opening of the spirocycle group (Scheme S1). In
Based on their superior photophysical properties, such as high
molar extinction coefficients, great photostability and excellent
fluorescence quantum yields, Rhodamine and its

contrast, other metal ions, such as heavy/transition metal ions
(Mn2+, Ni2+, Pb2+, Zn2+, Ag+, Au3+, Cd2+, Co2+, Cu2+ and Fe3+),
alkali-metal ions (K+, Na+), alkali-earth metal ions (Ca2+, Mg2+)
induced inconspicuous fluorescence enhancement (Figure 1a,
Figure S1a and S2a), indicating that probes L1-L3 were highly
selective. In order to obtain a fluorescent probe with high
selectivity and sensitivity for the detection of Hg2+, the
quantitative analytical behavior of L1-L3 for determination of
Hg2+ were studied. As shown in Figure 1b, Figure S1b and S2b,
with the increase of the concentration of Hg2+, the best
fluorogenic response was realized by L1. The analysis of the
titration data revealed that the emission intensity of L1 at 575 nm
increased linearly with the increasing concentrations of Hg2+ in
two dynamic ranges of 2-15 µM and 15-200 µM (Figure 2 and
Figure S3). The detection limit of L1 was 5.0 × 10-8 M based on
calculation (3σ/s; σ was the standard deviation of the blank
sample and s was the slope of the linear regression equation.) in
DMF-phosphate buffer solution (PBS, 25 mM, pH 7.4,
containing 40% DMF). Moreover, in accordance with the
obvious fluorescence enhancement, the absorbance intensity of
L1 at 534 nm gradually increased with the addition of Hg2+
(Figure S4), and the solution changed from colorless to pink
(Figure S5).
In order to study the potential interference with other metal
ions for Hg2+ detecting, the coexisting metal ions on the detection
of Hg2+ by L1 were subsequently detected. As shown in Figure 3,
The vast majority of tested metal species had negligible effect on
the Hg2+ detecting, and the minority had a weak effect. indicating
that L1 could also be suitable for selectively detecting Hg2+ even
though in the presence of other associated metal ions.
Y = 53.299 + 7.9332X
180
R = 0.9819
150
120
90
60
0
2
4
6
8
10 12 14 16
[Hg2+] * 10-6/M
In contrast, L2 showed no linear response for Hg2+ (0-1 mM)
and L3 can not have a linear response in the low concentration
range of Hg2+ (2-15 µM) (Figure S1b, S2b). Accordingly, we can
draw a conclusion that L1 was the best probe for the detection of
Hg2+ among the three probes, hence we selected L1 for further
investigations.
Figure 2. A plot of fluorescence intensity of L1 at 575 nm against Hg2+
concentration from 2 to 15 µM.
ens.
5
x10
6
a)
7000
6000
820
5000
4000
3000
2000
1000
0
2+
L1 + Hg
4
2
L1 + other metal ions
749.6149
679.1797
784.2735
0
650
700
750
800
550
600
650
700
Wavelength(nm)
Figure 3. Fluorescence response of L1 (5 µM) to the various species (200
equiv.) and Hg2+ (150 equiv.) in the DMF-phosphate buffer solution (25 mM,
0
b)
7000
6000
5000
4000
3000
2000
1000
0
pH 7.4, containing 40% DMF as a co-solvent).
0.5eq.
1.0eq
1.5eq.
2.0eq.
2.5eq.
3.0eq.
4.0eq.
5.0eq.
6.0eq.
8.0eq.
150 eq.
Hg2+
300
200
100
ntens.
5
x10
6
0eq.
0
550
600 650
Wavelength(nm)
700
1+
820.2480
4
550
600
Wavelength(nm)
650
700
2
749.6149
679.1797
784.2735
Figure 1. a) Fluorescence spectra of L1 (5 µM) with metal ions (200 equiv.)
and Hg2+ (200 equiv.) in the DMF-phosphate buffer solution (25 mM, pH 7.4,
containing 40% DMF as a co-solvent) Excitation at 500 nm. b) Fluorescence
spectra of L1 (5 µM) in DMF-phosphate buffer solution (25 mM, pH 7.4,
0
650
700
750
800
Figure 4. ESI experiment was carried out in DMF-phosphate buffer solutions,
L1 (5 µM); Hg2+ (1 mM).
containing 40% DMF as a co-solvent) in the presence of Hg2+
.

3
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Figure 5. Bright-field and fluorescence images of MCF-7 cells with L1: (a-c)
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(b) bright-field image; (c) overlay of (a) and (b); (d-f) bright-field and
fluorescence images of the cells incubated with L1 (5 µM) for 30 min, and
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In summary, a novel fluorescent probe (L1) based on indole
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selective and sensitive detection to Hg2+ species. The detection
limit of this new probe is as low as 5.0 × 10-8 M. Besides, the
probe L1 can display a good linear response toward Hg2+ in two
dynamic ranges of 2-15 µM and 15-200 µM in aqueous media,
which is useful for quantitatively detecting Hg2+ in low and high
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Acknowledgments
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We thank the financial support from the National Natural Science
Foundation of China (51578224) and the Natural Science
Foundation of Hunan Province (2015JJ4018).
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1. A novel light-up fluorescent probe (L1) based
on indole derivative and Rhodamine
fluorophore for Hg2+ species was developed.
2. The probe L1 can show a linear response to
Hg2+ with high sensitivity and selectivity, and
its detection limit was 5.0 × 10-8 M.
3. Futhermore, probe L1 can be successfully
applied to monitor Hg2+ species in living MCF-
7 cells by fluorescence imaging.

5
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Graphical Abstract
Toward a Highly Sensitive and Selective
Indole-Rhodamine-Based Light-up Probe
for Hg2+ and Its Application in Living Cells
Yao Tang, Guo-Fang Jiang*