A bright and specific fluorescent sensor for mercury in water, cells, and tissue

Article abstract of DOI:10.1002/anie.200701785

(Chemical Equation Presented) A bright idea: By restricting the rotation between receptor and reporter units of the fluorescent chemosensor Mercury Green 1 (MG1), a remarkably high quantum efficiency is achieved in its Hg ²⁺-ion-bound form in w

Full text of DOI:10.1002/anie.200701785

Communications
DOI: 10.1002/anie.200701785
Mercury Detection
A Bright and Specific Fluorescent Sensor for Mercury in Water, Cells,
and Tissue**
Sungho Yoon, Evan W. Miller, Qiwen He, Patrick H. Do, and Christopher J. Chang*
Mercury pollution pervades the globe and remains a danger
to human health and the environment. The US Environ-
mental Protection Agency (EPA)ꢀs estimates ofannual total
global mercury emissions from all sources—both natural and
human-generated—reach nearly 7500 tons per year.[1] Atmos-
pheric oxidation ofmercury vapor to water-soluble Hg 2+ ions
and its subsequent metabolism by aquatic microbes produces
methylmercury, a potent neurotoxin linked to many cognitive
and motion disorders.[2] Relevant cellular targets ofmethyl-
mercury include glutathione and other thiol-containing anti-
oxidants, but mechanistic details ofmercury toxicity at the
molecular level are still insufficiently understood.
date in water at physiological pH (F = 0.72), excellent
selectivity for Hg2+ ions over relevant competing metal ions,
a 44-fold turn-on response, and sensitivity to ppm–ppb levels
2+
ofHg
in complex aqueous solutions, cells, and tissue.
Furthermore, we show that because ofthese features, MG1 is
capable oftracking changes in mercury levels within living
cells and distinguishing safe and toxic amounts of mercury in
edible fish.
In a previous study we described Mercuryfluor-1 (MF1), a
fluorescein-like sensor for selective detection of aqueous
Hg2+ ions to ppm levels.[7b] This initial design is limited by the
relatively modest quantum yield for MF1 in its Hg2+-ion-
bound form (F = 0.16), which is in part due to the propensity
ofheavy metals, such as mercury, to quench fluorescent dyes
Fluorescent small-molecule sensors[3] and dosimeters,[4]
materials,[5] and biomolecules[6] offer an attractive approach
to tracing neurotoxic mercury. However,
despite widespread interest and recent
advances in this area, there are only a few
examples of fluorescent probes for successful
2+
detection ofHg
ions in biological sam-
ples,[7] as creating new synthetic molecules
that meet the criteria ofappropriate selec-
tivity, water solubility, and optical sensitivity
in natural settings remains an outstanding
challenge. In particular, a common limitation
for heavy-metal detection in the environ-
ment is the low quantum efficiency of metal-
bound dyes in water compared to that in
organic or in mixed aqueous-organic sol-
vents. We now report the synthesis and
applications ofMercury Green 1 (MG1), a
new Hg2+-ion-specific fluorescent chemosen-
sor that has the highest quantum efficiency to
Scheme 1. Synthesis of MG1. Ts=tosyl, TBDMS=tert-butyldimethylsilyl.
in aqueous media. Seeking to maintain high Hg2+-ion
specificity while increasing optical brightness values in
water, we reasoned that introducing an ortho-methyl group
on the phenylene linker would restrict its rotation with
respect to the xanthene reporter unit and potentially enhance
quantum efficiencies, an approach inspired by Tokyo Green
reporters of b-galactosidase activity.[8] Based on this restricted
rotation design, the new Hg2+-ion-responsive dye MG1 was
prepared as shown in Scheme 1.
[*] Dr. S. Yoon, E. W. Miller, Dr. Q. He, P. H. Do, Prof. C. J. Chang
Department of Chemistry
University of California, Berkeley
Berkeley, CA 94720-1460 (USA)
Fax: (+1)510-642-7301
E-mail: chrischang@berkeley.edu
Homepage: http://www.cchem.berkeley.edu/cjcgrp/
[**] This work was supported by the University of California, Berkeley,
the National Science Foundation (CAREER CHE-0548245), and the
Dreyfus, Beckman, Packard, and Sloan Foundations. E.W.M. was
supported by a Chemical Biology Interface Training Grant from the
NIH (T32 GM066698) and a Stauffer fellowship. We thank David
Crane and Martice Vasquez (California Dept. of Fish and Game) and
Richard Bauer (US EPA) for providing fish samples, and James
Sanborn (California Office of Environmental Health Hazard
Assessment) for helpful discussion.
Spectroscopic measurements for MG1 were performed in
aqueous solution buffered to pH 7 (20 mm 2-[4-(2-hydroxy-
ethyl)-1-piperazinyl]ethanesulfonic acid, HEPES) and they
establish the marked benefits of restricting rotation between
receptor and reporter units. Figure 1a displays the fluores-
cence turn-on response ofthe sensor to Hg 2+ ions. Metal-free
(apo) MG1 has a single visible absorption maximum at
485 nm (e = 2.9 ” 104 mÀ1 cmÀ1) and is weakly fluorescent
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
2+
(lem = 514 nm, F = 0.02). Addition ofHg
ions triggers a
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Figure 1b gives the relative responses ofMG1 and MF1 to
various aqueous Hg2+-ion levels in the ppm–ppb range and
shows that restricting rotation between reporter and receptor
units dramatically increases the absolute change in fluores-
cence turn-on upon Hg2+-ion detection. For example, at
10 ppb Hg2+ ion, MG1 is over 11 times more responsive than
its unrestricted congener MF1. Owing to its improved optical
brightness, MG1 is sensitive enough to detect environmen-
tally relevant concentrations ofHg 2+ ions in aqueous solution.
2+
Addition of2 ppb ofHg
ions, the maximum US EPA limit
for allowable levels of Hg2+ ions in drinking water, to a 1-mm
solution of MG1 affords a (72 Æ 10)% emission increase. In
contrast, the unrestrained analogue MF1 is not capable of
2+
detecting 2 ppb levels ofHg ions under similar conditions.
Finally, binding studies, using the method ofcontinuous
variations, point to a 1:1 Hg2+/MG1 complex being respon-
sible for the observed fluorescence turn-on with Kd = (895 Æ
85) nm.
[9]
Because ofits thioether-rich receptor,
MG1 retains
excellent selectivity for Hg2+ ions in the presence ofa range of
competing metal ions found in environmental and biological
settings (Figure 1c). MG1 exhibits excellent selectivity for
Hg2+ ions over alkali and alkaline-earth cations, including
millimolar concentrations ofLi +, Na+, K+, Mg2+, and Ca2+,
first-row transition-metal ions Mn2+, Fe2+, Fe3+, Co2+, Ni2+,
Cu+, and Cu2+ added at 300- to 500-fold excess, Group 12
metal ions Zn2+ and Cd2+, and the common heavy-metal
contaminant Pb2+. Ofnote are the observed selectivities of r
Hg2+ over Cu2+ and Pb2+, which are major competitors in real-
life field samples.
We explored opportunities for MG1 in environmental and
biological applications. Initial studies tested the ability ofthis
bright probe to track changes in mercury levels in living cells.
Live-cell imaging experiments utilized the acetoxymethyl
ester protected form of MG1 (MG1-AM) to enhance
membrane permeability. Confocal microscopy images of live
HEK 293T cells loaded with 1 mm ofMG1-AM ofr up to
60 min at 378C show relatively low levels ofbackground
intracellular fluorescence (Figure 2a), consistent with the
photoinduced-electron-transfer-quenched form of the apo
dye. In contrast, MG1-labeled cells exposed to 4 ppm ofHg 2+
for 30 min at 378C show increased intracellular fluorescence
compared to cells not treated with mercuric salts (Figure 2b).
Treatment with the heavy-metal chelator TPEN (N,N,N’,N’-
tetrakis(2-picolinyl)ethylenediamine) for 1 min at 258C
reduces the observed fluorescence enhancements (Figure 2c).
Brightfield measurements and Hoescht-33342 staining con-
firm that the cells are viable throughout the imaging studies
(Figure 2d and Supporting Information), and control experi-
ments on cells without dye give no fluorescence over back-
ground levels. These data establish that MG1 can respond
reversibly to increases or decreases in intracellular mercury
levels within living cells at ppm levels.
Figure 1. a) Fluorescence response of 1 mm MG1 to Hg2+ ions in
aqueous solution. Spectra shown are for Hg2+-ion concentrations of 0,
0.05, 0.1, 0.15, 0.2, 0.3, 0.4, 0.5, 0.6, 0.8, 1.0, 1.2, 1.8, 2.0, 3.2, 4.0, and
6.7 mm. The arrow indicates change with increasing Hg2+-ion concen-
tration. Spectra were acquired in 20 mm HEPES (pH 7) with excitation
at 480 nm. b) Relative responses of MG1 (black bars) and its unre-
stricted analogue MF1 (white bars) to ppb levels of Hg2+ ions. Data
were acquired under similar conditions to obtain an accurate reflection
of the relative brightness of the dyes in response to Hg2+ ions (1 mm
dye, 20 mm HEPES, pH 7). Excitation was provided at 495 nm, and the
emission was integrated over 505 to 700 nm. c) Fluorescence
responses of MG1 to various metal ions. Bars represent the final
integrated fluorescence response (If) over the initial integrated emis-
sion (Ii). Initial spectra were acquired in 20 mm HEPES, pH 7. White
bars represent the addition of an excess of the appropriate metal ion
(5 mm for Li+, Na+, K+, Mg2+, and Ca2+, 300 mm for Fe2+, Fe3+, and
Cu+, and 500 mm for all other cations) to a 1 mm solution of MG1.
Black bars represent the addition of 5 or 50 mm Hg2+ to solutions
containing MG1. Excitation was provided at 495 nm, and the emission
was integrated over 505 to 700 nm. 1) Hg2+; 2) Li+; 3) Na+; 4) K+;
5) Mg2+; 6) Ca2+; 7) Sr2+; 8) Mn2+; 9) Fe2+; 10) Fe3+; 11) Co2+; 12) Ni2+
13) Cu+; 14) Cu2+; 15) Zn2+; 16) Cd2+; 17) Pb2+
;
.
44-fold increase in integrated emission for MG1 with slight
shifts in excitation (494 nm, e = 4.8 ” 104 mÀ1 cmÀ1) and emis-
sion (513 nm) maxima. The quantum yield F = 0.72 for Hg2+-
We also investigated MG1 for Hg2+-ion detection in
complex tissue. Owing to widespread interest in determining
mercury contamination in edible fish, we applied MG1 to
analyze fish mercury levels. Experiments utilized a series of
fish collected from freshwater sources in greater Northern
California. Small tissue samples (< 100 mg) with mercury
bound MG1 is the highest reported value to date for a Hg2+
-
ion sensor in water. For comparison, the unrestricted ana-
logue MF1 has a Hg2+-bound quantum yield of0.16.
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Communications
In conclusion, we have described the synthesis and
properties of MG1, a bright and specific fluorescent sensor
for Hg2+ ions in water, cells, and tissue. MG1 features the
highest quantum efficiency to date for a Hg2+-bound sensor in
water (F = 0.72), excellent Hg2+-ion specificity, a 44-fold
turn-on response, and sensitivity to environmentally and
biologically relevant mercury levels in the ppm to ppb range
in complex natural samples. Additional experiments establish
the utility ofthis chemosensor of r tracking mercuric ions in
living cells as well as assaying safe and toxic levels of Hg2+
ions in fish according to US EPA standards. Because a
common limitation ofapplying small-molecule lfuorophores
to natural samples in the laboratory and the field is low
optical brightness in aqueous media, we anticipate that this
restricted-rotation approach should find utility in the future
design of metal-ion sensors with high quantum efficiencies in
water for a variety of chemical and biological applications.
Experimental Section
Experimental Details are provided in the Supporting Information
Figure 2. Confocal fluorescence imaging of Hg2+-ion levels using MG1
in live HEK 293T cells. a) Fluorescence image of cells incubated with
1 mm MG1-AM for 60 min at 378C. b) Fluorescence image of MG1-
loaded cells exposed to 4 ppm of HgCl2 for 30 min at 378C. c) Fluores-
cence image of cells in panel (b) treated with 2 mm of the competing
heavy-metal chelator TPEN for an additional 1 min at 258C. d) Bright-
field image of cells in panel (c), confirming their viability. Scale
bar=40 mm.
Received: April 23, 2007
Published online: July 30, 2007
Keywords: fluorescein · fluorescence · mercury · sensors
.
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