A chemodosimeter approach to fluorescent sensing and imaging of inorganic and methylmercury species

Article abstract of DOI:10.1039/b900380k

A highly sensitive fluorescent turn-on probe specific for methylmercury species as well as inorganic mercury ions has been developed on the basis of mercury ion-promoted hydrolysis of a fluorescein-derived aryl vinyl ether.

Full text of DOI:10.1039/b900380k

View Online / Journal Homepage / Table of Contents for this issue
COMMUNICATION
www.rsc.org/chemcomm | ChemComm
A chemodosimeter approach to fluorescent sensing and imaging
of inorganic and methylmercury speciesw
Mithun Santra,a Dowook Ryu,a Amrita Chatterjee,a Sung-Kyun Ko,b Injae Shin*b
and Kyo Han Ahn*a
Received (in Cambridge, UK) 8th January 2009, Accepted 3rd March 2009
First published as an Advance Article on the web 16th March 2009
DOI: 10.1039/b900380k
A highly sensitive fluorescent turn-on probe specific for methyl-
mercury species as well as inorganic mercury ions has been
developed on the basis of mercury ion-promoted hydrolysis of a
fluorescein-derived aryl vinyl ether.
of mammalian cells and vertebrate organisms treated with
methylmercury is also demonstrated for the first time.
The design of probe 1 is based on the mercury ion-promoted
hydrolysis reaction of vinyl ethers: the oxymercuration of vinyl
ether 1 generates the corresponding hemiacetal intermediate,
which undergoes fragmentation into alcohol 2 and aldehyde 3
(Scheme 1).
Mercury is a highly poisonous element and widespread
pollutant, occurring from natural and anthropogenic sources.
Organic forms of mercury, typically, methylmercury species
(CH3HgX; X = halides, etc.) are much more toxic than
inorganic mercury species (HgX2).1 Because of their lipid
solubility, methylmercury species readily cross the blood–
brain barrier, accumulate in the brain, and cause damage to
the central nervous system,1,2 as well as other organs. The
epidemics of Minamata Bay in Japan demonstrated the fatal
threat of methylmercury to human health.3
A variety of fluorescent molecular probes for the detection
of mercury ions with ease and high sensitivity have flourished
in recent years.4 Surprisingly, most of these probes only sense
inorganic mercury ions and those that sense methylmercury
are rare.5 Furthermore, none of the fluorescent probes can be
applied to evaluate the accumulation of methylmercury in
living species. For example, the rhodamine-based fluorescent
chemodosimeter reported by Tae and co-workers shows high
sensitivity and selectivity towards inorganic mercury ions, and
has been used to monitor them both in cells and in a living
vertebrate organism. However, the chemodosimeter showed a
weak response to CH3HgCl in contrast to HgCl2.4g,k
Such a mercury ion-promoted hydrolysis of vinyl ether 1 is
also expected to occur with methylmercury species (CH3HgX),
because a similar oxymercuration intermediate is envisioned
on the basis of the reaction mechanism. Because vinyl ether 1
is very weakly fluorescent, but alcohol 2 is highly fluorescent,
we may realize fluorescent turn-on sensing of methylmercury
as well as inorganic mercury ions in aqueous media.
The required vinyl ether 1 was directly synthesized from
known compound 4 through deallylation and vinylation
steps.7
A solution of probe 1 (5.0 mM) in PBS buffer (pH 7.4)
containing 5% DMSO became strongly fluorescent upon
addition of HgCl2 (Fig. 1(a)), as it is converted to fluorescein
2 (FF = 0.89)7a through hydrolysis. The generation of 2 was
confirmed by isolation and characterization of the final
product after titration. Importantly, the titration of probe 1
with HgCl2 (from 0 to 1.0 equiv.) showed saturation behavior
at 0.5 equiv. of HgCl2 (Fig. 1(b)), which can be explained by
the mechanism in Scheme 1. The mechanism suggests that an
organomercury species such as 3 also promotes the hydrolysis.
One plausible reason for the low sensitivity of the hetero-
atom-based molecular probes towards methylmercury may
arise from the weak molecular interactions between them.
To develop a fluorescent probe for the notoriously poisonous
methylmercury ions, we have designed a chemodosimeter
based on a chemical reaction that avoids the popular mercury
ion-coordination to heteroatoms.6 The chemodosimeter thus
designed is fluorescein-based vinyl ether, 1, which reacts only
with organomercury and inorganic mercury ions in the pre-
sence of other metal ions, and shows a ‘‘turn-on’’-type fluor-
escent response with high sensitivity. The fluorescent imaging
a Department of Chemistry and Center for Electro-Photo Behaviors in
Advance Molecular Systems, POSTECH, San 31 Hyoja-dong,
Pohang, 790-784, Republic of Korea. E-mail: ahn@postech.ac.kr
b Department of Chemistry, Yonsei University, Seoul 120-749,
Republic of Korea. E-mail: injae@yonsei.ac.kr
w Electronic supplementary information (ESI) available: Experimental
procedures for the synthesis of compounds and all the fluorescence
analysis data. See DOI: 10.1039/b900380k
Scheme 1 A plausible hydrolysis mechanism of probe 1 by HgCl2 and
RHgCl in water.
ꢀc
This journal is The Royal Society of Chemistry 2009
Chem. Commun., 2009, 2115–2117 | 2115

View Online
Probe 1 is specific towards mercury ions, while other metal
ions (Mg2+, Ca2+, Ba2+, Cr2+, Mn2+, Fe3+, Co2+, Ni2+
,
Cu2+, Zn2+, Cd2+, Pb2+ and Ag+) show essentially no
fluorescence change, owing to little hydrolysis of the vinyl
ether with these metals (Fig. 1(d)). Also, probe 1 shows full a
fluorescence response to the mercury ions, even in the presence
of all other metal ions.w Other mercury compounds, such as
Hg(OAc)2, gave the same results.
Next, we evaluated the effectiveness of probe 1 for fluore-
scence monitoring of both CH3HgCl-contaminated cells and a
living vertebrate organism, zebrafish. Human lung cancer cells
(A549 cells) incubated with probe 1 and CH3HgCl clearly
show green fluorescence, indicating that probe 1 can enter cell
membranes and react with the organomercury species to form
fluorescent 2 (Fig. 2(a)).
However, the cells do not exhibit fluorescence in the absence
of the external organomercury species (Fig. 2(b)). We explored
further whether 1 could be used to monitor organomercury
species in living organisms. For this study, a three-day-old
zebrafish was exposed to 1 in the presence and absence of
CH3HgCl. The results of the fluorescence microscopy
analysis show that the organomercury species in zebrafish is
fluorescently detected by 1 (Fig. 2(c) and (d)). Encouraged
by this result, we analyzed methylmercury accumulation in
grown zebrafish with probe 1. Interestingly, the methyl-
mercury accumulated in the eye, heart, fin, gall bladder, and
eggs, but not in the brain and liver under the experimental
conditions (Fig. 3). These preliminary in vivo studies clearly
demonstrate that the probe has potential for studying the
accumulation of methylmercury species in other cells and
organisms.
Fig. 1 (a) The time-dependent fluorescence change acquired for a
2 : 1 mixture of probe 1 and HgCl2; inset: a plot of the fluorescence
intensity change as a function of the reaction time. (b) The fluores-
cence intensity change of probe 1 as a function of equiv. of HgCl2 (’)
and CH3HgCl ( ), taken after 10 min for each addition. (c) A plot of
fluorescence intensity vs. [HgCl2] obtained for a 2 : 1 mixture of 1 and
HgCl2 for the range of 0.25–20 ppb [HgCl2], taken after 1 h of each
mixing. (d) The fluorescence change after 1 h acquired for a 2 : 1
mixture of probe 1 and various metal ions (Mg2+, Ca2+, Ba2+, Cr2+
,
Mn2+, Fe3+, Co2+, Ni2+, Cu2+, Zn2+, Cd2+, Pb2+, Ag+ and
Hg2+). All measurements were taken with 5.0 mM of probe 1 in PBS
buffer (pH 7.4) containing 5% DMSO (excitation at 480 nm; the
intensity was estimated by the peak height at l = 520 nm).
Certainly, probe 1 shows fluorescence recovery upon treat-
ment with CH3HgCl in the PBS buffer.w In this case, the
fluorescence saturation is reached with 1.0 equiv. of CH3HgCl
(Fig. 1(b)), which can be also explained by the mechanism.
Similar fluorescence titrations at different pH conditions
(pH 4.0–9.0) were also carried out.w In the absence of HgCl2,
probe 1 itself did not show any fluorescence change at pH 4.0,
even after 24 h, which indicates that the observed fluorescence
change is mainly due to catalysis by mercury species.
Comparing the fluorescence recovery time for the cases of
HgCl2 and CH3HgCl, the former reacts with probe 1 a little
faster than the latter.
In summary, we have devised a structurally simple yet
efficient fluorescent probe for organomercury as well as
inorganic mercury ions. The vinyl ether probe 1 thus shows
specific response to the mercury species and turn-on fluores-
cence with high sensitivity, as the vinyl ether undergoes
mercury ion-promoted hydrolysis to give the strongly
fluorescent 2. With this probe, the fluorescent imaging of
mammalian cells and organisms incubated with organo-
mercury species has been demonstrated for the first time.
This work was supported by grants from the EPB center
(R11-2008-052-01001) and NRL programs of KOSEF/MEST.
When 1 equiv. of HgCl2 was added to probe 1 in the PBS
buffer, the fluorescence saturation took about 1 h (about 70%
recovery after 10 min), while it took about 1.5 h in the case of
CH3HgCl under the same conditions.w However, it is not
necessary to wait for the full recovery of fluorescence for
quantification purposes, as we can obtain a linear relationship
between the concentration of mercury ions and the fluores-
cence intensity over an arbitrary time span. A linear relation-
ship between the fluorescence intensity and the concentration
of HgCl2 is obtained for
a wide concentration range
(1.0 ꢁ 10ꢂ9–1.0 ꢁ 10ꢂ5 M; 0.2–2000 ppb, a lower concentration
part of which is shown in Fig. 1(c)), and a detection limit of
below 1 ppb level is obtained.wz If an assay of fast and full
response is needed, the molar ratio of the probe to the mercury
ions can be increased, because an excess amount of the probe
gives negligible fluorescence and hence does not affect quantifi-
cation of the data. This is an advantageous property of probe 1.
Fig. 2 Cells and organisms incubated with 50 mM probe 1 and
100 mM CH3HgCl in 5% CH3CN–water. Images of A549 cells (a) in
the presence and (b) absence of CH3HgCl. Images of three-day-old
zebrafish (c) in the presence and (d) absence of CH3HgCl.
ꢀc
This journal is The Royal Society of Chemistry 2009
2116 | Chem. Commun., 2009, 2115–2117

View Online
Fig. 3 (a) Images of zebrafish organs treated with 200 nM of CH3HgCl and 50 mM of probe 1 (top: microscope images, bottom: fluorescence
images). (b) Images of zebrafish organs treated with 50 mM of probe 1 in the absence of external CH3HgCl (top: microscope images, bottom:
fluorescence images).
X. Yang, Z. Shuai and D. Zhu, Chem. Commun., 2005, 2161;
(j) B. Liu and H. Tian, Chem. Commun., 2005, 3156; (k) S.-K. Ko,
Y.-K. Yang, J. Tae and I. Shin, J. Am. Chem. Soc., 2006, 128, 14150;
Notes and references
z The maximum concentration level of mercury ions in drinking water
set by the US EPA is 2 ppb.
(l) H. Zheng, Z.-H. Qian, L. Xu, F.-F. Yuan, L.-D. Lan and
J.-G. Xu, Org. Lett., 2006, 8, 859; (m) X.-J. Zhu, S.-T. Fu,
W.-K. Wong, J.-P. Guo and W.-Y. Wong, Angew. Chem., Int.
Ed., 2006, 45, 3150; (n) S. Yoon, E. W. Miller, Q. He, P. H. Do
and C. J. Chang, Angew. Chem., Int. Ed., 2007, 46, 6658;
(o) H. Yang, Z. Zhou, K. Huang, M. Yu, F. Li, T. Yi and
C. Huang, Org. Lett., 2007, 9, 4729; (p) W. Shi and H. Ma, Chem.
Commun., 2008, 1856; (q) X.-Q. Zhan, Z.-H. Qian, H. Zheng,
B.-Y. Su, Z. Lan and J.-G. Xu, Chem. Commun., 2008, 1859.
5 A chromogenic chemodosimeter appeared that senses methylmer-
cury species with low sensitivity: O. del Campo, A. Carbayo,
1 (a) ATSDR, Toxicological Profile for Mercury, U.S. Department of
Health and Human Services, Atlanta, GA, 1999; (b) ATSDR,
ToxProfiles: Mercury, U.S. Department of Health and Human
Services, Atlanta, GA, 2005.
2 (a) P. Jitaru and F. Adams, J. Phys. IV France, 2004, 121, 185;
(b) T. W. Clarkson and L. Magos, Off. Rev. Toxicology, 2006, 36, 609.
3 F. Bakir, S. F. Damluji, L. Amin-Zaki, M. Murtadha, A. Khalidi,
N. Y. Al-Rawi, S. Tikriti, H. I. Dhahir, T. W. Clarkson, J. C. Smith
and R. A. Doherty, Science, 1973, 181, 230.
4 For a recent review, see: (a) E. M. Nolan and S. J. Lippard, Chem.
Rev., 2008, 108, 3443; (b) for selected examples, see: A. B. Descalzo,
´ ´
R. Martınez-Manez, R. Radeglia, K. Rurack and J. Soto, J. Am.
Chem. Soc., 2003, 125, 3418; (c) E. M. Nolan and S. J. Lippard,
J. Am. Chem. Soc., 2003, 125, 14270; (d) X. Guo, X. Qian and L. Jia,
´
J. Am. Chem. Soc., 2004, 126, 2272; (e) A. Caballero, R. Martınez,
J. V. Cuevas, Z. Munoz, G. Garcı
E. Ballesteros, S. Basurto, T. Gomez and T. Torroba, Chem.
Commun., 2008, 4576.
´
a-Herbosa, D. Moreno,
´
6 During the publication process of this manuscript, a fluorogenic
probe for inorganic mercury ions on the basis of alkyne oxymer-
curation appeared, which required heating at 90 1C: F. Song,
S. Watanabe, P. E. Florencig and K. Koide, J. Am. Chem. Soc.,
2008, 130, 16460.
7 (a) F. Song, A. L. Garner and K. Koide, J. Am. Chem. Soc., 2007,
127, 12354; (b) B. A. Sparano, S. P. Shahi and K. Koide, Org. Lett.,
2004, 6, 1947.
V. Lloveras, I. Ratera, J. Vidal-Gancedo, K. Wurst, A. Tarraga,
´
P. Molina and J. Veciana, J. Am. Chem. Soc., 2005, 127, 15666;
(f) S. Yoon, A. E. Albers, A. P. Wong and C. J. Chang, J. Am.
Chem. Soc., 2005, 127, 16030; (g) Y.-K. Yang, K.-J. Yook and
J. Tae, J. Am. Chem. Soc., 2005, 127, 16760; (h) B. Liu and H. Tian,
Chem. Commun., 2005, 3156; (i) G. Zhang, D. Zhang, S. Yin,
ꢀc
This journal is The Royal Society of Chemistry 2009
Chem. Commun., 2009, 2115–2117 | 2117