Screening mercury levels in fish with a selective fluorescent chemosensor

Article abstract of DOI:10.1021/ja0557987

Societal concerns over toxic mercury accumulation in humans from fish and other dietary and environmental sources provide motivation to develop new tools and tactics for mercury detection in a wide range of laboratory and field settings. Here we report th

Full text of DOI:10.1021/ja0557987

Published on Web 10/28/2005
Screening Mercury Levels in Fish with a Selective Fluorescent Chemosensor
Sungho Yoon, Aaron E. Albers, Audrey P. Wong, and Christopher J. Chang*
Department of Chemistry, UniVersity of California, Berkeley, California 94720
Received August 30, 2005; E-mail: chrischang@berkeley.edu
Mercury is a dangerous and widespread global pollutant.1 The
Scheme 1. Synthesis of Mercuryfluor-1 (MF1)
long atmospheric residence time of Hg0 vapor and its oxidation to
soluble inorganic Hg2+ provides a pathway for contaminating vast
amounts of water and soil.2 A significant problem stemming from
this ecological oxidation chemistry is that bacteria living in the
sediments of aqueous environments transform inorganic Hg2+ into
methylmercury, a potent neurotoxin that concentrates through the
food chain in the tissues of fish and marine mammals. Subsequent
ingestion of methylmercury by humans from seafood and other
dietary and environmental sources is connected to serious sensory,
motor, and cognitive disorders.3
Concerns over toxic exposure to mercury provide motivation to
explore new methods for monitoring aqueous Hg2+ from biological
Spectroscopic measurements under simulated physiological
and environmental samples. Current techniques for mercury screen-
conditions (20 mM HEPES buffer, pH 7) reveal that the optical
ing, including atomic absorption/emission spectroscopy4 and in-
properties of MF1 are dominated by the fluorescein chromophore.
ductively coupled plasma mass spectrometry,5 often require ex-
pensive and sophisticated instrumentation and/or sample preparation.
Fluorescence detection with Hg2+-responsive chemosensors offers
a promising approach for simple and rapid tracking of mercury
ions for biological, toxicological, and environmental monitoring.
An important practical challenge to achieving this goal is devising
water-soluble fluorescent dyes that report Hg2+ selectively over
competing metal ion contaminants. Several types of small
molecules,6-18 DNAzymes,19 and protein20 or oligonucleotide21
platforms have been examined for fluorescence Hg2+ detection.
However, none of these probes have been utilized successfully for
sensing Hg2+ in natural samples, as available Hg2+-responsive
fluorophores are often limited by nonspecific interference from
Cu2+, Pb2+, and other competing metal ions, incompatibility with
aqueous media, and/or delayed or irreversible Hg2+ response. We
now present the synthesis and properties of Mercuryfluor-1 (MF1),
a new water-soluble fluorescent chemosensor for screening mercury
levels in fish. This reagent features excellent selectivity for Hg2+
over competing analytes, including common metal ion contaminants
Cu2+ and Pb2+, and the largest fluorescence enhancement to date
for sensing Hg2+ in water (>170-fold). Experiments with fish
collected from field studies show that MF1 is capable of reliably
detecting mercury levels in fish over a range of 0.1 to 8 ppm,
establishing the utility of this probe for assaying fish for safe human
consumption according to guidelines suggested by the U.S. EPA.1
MF1 combines a fluorescein reporter22 having desirable optical
properties and water solubility with a thioether-rich crown receptor
to favor selective and stable binding of soft Hg2+ in water.23 Scheme
1 outlines the synthesis of MF1. Reaction of 4-bromo-N,N-bis(2-
hydroxyethyl)aniline 1 and p-toluenesulfonyl chloride affords
ditosylate 2 in 71% yield. Conversion of 2 with sodium iodide
proceeds smoothly to generate the corresponding diiodo compound
3 in 75% yield. Cyclization of diiodo 3 and 3,6-dithiaoctane-1,8-
dithiol 4 with Cs2CO3 under high-dilution conditions produces the
azathiacrown receptor 5 in low yield (9%). Lithium-mediated
coupling24 of macrocycle 5 and 3,6-bis[[(1,1-dimethylethyl)di-
methylsilyl]oxy]-9H-xanthen-9-one 6 furnishes MF1 7 in 37% yield.
In the absence of Hg2+, MF1 has a visible absorption band centered
at 485 nm (ꢀ ) 2.0 × 104 M-1 cm-1) with a corresponding emission
maximum at 514 nm. MF1 is virtually nonfluorescent in its apo
state (Φ < 0.001), indicative of efficient photoinduced electron
transfer (PET) quenching of the fluorophore by the azathiacrown
receptor. Upon addition of Hg2+, the fluorescence intensity of MF1
increases by over 170-fold (Φ ) 0.16, Figure 1A). This dramatic
turn-on response is accompanied by red shifts in both excitation
(495 nm, ꢀ ) 4.9 × 104 M-1 cm-1) and emission (517 nm) maxima.
Binding analysis using the method of continuous variations
establishes that a 1:1 Hg2+:MF1 complex is responsible for the
observed fluorescence enhancement, and the EC50 for 1 µM MF1
is 700 nM. Using 1% of the total dynamic range as a cutoff (1.7-
fold fluorescence increase), a 60 nM detection limit for aqueous
Hg2+ is obtained for MF1.
MF1 is highly selective for Hg2+ over competing metal ion
analytes in aqueous solution. Figure 1B depicts the fluorescence
responses of a 1 µM solution of MF1 to the presence of various
environmentally relevant metal ions. The emission profiles of MF1
or its Hg2+-bound form are unperturbed by millimolar concentra-
tions of Li+, Na+, K+, Mg2+, and Ca2+, indicating excellent
selectivity over these alkali and alkaline earth cations. MF1 is also
selective for Hg2+ over first-row transition metal ions Mn2+, Fe2+
,
Co2+, Ni2+, and Cu2+ at 67-fold excess. The observed selectivity
of MF1 for Hg2+ over Cu2+ is notable and reveals a greater affinity
of the NS4 macrocycle for the former.23 In addition, MF1 is also
selective for Hg2+ over group 12 ions Zn2+ and Cd2+, as well as
the common heavy metal ion pollutant Pb2+
.
With a firm understanding of the spectroscopic properties and
Hg2+ responses of MF1 in hand, the fluorescein-based probe was
applied to provide a rapid screen for total mercury content in fish.
Assays employed fish collected from field studies whose mercury
content was verified by atomic absorption spectroscopy. Tissue
samples (100-200 mg) were subjected to microwave digestion in
nitric acid, and the resulting solutions were directly basified, brought
to pH 7 in 20 mM HEPES buffer, and analyzed with MF1. Overall
sample processing takes less than 15 min, and the assay is amenable
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C O M M U N I C A T I O N S
selectivity for Hg2+ over competing environmentally relevant metal
ions and the largest turn-on response to date for detecting this ion.
Furthermore, MF1 is capable of measuring mercury levels in fish
well within the safe edible limit. While demonstrated in fish, this
method provides a useful starting point for developing new mercury
contamination screens for a wide range of biological, toxicological,
and environmental samples. The combined gains in selectivity and
dynamic range presage many opportunities for MF1 and related
Hg2+ chemosensors in laboratory and field applications.
Acknowledgment. We thank Richard Bauer (U.S. EPA), David
Crane and Martice Vasquez (California Department of Fish and
Game), and James Sanborn (California Office of Environmental
Health Hazard Assessment) for providing fish samples. The
University of California, the Camille and Henry Dreyfus Founda-
tion, and the Arnold and Mabel Beckman Foundation generously
supported this work. A.E.A. is a trainee of the Chemical Biology
Graduate Program (T32 GM066698) and acknowledges a Berkeley
Chancellor’s Opportunity Fellowship. C.J.C. thanks Prof. Stephen
Lippard and Ms. Elizabeth Nolan for many stimulating intellectual
and practical discussions that inspired this research.
Supporting Information Available: Synthetic and experimental
details (PDF). This material is available free of charge via the Internet
at http://pubs.acs.org.
References
(1) U.S. EPA, Regulatory Impact Analysis of the Clean Air Mercury Rule:
EPA-452/R-05-003, 2005.
Figure 1. (A) Fluorescence response of 1 µM MF1 to Hg2+ in aqueous
solution. Spectra shown are for Hg2+ concentrations of 0, 0.1, 0.2, 0.3, 0.4,
0.5, 0.6, 0.8, 1.0, 1.2, 1.6, and 2.0 µM. Spectra were acquired in 20 mM
HEPES (pH 7) with excitation at 480 nm. (B) Fluorescence responses of
MF1 to various metal ions. Bars represent the final integrated fluorescence
response (Ff) over the initial integrated emission (Fi). Initial spectra were
acquired in 20 mM HEPES, pH 7. White bars represent the addition of an
excess of the appropriate metal ion (1 mM for Li+, Na+, K+, Mg2+, and
Ca2+, 67 µM for all other cations) to a 1 µM solution of MF1. Gray bars
represent the addition of 6.7 µM Hg2+ to the solution. Excitation was
provided at 495 nm, and the emission was integrated over 505-700 nm. 1.
(2) Boening, D. W. Chemosphere 2000, 40, 1335-1351.
(3) Clarkson, T. W.; Magos, L.; Myers, G. J. N. Engl. J. Med. 2003, 349,
1731-1737.
(4) Bloom, N.; Fitzgerald, W. F. Anal. Chim. Acta 1988, 208, 151-161.
(5) Moreton, J. A.; Delves, H. T. J. Anal. Atom. Spectrosc. 1998, 13, 659-
665.
(6) Chae, M. Y.; Czarnik, A. W. J. Am. Chem. Soc. 1992, 114, 9704-9705.
(7) Hennrich, G.; Sonnenschein, H.; Resch-Genger, U. J. Am. Chem. Soc.
1999, 121, 5073-5074.
(8) Rurack, K.; Kollmannsberger, M.; Resch-Genger, U.; Daub, J. J. Am.
Chem. Soc. 2000, 122, 968-969.
(9) Prodi, L.; Bargossi, C.; Montalti, M.; Zaccheroni, N.; Su, N.; Bradshaw,
J. S.; Izatt, R. M.; Savage, P. B. J. Am. Chem. Soc. 2000, 122, 6769-
6770.
Hg2+; 2. Li+; 3. Na+; 4. K+; 5. Mg2+; 6. Ca2+; 7. Sr2+; 8. Mn2+; 9. Fe2+
;
10. Co2+; 11. Ni2+; 12. Cu2+; 13. Zn2+; 14. Cd2+; 15. Pb2+. (C) Fluorometric
analysis of mercury in fish using MF1. Fish were taken from California
waters and digested with microwave irradiation, and emission responses
were calibrated versus independent measurement of mercury content by
atomic absorption spectroscopy: Lime Saddle Marina (bluegill, 0.1 ppm
Hg), Calero Resevoir (bass, 1.0 ppm Hg), Lake Almaden (bass, 2.5 ppm
Hg), Almaden Resevoir (bass, 5.5 ppm Hg), Guadelupe Resevoir (bass,
7.5 ppm Hg). Excitation was provided at 495 nm, and the emission was
integrated over 505-700 nm.
(10) Sakamoto, H.; Ishikawa, J.; Nakao, S.; Wada, H. Chem. Commun. 2000,
2395-2396.
(11) Al Shihadeh, Y.; Benito, A.; Lloris, J. M.; Martinez-Manez, R.; Pardo,
T.; Soto, J.; Marcos, M. D. Dalton Trans. 2000, 1199-1205.
(12) Descalzo, A. B.; Martinez-Manez, R.; Radeglia, R.; Rurack, K.; Soto, J.
J. Am. Chem. Soc. 2003, 125, 3418-3419.
(13) Nolan, E. M.; Lippard, S. J. J. Am. Chem. Soc. 2003, 125, 14270-14271.
(14) Guo, X.; Qian, X.; Jia, L. J. Am. Chem. Soc. 2004, 126, 2272-2273.
(15) Ros-Lis, J. V.; Marcos, M. D.; Martinez-Manez, R.; Rurack, K.; Soto, J.
Angew. Chem., Int. Ed. 2005, 44, 4405-4407.
(16) Liu, B.; Tian, H. Chem. Commun. 2005, 3156-3158.
(17) Mello, J. V.; Finney, N. S. J. Am. Chem. Soc. 2005, 127, 10124-10125.
(18) Nolan, E. M.; Lippard, S. J. J. Mater. Chem. 2005, 15, 2778-2783.
(19) Thomas, J. M.; Ting, R.; Perrin, D. M. Org. Biomol. Chem. 2004, 2, 307-
312.
to parallel high-throughput screening methods. In addition, the small
sample sizes employed are compatible with catch-and-release
programs used for field studies25 with no need for fish euthanization.
The data collected in Figure 1C show a good linear correlation
between emission response and total mercury content over a range
of 0.1 to 8 ppm, establishing that MF1 is capable of distinguishing
safe and toxic levels of mercury in edible fish samples according
to the 0.55 ppm U.S. EPA standard.1 This fluorescence method is
complementary to colorimetric assays described previously.25,26
In closing, we have described the synthesis, properties, and
environmental applications of MF1, a unique fluorescent chemo-
sensor for screening mercury in fish. MF1 exhibits excellent
(20) Chen, P.; He, C. J. Am. Chem. Soc. 2004, 126, 728-729.
(21) Ono, A.; Togashi, H. Angew. Chem., Int. Ed. 2004, 43, 4300-4302.
(22) Lakowicz, J. R. Principles of Fluorescence Spectroscopy, 2nd ed.; Kluwer
Academic/Plenum: New York, 1999.
(23) Caltagirone, C.; Bencini, A.; Demartin, F.; Devillanova, F. A.; Garau,
A.; Isaia, F.; Lippolis, V.; Mariani, P.; Papke, U.; Tei, L.; Verani, G.
Dalton Trans. 2003, 901-909.
(24) Minta, A.; Kao, J. P. Y.; Tsien, R. Y. J. Biol. Chem. 1989, 264, 8171-
8178.
(25) Brummer, O.; La Clair, J. J.; Janda, K. D. Bioorg. Med. Chem. 2001, 9,
1067-1071.
(26) Brummer, O.; La Clair, J. J.; Janda, K. D. Org. Lett. 1999, 1, 415-418.
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