A rhodamine derivative for Hg2+-selective colorimetric and fluorescent sensing and its application to in vivo imaging

Article abstract of DOI:10.1016/j.cclet.2016.04.001

A rhodamine-based sensor has been developed for the detection of mercuric ions. The colorimetric and fluorescence responses, allowing naked-eye detections, are based on Hg²⁺-induced opening of the rhodamine spirocycle. Among all the testes ions, only Hg²⁺ generated a significant fluorescence enhancement of up to 300-fold, with a bright yellow-green emission. This sensor was a low toxic compound, and was successfully applied in the in vivo imaging of Hg²⁺ in Spill 2 cells and C. elegans. This approach provides a sensitive and accurate method for the estimation of Hg²⁺ in environmental, tobacco and biological applications.

Full text of DOI:10.1016/j.cclet.2016.04.001

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Chinese Chemical Letters xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Chinese Chemical Letters
journal homepage: www.elsevier.com/locate/cclet
Original article
2
+
A rhodamine derivative for Hg -selective colorimetric and fluorescent
sensing and its application to in vivo imaging
a
b
a
b
c
Xue-Mei Li , Rui-Rui Zhao , Yu-Ling Wei , Dan Yang , Zhang-Jian Zhou ,
c,
b,
Jun-Feng Zhang *, Ying Zhou *
a
Department of Safety Evaluation, Technology Center of China Tobacco Yunnan Industrial Co., Ltd., Kunming 650106, China
b
College of Chemical Science and Technology, Yunnan University, Kunming 650091, China
c
College of Chemistry and Chemical Engineering, Yunnan Normal University, Kunming 650500, China
A R T I C L E I N F O
A B S T R A C T
Article history:
A rhodamine-based sensor has been developed for the detection of mercuric ions. The colorimetric and
Received 2 February 2016
Received in revised form 15 March 2016
Accepted 21 March 2016
Available online xxx
2+
fluorescence responses, allowing naked-eye detections, are based on Hg -induced opening of the
2+
rhodamine spirocycle. Among all the testes ions, only Hg generated a significant fluorescence
enhancement of up to 300-fold, with a bright yellow–green emission. This sensor was a low toxic
2
+
compound, and was successfully applied in the in vivo imaging of Hg in Spill 2 cells and C. elegans. This
2
+
approach provides a sensitive and accurate method for the estimation of Hg in environmental, tobacco
and biological applications.
Keywords:
Chemsensor
Mercury
ß 2016 Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of Medical Sciences.
Published by Elsevier B.V. All rights reserved.
Rhodamine
Colorimetric
Fluorescence
In vivo imaging
1
. Introduction
coordination capacity required to chelate mercury ions. Sensor 1
2+
showed colorimetric and fluorescent selectivity for Hg in DMSO–
HEPES buffer (0.02 mol/L, pH 7.4; v/v = 6:4) solution over other
common physiologically important metal ions. It showed that
sensor 1 was a low toxic compound, and was successfully applied
Mercury is one of the most toxic heavy metals found in aquatic
systems, and it has become a worldwide issue in recent years due
to its severe risk to human health and to the environment.
Compared with many of the current techniques for mercury
detection, such as atomic absorption spectrometry, inductively
coupled plasma mass spectroscopy, spectrophotometry, neutron
activation analysis, anodic stripping voltammetry and X-ray
fluorescence spectrometry, fluorescence sensing provide simple,
safe, effective and rapid detection of Hg , paving the way for its
applications [1]. Even a number of research groups have reported
their recent achievements on important ion-targeted or anion-
targeted fluorescent sensors [2], the development of organic
probes for sensing of environmentally hazardous Hg ions is still
of great importance due to their implications in broad areas.
Herein, we have designed and synthesized a simple and easy to
prepare fluorescent chemosensor (1) for the Hg ion sensing. In
molecule 1, rhodamine group acts as a fluorophore group, with a
hydroxyquinoline group as a recognition group, allowing the
2
+
in the in vivo imaging of Hg in Spill 2 cells and C. elegans.
2. Experimental
2+
2.1. Reagents and chemicals
All reagents were purchased from commercial sources and were
used without further purification. Flash chromatography was
1
2+
carried out on silica gel (230–400 mesh). H NMR spectra (DMSO-
TM
13
d
6
) were recorded using Ascend
400 spectrometer; C NMR
III
spectra (DMSO-d
6
) were recorded using Avance 400 spectrome-
2+
ter; mass spectrometry was recorded with Xevo TQ-S mass
spectrometer. The UV–vis spectrum was obtained using UV-
240IPC spectrophotometer. The fluorescence spectra were
obtained with F-4500 FL spectrometer with a 1 cm standard
quartz cell. The cells were imaged using an Olympus IX71 inverted
fluorescence microscopy. The mounted nematodes were imaged
using an Olympus BX51 inverted fluorescence microscopyll
reagents were of analytical grade or the best grade commercially
*
Corresponding authors.
E-mail addresses: junfengzhang78@aliyun.com (J.-F. Zhang),
yingzhou@ynu.edu.cn (Y. Zhou).
http://dx.doi.org/10.1016/j.cclet.2016.04.001
1
001-8417/ß 2016 Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of Medical Sciences. Published by Elsevier B.V. All rights reserved.
2
+
Please cite this article in press as: X.-M. Li, et al., A rhodamine derivative for Hg -selective colorimetric and fluorescent sensing and its
application to in vivo imaging, Chin. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.cclet.2016.04.001

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X.-M. Li et al. / Chinese Chemical Letters xxx (2016) xxx–xxx
2
+
2+
2+
available, and were put into use without further purification.
Deionized water was used throughout. HEPES buffer solutions
buffer (0.02 mol/L, pH 7.4, v/v = 6:4) solutions. Cd , Ni , Zn ,
3
+
3+
3+
+
2+
2+
2+
Fe , Cr , Al , Ag , Co , Cu , and Hg were used to measure the
selectivity of probe 1. All spectra were recorded after three minutes
upon addition of 25 equiv. of each of these ions. As shown in Fig. 1,
compound 1 exhibits no major absorption band. Upon addition of
different metal ions, only the presence of Hg could lead to an
obvious absorption increase at 533 and 662 nm (Fig. 1). It revealed
(
0.02 mol/L, pH 7.4) were prepared in deionized water. Analyte
3+
+
2+
2+
2+
2+
2+
solutions of the perchlorate of Al , Na , Co , Ni , Cu , Zn , Pb
Cd , Ag , Fe , Hg and Cr were prepared by dissolving the salts
in distilled water to final concentrations of 0.1 mol/L.
,
2+
+
3+
2+
3+
2
+
2
+
2.2. Synthesis of probe 1
that 1 had a good selectivity toward Hg in the absorption among
2+
the tested ions. In the titration tests, with the addition of Hg , the
absorbance at 533 and 662 nm increased sharply, which induced a
color change from colorless to pink (Fig. 2c).
Compound 2 was synthesized from 2-methyloxine by the
procedure published in literature [3]. Compound 3, as a known
rhodamine 6G derivative, was synthesized as described previously
2
+
2+
2+
+
3+
3+
3+
2+
+
2+
2+
Then, Cd , Ni , Zn , Na , Fe , Cr , Al , Pb , Ag , Co , Cu
2
+
[
4]. Compounds 2 (210 mg, 1.2 mmol) and 3 (0.307 mg, 0.8 mmol)
were mixed in boiling ethanol with three drops of acetic acid
Scheme 1). After 8 h of stirring, the pink precipitate formed was
and Hg
compound
v/v = 6:4) solutions. From the fluorescence experiments (Fig. 3a),
were used to evaluate fluorescent selectivity of
1
in DMSO–HEPES buffer (0.02 mol/L, pH 7.4,
(
2
+
removed by filtration, washed with ethanol/diethyl ether (1:1),
and purified by silica gel column chromatography to afford
clear ‘‘off-on’’ fluorescence changes of 1 to Hg were observed.
Among the tested metal ions (30 equiv.), 1 showed a selective
fluorescence enhancement only with Hg , indicating that 1
1
2+
orange–yellow solid product 1 (230 mg, 55%). H NMR (400 MHz,
2
+
DMSO-d
.98–7.96 (d, 1H, J = 7.2 Hz), 7.87–7.85 (d, 1H, J = 8.8 Hz), 7.62–7.57
m, 2H), 7.40–7.38 (d, 2H, J = 7.6 Hz), 7.33–7.31 (d, 1H, J = 8.0 Hz),
6
):
d
9.87 (s, 1H), 8.69 (s, 1H), 8.24–8.22 (d, 1H, J = 8.8 Hz),
displayed a high Hg selectivity (Fig. 3b).
2
+
7
(
Compound 1 displays almost no fluorescence. When Hg was
added to the solution, a significant increase of the fluorescence
2
+
7
.09–7.05 (t, 2H, J = 7.2 Hz), 6.40 (s, 2H), 6.27 (s, 2H), 5.10 (s, 2H),
intensity of 556 nm, which was attributed to the Hg -induced ring
1
3
2+
3
.17–3.12 (m, 4H), 1.85 (s, 6H), 1.23–1.19 (t, 6H, J = 7.0 Hz);
): 168.87, 153.90, 152.51, 152.39,
51.26, 148.35, 145.98, 138.54, 137.00, 134.80, 129.29, 129.24,
C
opening of the spirolactam moiety, was observed. Hg generated a
NMR (100 MHz, DMSO-d
6
d
significant fluorescence enhancement of up to 300-fold, with a
bright yellow–green emission (Fig. 4). These results suggested that
1 has high fluorescence selectivity for Hg compared to the other
1
2
+
1
28.71, 127.96, 126.93, 124.16, 123.82, 118.86, 118.26, 117.46,
1
12.79, 105.10, 96.58, 66.02, 32.00, 17.44, 14.64. HRMS (ESI, m/z):
ions.
+
+
2+
calcd. for C36
5
H
33
N
5
O
3
584.2583 [M+H] , 606.2476 [M+Na] , found
The proposed binding mechanism of compound 1 with Hg is
shown in the Scheme 2. The spirolactam moiety of the rhodamine
group acts as
fluorescence-off state of 1 converts to the Hg -promoted ring-
opened amide form with a fluorescence-on state. Moreover, 1 is
most likely to bind Hg via the imide N and quinoline O atoms like
+
+
84.2650 [M+H] ; 606.2477 [M+Na] .
2+
a signal switcher, when 1 binds Hg , the
2
+
3
. Results and discussion
2
+
In order to clarify the interaction of 1 with metal ions, the UV–
vis absorption spectra of 1 were first studied in DMSO–HEPES
other reported researches [5]. The detection limit was calculated
Scheme 1. Synthetic route of probe 1.
(
a) 0.8
(
b) 0.8
2
+
Hg
0
0
.4
.0
0
.4
2+
Ni
1
4
00
500
600
700
800
0.0
1
a
b
c
d
e
f
g
h
i
Wavelength (nm)
À5
3+
3+
2+
2+
+
3+
2+
2+
2+
Fig. 1. (a) Absorption spectra of 1 (2.0 Â 10 mol/L) in DMSO–HEPES buffer (0.02 mol/L, pH 7.4, v/v = 6:4) with 25 equiv. of Fe , Cr , Co , Ni , Na , Al , Cd , Zn , Hg ; (b)
À5
2+
2+
2+
3+
3+
+
2+
3+
2+
Absorbances of 1 (2.0 Â 10 mol/L) at 533 nm after addition of 25 equiv. selected ions (1: blank, a: Zn , b: Ni , c: Cd , d: Fe , e: Al , f: Na , g: Hg , h: Cr , i: Co ).
2
+
Please cite this article in press as: X.-M. Li, et al., A rhodamine derivative for Hg -selective colorimetric and fluorescent sensing and its
application to in vivo imaging, Chin. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.cclet.2016.04.001

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3
À5
2+
Fig. 2. (a) Absorption titration spectra of 1 (2.0 Â 10 mol/L) in the presence of varying concentrations of Hg in DMSO–HEPES buffer (0.02 mol/L, pH 7.4; v/v = 6:4). (b)
À5
2+
Absorbance of 1 (2.0 Â 10 mol/L) at 533 nm as a function of varying concentrations of Hg . (c) Picture of compound 1 (left) and compound 1 upon the addition of Hg
2+
À5
(
50 Â 10 mol/L) (right).
(
a) 4500
(
b) 4500
2+
Hg
3
2
1
375
250
125
0
3
2
1
375
250
125
0
1
5
60
600
640
680
Wa ve length (nm)
1
a
b
c
d
e
f
g
h
i
j
k
l
À5
2+
Fig. 3. (a) Fluorescence emission spectra of 1 (2.0 Â 10 mol/L) in DMSO–HEPES buffer (0.02 mol/L, pH 7.4, v/v = 6:4) with 30 equiv. of Hg ; (b) fluorescence intensity of 1
À5
2+
2+
2+
+
3+
3+
3+
2+
+
2+
2+
2+
(
2.0 Â 10 mol/L) at 556 nm after addition of 30 equiv. selected ions. (1: blank, a: Cd , b: Ni , c: Zn , d: Na , e: Fe , f: Cr , g: Al , h: Pb , i: Ag , j: Co , k: Cu , l: Hg ).
based on the method reported in the previous literature [6]. The
fluorescence intensity at 556 nm was plotted as a concentration of
concentration of 1, the corresponding concentration is EC50
(effective concentration). In this experiment, EC50 is used to
represent the cytotoxicity of compound 1 toward cells. As shown in
Table S1 in Supporting information, it proved that 1 is low toxic
and safe for the in vivo tests.
To demonstrate the feasibility of 1 for its application in in vivo
imaging, fluorescence imaging tests were performed in Spill 2 cells
and C. elegans. Previously, the author successfully designed a
pyrene-bearing probe to test and evaluate the toxicity levels of
heavy metal mercury in BEAS-2B cells, CHO cells, C. elegans, and
2+
–6
Hg , and detection limit was calculated to be 6.79 Â 10 L/mol by
using detection limit 3 /k: Where is the standard deviation of
blank measurement, k is the slope between the fluorescence
s
s
2+
intensity versus Hg concentration.
To investigate the cytotoxicity of compound 1, CCK8 tests were
performed in HaCaT cells, L6 cells, MA104 cells, RAEC cells, Ins-
1
cells, HepG2 cells, A549 cells and SKOV cells. When 50% of same
type experimental cells produce a particular response in specific
À5
2+
Fig. 4. (a) Fluorescence titration spectra of 1 (2.0 Â 0 mol/L) in the presence of varying concentrations of Hg in DMSO–HEPES buffer (0.02 mol/L, pH 7.4; v/v = 6:4).
À5
2+
Excitation wavelength was 528 nm. (b) Fluorescence intensity of 1 (2.0 Â 0 mol/L) at 556 nm as a function of varying concentrations of Hg . (c) Fluorescence picture of
2
+
À5
compound 1 (left) and compound 1 upon the addition of Hg (50 Â 10 mol/L) (right).
2
+
Please cite this article in press as: X.-M. Li, et al., A rhodamine derivative for Hg -selective colorimetric and fluorescent sensing and its
application to in vivo imaging, Chin. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.cclet.2016.04.001

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2
+
concentration of Hg reached 600
mmol/L (Fig. 6c). The above-
mentioned investigations confirmed that 1 could stain and sense
2
+
Hg in C. elegans.
4. Conclusion
In conclusion, a rhodamine derivative, 1, was synthesized as a
2
+
fluorescent sensor for Hg , with a binding process marked by a
distinct ‘‘off/on’’ fluorescent change. Hg - induced ring opening of
2
+
2+
Scheme 2. The proposed binding mechanism of compound 1 with Hg
.
the spirolactam moiety and generated a significant fluorescence
enhancement of up to 300-fold, with a bright yellow–green
emission. Compound 1 was a low toxic compound, and was
successfully applied in the in vivo imaging of Hg in Spill 2 cells
and C. elegans, which supported its further utility in environmental
zebrafish [7]. In this study, the application of 1 in in vivo imaging
2+
2+
was first evaluated by visualizing the distribution of Hg in cells
2+
incubated with different amount of Hg for 2 h. As shown in Fig. 5,
2+
with the dose of Hg was increased, fluorescence emission became
brighter and brighter. Green fluorescence emission colors were
mainly observed in the cell cytoplasm, whereas the cell nucleus
and tobacco samples.
Acknowledgments
2+
showed weak fluorescence. This suggests that the 1-Hg agent
showed a good bonding capacity for the cytoplasm and a weak
binding capacity for nucleic acids in the tested cells [8].
This work was supported by the fund of China Tobacco Yunnan
Industrial Co. (No. 2015JC05), the Foundation of the Department of
Science and Technology of Yunnan Province of China (Nos.
2013HB062, 2014HB008), and Training Project (No. XT412003)
of Yunnan University.
The application of 1 in in vivo imaging was evaluated by
2+
visualizing the distribution of Hg in nematodes previously
2+
incubated with various concentrations of Hg for 5 h. C. elegans
larvae at developmental stage 4 (L4) were then incubated in Petri
dishes filled with M9 buffer, which contained 1 (20
m
mol/L) at
Appendix A. Supplementary data
o
2
0 C for 1 h. Almost no fluorescence could be observed, when the
2+
concentration of Hg is around 300
mmol/L (Fig. 6b). Very weak
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.cclet.2016.04.001.
fluorescence was observed in the pretreated nematodes until the
References
[
1] (a) K. Bera, A.K. Das, M. Nag, S. Basak, Development of a rhodamine-rhodanine-
based fluorescent mercury sensor and its use to monitor real-time uptake and
distribution of inorganic mercury in live zebrafish larvae, Anal. Chem. 86 (2014)
2740–2746;
(b) Y. Zhu, H. Li, B. Shi, et al., A reversible fluorescent chemosensor for the rapid
detection of mercury ions (II) in water with high sensitivity and selectivity, RSC
Adv. 4 (2014) 61320–61323;
(
c) Y. Liu, E.B. Yang, R. Han, et al., A new rhodamine-based fluorescent chemo-
sensor for mercury in aqueous media, Chin. Chem. Lett. 25 (2014) 1065–1068;
d) Z. Wang, Z. Yang, T. Gao, et al., An Hg2+-selective chemosensor based on the
(
self-assembly of a novel amphiphilic block copolymer bearing rhodamine 6G
derivative moieties in purely aqueous media, Anal. Methods 7 (2015) 2738–2746.
2] (a) J.F. Zhang, Y. Zhou, J. Yoon, J.S. Kim, Recent progress in fluorescent and
colorimetric chemosensors for detection of precious metal ions (silver, gold and
platinum ions), Chem. Soc. Rev. 40 (2011) 3416–3429;
[
[
2
+
Fig. 5. Fluorescent imaging (top) and phase contrast (bottom) for Hg in Spill 2
À5
À5
2+
(b) Y. Zhou, J.F. Zhang, J. Yoon, Fluorescence and colorimetric chemosensors for
fluoride-ion detection, Chem. Rev. 114 (2014) 5511–5571;
cells. (a) Probe 1 (2.0 Â 10 mol/L) only. (b) Probe 1 (2.0 Â 10 mol/L) and Hg
À5
À5
2+
À5
(
30 Â 10 mol/L). (c) Probe 1 (2.0 Â 10 mol/L) and Hg (60 Â 10 mol/L).
(
c) N. Kumar, V. Bhalla, M. Kumar, Resonance energy transfer-based fluorescent
2+ 2+ 2+ 3+
probes for Hg , Cu and Fe /Fe ions, Analyst 139 (2014) 543–558.
3] (a) L. Huang, F. Hou, J. Cheng, et al., Selective off-on fluorescent chemosensor
for detection of Fe 3+ ions in aqueous media, Org. Biomol. Chem. 10 (2012)
9634–9638;
(b) N.R. Chereddy, K. Saranraj, A.K. Barui, et al., Donor atom selective coordination
of Fe3+ and Cr3+ trigger fluorophore specific emission in a rhodamine–naphtha-
limide dyad, RSC Adv. 4 (2014) 24324–24327.
[
[
[
[
[
4] (a) L.E. Guo, X.Y. Liu, H. Wang, et al., Iron(III)-selective chelation-enhanced
fluorescence sensing for in vivo imaging applications, Chem. Asian J. 10 (2015)
1
898–1902.
5] Z. Dong, X. Tian, Y. Chen, et al., A highly selective fluorescent chemosensor for Hg
based on rhodamine B and its application as a molecular logic gate, Dyes Pigm. 97
2013) 324–329.
2+
(
6] B. Zhu, C. Gao, Y. Zhao, et al., A 4-hydroxynaphthalimide-derived ratiometric
fluorescent chemodosimeter for imaging palladium in living cells, Chem. Commun.
4
7 (2011) 8656–8658.
7] G.K. Wang, Q.L. Mi, L.Y. Zhao, et al., A pyrene derivative for Hg
fluorescent sensing and its application in in vivo imaging, Chem. Asian J. 9
2014) 744–748.
2+
-selective
(
8] L.Y. Zhao, Q.L. Mi, G.K. Wang, et al., 1,8-Naphthalimide-based ‘turn-on’ fluorescent
sensor for the detection of zinc ion in aqueous media and its applications for
bioimaging, Tetrahedron Lett. 54 (2013) 3353–3358.
2
+
Fig. 6. Fluorescent imaging (top) and phase contrast (bottom) for Hg in C. elegans.
À5
À5
2+
(
a) with 2.0 Â 10 mol/L of 1 only; (b) with 30 Â 10 mol/L Hg for 5 h and
À5
À5
2+
2
2
.0 Â 10 mol/L of
1
for 1 h; (c) with 60 Â 10 mol/L Hg
for 5 h and
À5
.0 Â 10 mol/L of 1 for 1 h.
2
+
Please cite this article in press as: X.-M. Li, et al., A rhodamine derivative for Hg -selective colorimetric and fluorescent sensing and its
application to in vivo imaging, Chin. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.cclet.2016.04.001