Fluorescent detection of methylmercury by desulfurization reaction of rhodamine hydrazide derivatives

Article abstract of DOI:10.1039/b915723a

Exposure to methylmercury causes severe damage to various tissues and organs in humans. Although a variety of fluorescent chemosensors have been exploited, only few biological monitoring systems for organomercury species have been described to date. In th

Full text of DOI:10.1039/b915723a

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Fluorescent detection of methylmercury by desulfurization reaction of
rhodamine hydrazide derivatives†
Young-Keun Yang, Sung-Kyun Ko, Injae Shin* and Jinsung Tae*
Received 31st July 2009, Accepted 10th September 2009
First published as an Advance Article on the web 21st September 2009
DOI: 10.1039/b915723a
Exposure to methylmercury causes severe damage to various
tissues and organs in humans. Although a variety of fluores-
cent chemosensors have been exploited, only few biological
monitoring systems for organomercury species have been
described to date. In this report, we describe an irreversible
rhodamine chemosensor for the detection of methylmercury
and real-time monitoring of methylmercury in living cells and
organisms.
Scheme 1 Hg2+-induced desulfurization of rhodamine thiosemicar-
bazide 1.
Mercury exists in the environment as inorganic mercury species
rhodamine hydrazide system could serve as a CH3Hg+ probe
too. Herein, we report our results on the fluorescent sensing of
methylmercury in aqueous solutions and applications to biological
imaging by using the rhodamine thiosemicarbazides.
In addition to the previously reported rhodamine thiosemi-
carbazide 1, we also prepared rhodamine thiosemicarbazide
derivatives containing p-NO2 (4) and p-OMe (5) groups to see the
reactivity differences caused by the electron density on the aniline
ring. The probes were synthesized from the known rhodamine 6G
hydrazide 3 and aryl isothiocyanates in good yields (Scheme 2).
(Hg0, Hg2+) and organic mercury (e.g. RHg+, R2Hg, where
R = typically Me, Et).1 Methylation of inorganic mercury
species by aquatic microorganisms can produce methylmercury
compounds.2 The biological targets and toxicity profile of mercury
species depend on their chemical composition.3 Methylmercury
species (CH3HgX), which can readily pass through biological
membranes,4 are powerful neurotoxicants5 to fish, animals, and
humans.6 Neurological damages7 associated with methylmercury
intoxication are manifold and include prenatal brain damage,
cognitive and motion disorders, vision and hearing loss, and
Minamata disease.8 Several targets, such as the BBB (blood
brain barrier), axonal transport, neurotransmission, synthesis of
protein, DNA, and RNA, have been proposed as sites sensitive to
methylmercury.9 The ramifications of long-term or short-term and
low-level exposure to methylmercury are less clear and warrant
thorough toxicological investigations. Therefore, the biological
effects of methylmercury species have been considerably studied.10
Fluorescent sensors based on small molecules,11 polymeric
materials,12 nanoparticles,13 and dosimeters14 have served as trac-
ing tools for neurotoxic Hg2+. Recently, the sensing and imaging
of Hg2+ in living biological systems have also been realized.15,16
Fluorescent detection of methylmercury in the environment and
biological systems using chemosensor techniques has not been
studied well. Recently, Ahn’s group has reported a fluorescein-
based vinyl ether probe for detection of Hg2+ and MeHgX.17 They
utilized fluorescence changes associated with methylmercury-
promoted hydrolysis of vinyl ether. With this “turn-on”-type green
fluorescent probe, they also demonstrated imaging of cells and
zebrafish. From the previous experiments on the Hg2+-selective
chemosensor utilizing the Hg2+-induced desulfurization reaction
(Scheme 1),16 we observed that a similar desulfurization reaction
could be promoted by CH3Hg+ too, but less efficiently. Although
CH3Hg+ is less thiophilic than Hg2+, we expected the same
Scheme 2 Synthesis of rhodamine thiosemicarbazides.
Upon addition of CH3Hg+ to solutions (10 mM) of the
rhodamine thiosemicarbazides (1, 4, or 5) in water (DMSO 1%)
at 25 ◦C, the fluorescence intensities (at 560 nm) of the solutions
increased gradually as shown in Fig. 1. While the probe 1 showed
strong fluorescence intensity changes (Fig. 1A), the rhodamine
derivatives with an electron withdrawing group (4) or an electron
donating group (5) exhibited weak fluorescence intensity changes
under the same conditions (Fig. 1C, D). As expected, probe 1
required greater amounts of CH3Hg+ (needs 6–8 equiv., Fig. 1B)
than Hg2+ (needs 1 equiv.)16a for the saturation of the fluorescence
intensity.
The detection limit of probe 1 for CH3Hg+ was evaluated by
monitoring the fluorescence titration curves of 1 (10-6 M) with
CH3Hg+ at nanomolar (nM) levels (Fig. 2). The fluorescence
intensity of 1 was nearly proportional to the amount of CH3Hg+
added (Fig. 2B) and detection was possible at 200 nM of CH3Hg+
in water (DMSO 1%) at 25 ◦C. The fluorescence response of 1 to
Department of Chemistry, Yonsei University, Seoul 120-749, Korea.
E-mail: jstae@yonsei.ac.kr, injae@yonsei.ac.kr; Fax: +82 2-364-7050;
Tel: +82 2-2123-2603
† Electronic supplementary information (ESI) available: Experimental and
copies of 1H and 13C NMR spectra. See DOI: 10.1039/b915723a
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Scheme 3 A proposed mechanism for the CH3Hg+-induced desulfuriza-
tion reaction of 1.
Fig. 1 Fluorescence titration curves of the chemosensors with methylmer-
cury. Fluorescence responses of 1 (A), 4 (C), and 5 (D) (10 mM) upon
additions of CH3Hg+ (0–8.0 equiv.) in water (DMSO 1%) at 25 ◦C
(excitation at 500 nm; emission at 560 nm). Each spectrum was acquired
5 min after each addition of CH3Hg+. (B): Plot of fluorescence intensity
(at 560 nm) versus equivalents of CH3Hg+ shown in the titration curve A.
Fig. 2 (A) Fluorescence emission changes of 1 (10-6 M) upon additions of
CH3Hg+ (by 200 nM) in water (DMSO 1%) at 25 ◦C. (B) The fluorescence
intensity changes at 550 nm (excitation at 500 nm) versus the concentration
of CH3Hg+.
CH3Hg+ in PBS-buffer solutions (DMSO 1%, at pH 7.4) showed
similar trends to those observed in aqueous solutions (DMSO
1%). Reaction of 1 with CH3Hg+ is slow and requires an excess of
CH3Hg+ for completion of the reaction. Typically, ~10 equiv. of
CH3Hg+ is required to meet the fluorescence enhancement induced
by 1 equiv. of Hg2+ in the given reaction time period (see ESI†).
The fluorescent product obtained from the reaction of 1 with
CH3HgCl proved to be the 1,3,4-oxadiazole compound 2 which is
observed from the reaction 1 with HgCl2.16 Therefore, a similar
desulfurization reaction mechanism could be responsible for
the fluorescent detection of methylmercury by 1. In this case,
CH3HgSH could be eliminated as the result of the desulfurization
reaction as proposed in Scheme 3.
Fig. 3 Fluorescence images of methylmercury in live HeLa cells. Mi-
croscopic (a) and fluorescence (e) images of HeLa cells treated with 1
(20 mM) in the absence of CH3Hg+. Microscopic (b–d) and fluorescence
(f–h) images of HeLa cells treated with both CH3Hg+ (b, f—5 mM,
c, g—10 mM, d, h—20 mM) and 1 (20 mM).
We then evaluated bio-imaging applications of 1 for detection
of organomercury species in biological systems. HeLa cells were
incubated with 20 mM of 1 for 10 min at 37 ◦C, washed with PBS-
buffer (pH 7.4) to remove the remaining chemosensors, then the
treated cells were incubated with 5–20 mM of CH3HgCl in culture
in live HeLa cells. We were able to detect 300 nM of CH3HgCl in
HeLa cells by this method (see ESI†).
Next, time-dependent uptake of methylmercury in live cells was
determined by incubating cells with probe 1 and methylmercury
while measuring the fluorescence intensity changes as a function
of time. The HeLa cells and A549 cells were incubated with 1
(20 mM) for 30 min, washed with PBS to remove the remaining
sensors, then treated with 0–200 mM of CH3HgCl in the culture
media. The fluorescence intensity changes were continuously
◦
medium for 10 min at 37 C. While the HeLa cells treated with
only 1 did not show any fluorescence (Fig. 3e), the HeLa cells
treated with both 1 and CH3HgCl displayed strong fluorescence
intensity (Fig. 3f–h). The microscopic and fluorescent images
clearly indicate that probe 1 can detect 5–20 mM of methylmercury
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monitored by using a fluorescence microplate reader. Although
full saturations of the fluorescence intensity were not observed in
the live cell uptake experiments, methylmercury can clearly enter
the cells within 30–40 min as shown in Fig. 4.
demonstrated the detection of methylmercury in living cells and
organisms.
Acknowledgements
This work is funded by the Korea Science and Engineering
Foundation (KOSEF) grant funded by the Korea govern-
ment (MEST) (No. R01-2008-000-10245-0 and R32-2008-000-
10217-0).
Notes and references
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Fig. 4 Real-time monitoring of methylmercury uptake in live cells.
Real-time monitoring of CH3Hg+ (0 (ꢀ), 10 (᭹), 20 (ꢀ), 40 (᭢), 100
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Encouraged by the live cell experiments, we examined if
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In summary, we have described an irreversible chemosensor
for the detection of methylmercury species in live cells and
zebrafish. The rhodamine thiosemicarbazide probe, which reacts
irreversibly with methylmercury via a desulfurization reaction, can
detect methylmercury with high sensitivity in aqueous media.
Fluorescent imaging of HeLa cells and zebrafish successfully
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