A new fluorescent chemodosimeter for Hg2+: Selectivity, sensitivity, and resistance to Cys and GSH

Article abstract of DOI:10.1021/ol902590g

[Chemical equation presented] On the basis of the mechanism of Hg ²⁺-promoted hydrolysis, a new fluorescent chemodosimeter (Rho-Hg1) is reported for single-selective and parts per billion level-sensitive detection of Hg²⁺ in natural

Full text of DOI:10.1021/ol902590g

ORGANIC
LETTERS
2010
Vol. 12, No. 3
476-479
A New Fluorescent Chemodosimeter for
Hg2+: Selectivity, Sensitivity, and
Resistance to Cys and GSH
Jianjun Du,† Jiangli Fan,† Xiaojun Peng,*,† Pingping Sun,‡ Jingyun Wang,*,‡
Honglin Li,† and Shiguo Sun†
State Key Laboratory of Fine Chemicals, Dalian UniVersity of Technology, 158 Zhongshan
Road, Dalian 116012, P.R. China, and Department of Bioscience and Biotechnology,
Dalian UniVersity of Technology, 2 Linggong Road, Dalian 116024, P.R. China
pengxj@dlut.edu.cn
Received November 20, 2009
ABSTRACT
On the basis of the mechanism of Hg2+-promoted hydrolysis, a new fluorescent chemodosimeter (Rho-Hg1) is reported for single-selective
and parts per billion level-sensitive detection of Hg2+ in natural waters. Moreover, the fluorescence response of Rho-Hg1 to Hg2+ has little
interference from sulfur compounds such as cysteine and glutathione and could be used in the Hg2+ imaging in living cells.
Up to the present, many good fluorescent probes for
multifarious targets have been described.1 Recently, as the
demand of sensitivity and high selectivity is continuously
increasing, some fascinating chemodosimeters have been
designed to detect analyte utilizing chemical events. They
have played significant roles in the detection of ion and small
molecules, especially in monitoring heavy and transition
metal ions.2 Mercury is one of the most toxic and dangerous
heavy metal elements because of its high affinity for thiol
groups in proteins and enzymes, leading to the dysfunction
of cells and consequently causing many health problems in
the brain, kidney, and central nervous system.3 Thus,
concerns over toxic damage of mercury provide motivation
to explore selective reactions to monitor Hg2+ in biological
and environmental samples.
At present, most of the reported chemodosimeters for Hg2+
are based on thiophilic specialty.4 However, mercury is
abundant in the sulfur-rich environments.5 Unfortunately, few
literatures discuss how to avoid the potential interference from
mercapto-chemicals in organism and sulfur-rich environments
† State Key Laboratory of Fine Chemicals.
‡ Department of Bioscience and Biotechnology.
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10.1021/ol902590g  2010 American Chemical Society
Published on Web 01/12/2010

in the detection of Hg2+ until the recent report from Koide who
developed a chemodosimeter based on Hg-catalyzed hydration
of alkynes into ketones at 90 °C.6 The high temperature,
however, could not be used in thermosensitive systems such
as living cells and other biosamples. Therefore, mild reactions
for selective and sensitive Hg2+-signaling without interference
from organo-sulfide are very important.
Scheme 2
.
Hg2+-Promoted Ring Opening and Hydrolysis of
Rho-Hg1
It is known that Hg2+ promoted an irreversible hydrolysis
of isopropenyl acetate in mild conditions7 (Scheme 1), and
Scheme 1
.
Mercury-Promoted Hydrolysis of Isopropenyl
Acetate
synthesized from Rhodamine B by a three-step procedure
in a total yield of 45.6% (Scheme S1 in Supporting
Information (SI)). We anticipated that it would undergo
hydrolysis reaction when a similar but modified molecular
moiety of isopropenyl acetate was liberated by Hg2+-
facilitated ring opening of the spirocycle group (Scheme 2).
Here, Hg2+ acts not only as an analyte but also as the
promoter for the hydrolytic reaction. Compared with other
Hg2+ fluorescent probes, Rho-Hg1 shows two advantages:
first, Rho-Hg1 has single selectivity and parts per billion (ppb)
level sensitivity for Hg2+ detection in natural waters at room
temperature; second, Rho-Hg1 realizes the avoidance of
potential interference from sulfide such as cysteine and glu-
tathione in the detection of Hg2+ and then is successfully applied
in the fluorescence imaging of Hg2+ in living cells.
rhodamines undergo a great fluorescence enhancement via
their structure change from spirocyclic (nonfluorescent and
colorless) to ring-open (fluorescent and colorated) states
induced by specific chemical environments at room temper-
ature.8 We envisioned that combining the spirolactam ring
opening of rhodamine derivatives with the mild Hg2+-
promoted hydrolysis reaction of isopropenyl acetate would
serve as the fundamental reactions for a novel chemodosim-
eter for Hg2+.
Herein, a new rhodamine-based fluorescent Hg2+ chemo-
dosimeter Rho-Hg1 is presented, which has a similar
structure with isopropenyl acetate (Scheme 2). It was facilely
To explore the mechanism, Rho-Hg1/Hg2+ complexes
were detected by the ESI-TOF high resolving mass spectrum
analysis. As shown in Scheme 2, a peak at m/z 788.3576
corresponding to [Rho-Hg1 + Hg2+ - H+]+ was observed
after the addition of Hg(ClO4)2 to the ethanol solution of
Rho-Hg1 (Figure S2, SI). At the same time, the complex
exhibited obvious fluorescence emission at 580 nm and color
of amaranth with absorption at 554 (Table S1 and Figure
S1, SI). After addition of water (ethanol/water, 1/1, v/v), the
peak at m/z 788.3576 disappeared, and then a new peak of
m/z 443.2709 appeared which was confirmed as Rhodamin
B (Figure S2, SI). In the absence of Hg2+, Rho-Hg1 is
colorless and nonfluorescent due to the closed spirolactam
ring. In the presence of Hg2+, the complex of Rho-Hg1 -
Hg2+ forms in the first equilibrium resulting in ring opening,
and then the complex further hydrolyzed to brilliant pink
and strong fluorescent Rhodamine B in the presence of water.
An ethanol/water (1/1, v/v, pH 7.0) solution was selected
as a testing system to investigate the chemical response of
Rho-Hg1 to Hg2+ at room temperature. A time course study
revealed the recognizing event could complete in 8 min
(Figure S3, SI).
One challenge for the Hg2+ probe is to obtain systems
that are selective to Hg2+ over a wide range of potentially
competing ions, such as alkali or alkaline-earth metals (Na+,
K+, Mg2+, and Ca2+) and heavy and transition metal ions
(Co2+, Cr3+, Ni2+, Zn2+, Cd2+, Pb2+, Ag+, Fe3+, and Cu2+).
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Org. Lett., Vol. 12, No. 3, 2010
477

The Rho-Hg1 exhibited no obvious fluorescence and absorp-
tion because it mostly exists in the spirocyclic form. Only
after addition of Hg2+, the intensity of fluorescence emission
was significantly enhanced by over 370-fold at 579 nm with
a quantum yield of 0.75 (Figure 1a,b), and also, an absorption
of Rho-Hg1 in the absence and presence of Hg2+ in different
pH values were evaluated (Figure 2). The pKa of Rho-Hg1
Figure 2. Effect of pH on the fluorescence intensity of Rho-Hg1 (5
µM) and Rho-Hg1/Hg2+ (5 µM/75 µM) in the aqueous solution
(ethanol/water ) 1/1, v/v, pH 7.0). Excitation wavelength is 510 nm.
alone is 3.68 from the sigmoidal curve. In the pH range from
5.0 to 8.0, Rho-Hg1 can respond to Hg2+ without any
interference by protons (Figure 2 inset).
Figure 1. (a) Emission spectra of Rho-Hg1 (5 µM) in the presence
of increasing concentrations of Hg2+ (0, 15, 25, 30, 35, 40, 45, 50,
55, 60, 65, 70, 75, 80 µM) in aqueous solution (ethanol/water )
1/1, v/v). (b) The fluorescence intensity at 580 nm of Rho-Hg1 (5
µM each) in the presence of different metal ions (Hg2+ is 75 µM
Generally, one of the most important and useful applica-
tions for a fluorescent probe is the detection of Hg2+ in
natural water samples. Owing to its excellent spectroscopic
properties, Rho-Hg1 is sensitive enough to detect relevant
concentrations of Hg2+ in natural water samples. We chose
natural water samples from three different sources: seawater
from Yellow Sea (Dalian), pool water, and tap water (in
campus). The U.S. EPA standard for the limit of inorganic
Hg in drinking water is 2 ppb. As shown in Figure 3 and
and other metal ions are 250 µM: Ca2+, K+, Mg2+, Na+, Cd2+
,
Cu2+, Ni2+, Pb2+, Zn2+, Co2+, Ag+, Cr3+, and Fe3+). Inset: the
changes of fluorescence intensity of Rho-Hg1 (5 µM) upon addition
of Hg2+ (1-5 ppb). (c) The absorption spectra of Rho-Hg1 (5 µM)
in the absence and presence of different metal ions (mentioned
above). (d) Color (up) and fluorescence (down) change (from left
to right: Rho-Hg1 only; Rho-Hg1 + Hg2+; Rho-Hg1 + Hg2+
+
X; Rho-Hg1 + X; Rhodamine B + Hg2+; X denotes miscellaneous
-
cations mentioned above and anions including NO3-, SO4-, ClO4
,
CO32-, Cl-, H2PO4-, SCN-, and CH3COO-).
band centered at 554 nm appeared obviously (Figure 1c).
The competition experiments revealed that the Hg2+-induced
fluorescence and absorption response were unaffected in the
background of metal ions mentioned above and the anions
-
-
-
including NO3 , SO42-, ClO4 , CO32-, Cl-, H2PO4 , SCN-,
and CH3COO- (Figure 1d).
The U.S. EPA (Environmental Protection Agency) stan-
dard for the maximum allowable level of inorganic Hg in
drinking water is no more than 2 ppb.9 When Rho-Hg1 was
added to a solution containing 2 ppb of Hg2+, fluorescence
enhanced nearly 400%, and the F/F0 was proportional to the
amount of Hg2+ added in ppb level with a detection limit of
0.91 ppb10 (Figure 1b inset).
Normally, the spirolactam ring of the rhodamine derivative
is open in acidic media and then shows the fluorescence of
rhodamine. To apply Rho-Hg1 in more complicated systems,
like in organism and environment, the fluorescence responses
Figure 3. Linear fluorescence enhancement (F/F0) of Rho-Hg1 (1
µM) upon addition of Hg2+ (1-5 ppb) to seawater. The response
(F) is normalized to the emission of the free Rho-Hg1 (F0). The
samples were excited at 510 nm, and the emission intensities were
recorded at 580 nm.
Figures S4 and 5 (SI), about 2.8-fold, 1.7-fold, and 1.5-fold
enhancement of fluorescence intensity were displayed when
2 ppb of Hg2+ was added into those waters with Rho-Hg1.
Furthermore, the F/F0 is well proportional to the amount of
Hg2+ (1-5 ppb). The result shows that Rho-Hg1 is capable
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1996, 68, 1414–1418.
478
Org. Lett., Vol. 12, No. 3, 2010

of distinguishing between the safe and toxic levels of Hg2+
in more complicated natural water systems.
It is known that there are some important mercapto
biomolecules in organisms, such as cysteine, homocysteine,
and glutathione,11which could interact with Hg2+. Therefore,
for more practical application, we investigated the effect of
cysteine and glutathione on the detection of Hg2+ in ethanol/
HEPES buffer solutions (1/1, v/v, pH 7.0, 50 mM). As shown
in Figure 4, the addition of cysteine (100 µM) could induce
Figure 5. Fluorescence image of Hela cells incubated with 10 µM
Rho-Hg1 for 30 min (a) and then further incubated with 10 µM
Hg2+ for 30 min (b). (c) Bright-field transmission image of cells in
(b). The excited light is WB510-570 nm (Nikon eclipase TE 2000-
5, 32 × objective lens).
After three times washing with PBS buffer, the cells were
incubated with Hg2+ (10 µM) in the medium for another 30
min at 37 °C, and the fluorescence in living cells was much
brighter (Figure 5b). A bright-field transmission image of cells
treated with Rho-Hg1 and Hg2+confirmed that the cells were
viable throughout the imaging experiments (Figure 5c). It is
proved that Rho-Hg1 is cell-permeable and primarily nontoxic
to the cell culture.
In summary, a highly selective chemodosimer Rho-Hg1
for Hg2+ based on hydrolysis was reported. It exhibits ultra
sensitivity for Hg2+ detection in natural waters, even in the
presence of cysteine and glutathione. Moreover, fluorescence
imaging for Hg2+ in living cells shows Rho-Hg1 has ideal
chemical and spectroscopic properties that satisfy the criteria
for further biological applications.
Figure 4.
Hg2+ (2 µM) detection in the presence of L-cysteine and
glutathione (100 µM) in ethanol/HEPES buffer (1/1, v/v, pH 7.0,
50 mM), [Rho-Hg1] ) 0.3 µM. Excitation wavelength is 510 nm.
little fluorescence change (red bar) compared with Rho-Hg1
(black bar); when Hg2+ (2 µM) is added into the mixed
solution of Rho-Hg1 (0.3 µM) and cysteine (100 µM), the
obvious fluorescence enhancement (green bar) was observed,
which is very close to the enhancement induced by Hg2+
only (blue bar). There is a similar result in the glutathione
testing. Therefore, Rho-Hg1 could detect Hg2+ in the sulfur-
rich environment insusceptibly.
Acknowledgment. This work was supported by NSF of
China (20725621, 20706008, and 20876024), National Basic
Research Program of China (2009CB724706), and Ministry
of Education of China (Program for Changjiang Scholars
and Innovative Research Team in University, IRT0711; and
Cultivation Fund of the Key Scientific and Technical
Innovation Project, 707016).
Cultured Hela cells were incubated with Rho-Hg1 in culture
medium for 30 min at 37 °C, and very weak fluorescence of
Rho-Hg1 inside the living Hela cells was observed (Figure 5a).
Supporting Information Available: Experimental pro-
cedure, structural data, spectral property in ethanol, time
course, TOF-MS data, and the testings for Hg2+ in pool and
tap water are provided. This material is available free of
charge via the Internet at http://pubs.acs.org.
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