Use of selenium to detect mercury in water and cells: An enhancement of the sensitivity and specificity of a seleno fluorescent probe

Article abstract of DOI:10.1002/chem.200802165

Inspired by the antitoxic function of selenium towards heavymetal ions, we designed an organoselenium fluorescent probe (FSe-1) for mercury. The reaction of FSe-1 and Hg²⁺ is an irreversible deselenation mechanism based on the selenophilic char

Full text of DOI:10.1002/chem.200802165

FULL PAPER
DOI: 10.1002/chem.200802165
Use of Selenium to Detect Mercury in Water and Cells: An Enhancement of
the Sensitivity and Specificity of a Seleno Fluorescent Probe
Bo Tang,* Baiyu Ding, Kehua Xu, and Lili Tong[a]
Abstract: Inspired by the antitoxic
function of selenium towards heavy-
metal ions, we designed an organosele-
nium fluorescent probe (FSe-1) for
mercury. The reaction of FSe-1 and
Hg2+ is an irreversible deselenation
mechanism based on the selenophilic
character of mercury. FSe-1 exhibits an
ultrahigh selectivity and sensitivity for
Hg2+ detection only for reactive seleni-
um atom sites due to the strong affinity
between Se and Hg. The experimental
results proved that FSe-1 was selective
for Hg2+ ions over other relevant
metal ions and bioanalytes, and also
showed an enhancement in sensitivity
of up to 1.0 nm, which is lower than the
current
Environmental
Protection
Agency standard for drinking water.
Furthermore, the new probe has been
successfully applied to the imaging of
mercury ions in RAW 264.7 cells (a
mouse macrophage cell line) with high
sensitivity and selectivity.
Keywords: biological applications ·
fluorescent probes · imaging agents ·
mercury · selenium
Introduction
specificity. Thus, fluorescent probes for metal ions have
been widely researched. As is known, effective metal-re-
sponsive groups are crucial in defining the sensitivity and se-
lectivity of fluorescent probes. Up to now, several types of
small organic molecules,[5–7] oligonucleotides,[8] proteins,[9]
DNA,[10] and DNAzyme[11] platforms have been examined
for potential as Hg2+-responsive groups in fluorescent
probes. However, most of these probes showed quenched
emission, required the involvement of an organic solvent,
and had limitations on selectivity and sensitivity. Therefore,
not all these reported fluorescent probes could successfully
detect Hg2+ ions in biological samples, only a few probes
based on small organic molecules have achieved this goal
and given fluorescence images of biological samples.[7] We
observed that all the reported probes that have been applied
to biological samples have one thing in common, that is, all
of them are based on noncovalent/covalent interactions of
Hg toward S or N atoms. As we know, S and N atoms are
often applied as reactive sites for the selective recognition
of other metals, such as CuII, NiII, and ZnII. As a result, it is
difficult to make the probe respond to a specific target with
high selectivity and sensitivity. Thus, we report a new route
for improving sensitivity and selectivity in the detection of
Hg2+ ions in biological and environmental systems.[12]
Mercury is a heavy-metal element with environmental and
physiological toxicities that are recognizable even at very
low concentrations.[1] Long-term exposure to this metal
leads to permanent deterioration of the central nervous and
endocrine system in the human body because mercury
passes easily through biological membranes, such as skin,
respiratory, and gastrointestinal tissues.[2] Therefore, mercury
is widely studied in terms of its bioavailability, bioaccumula-
tion, and cellular toxicity.[3]
Compared with sophisticated analytical techniques[4] for
mercury screening, such as atomic absorption/emission spec-
troscopy and inductively coupled plasma mass spectrometry,
the fluorescence method demonstrates obvious advantages
in biological and environmental monitoring on account of
its simple, nondestructive character and high sensitivity and
[a] Prof. B. Tang, Dr. B. Ding, Dr. K. Xu, Dr. L. Tong
College of Chemistry, Chemical Engineering and Materials Science
Engineering Research Center of Pesticide and Medicine
Intermediate Clean Production, Ministry of Education
Key Laboratory of Molecular and Nano Probes
Ministry of Education
Selenium is an essential element in vivo[13] and mainly
functions through selenoprotein, a vitally important protein
in biological defense systems due to its antioxidative[14] and
antitoxic actions towards heavy-metal ions.[13] Ebselen (2-
phenyl-1,2-benzisoselenazol-3(2H)-one), a known mimetic
Shandong Normal University, Jinan, 250014 (P.R. China)
Fax : (+86)531-8618-0017
E-mail: tangb@sdnu.edu.cn
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.200802165.
Chem. Eur. J. 2009, 15, 3147 – 3151
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3147

of glutathione peroxidase (GPx), has been developed as an
antioxidative agent.[15] In a previous study, this inspired us to
À
design a fluorescent probe that contained a Se N bond for
detecting thiols in living cells.[16] In addition, the antitoxic
action of selenium toward toxic heavy-metal ions should
ˇ
also be paid much attention. As early as 1967, Paꢁzek and
ˇ
Ostꢂꢃdalovꢃ described how selenite shows a protecting prop-
erty against the toxicity of inorganic mercury.[17] In 1997,
Yoneda and Kazuo showed that toxicity of mercury can be
reduced by coadministration with selenium.[13] Our strategy
is to take advantage of the potential protective effect of Se
against Hg toxicity[14] and design and synthesize a novel or-
ganoselenium fluorescent probe (FSe-1), which is nonfluor-
escent in aqueous solution but gives a fluorescence turn-on
response upon HgII addition. Unlike conventional chemo-
sensors that operate by binding to Hg2+ ions, FSe-1, which
utilizes a deselenation reaction, features ultrahigh sensitivity
and selectivity for mercury over other metal ions and relat-
ed bioanalytes.
Results and Discussion
The organoselenium fluorescent probe was constructed by
using fluorescein as a fluorophore and the P-phenyphosphi-
noselenoic group as a reactive target for mercury ions. The
synthesis of compound FSe-1 is described in detail in
Scheme 1a and the reaction of FSe-1 and Hg2+ ions is out-
*
Figure 1. a) Excitation ( ) and emission (c) spectra of FSe-1 and fluo-
rescein (lex/lem =492/513 nm). The fluorescence response of FSe-1
(2.5 mm) to Hg2+ (1.0 mm) ions was investigated in HEPES buffer solution
(pH 7.4, 20 mm). The fluorescence intensity of solution increased 156-
fold. b) The XRD pattern of HgSe shows all peaks at scattering angles
(2q) of 25.3, 29.9, 42.0, 49.6, 61.8, and 67.28.
stoichiometric response of FSe-1 to mercury ions by keeping
the concentration of FSe-1 the same (2.5 mm) and changing
the molar ratio of [Hg2+]/
ACTHNUTRGNE[NUG FSe-1] from 0.05 to 3.50. The
fluorescence intensity at lem was directly proportional to the
amount of Hg2+ up to two equivalents, after which the fluo-
rescence intensity stopped increasing (Figure 2). This behav-
ior indicates that FSe-1 reacts with Hg2+ in 1:2 stoichiome-
try. The data described above completely confirmed that the
reaction mechanism of FSe-1 with mercury ions is a mercu-
ry-induced irreversible deselenation reaction.
Scheme 1. a) Synthesis of FSe-1. b) Deselenation reaction mechanism of
FSe-1 with mercury ions.
Fluorescence spectroscopic measurements for this system
were recorded at 258C in aqueous solution buffered to
pH 7.4 [20 mm HEPES buffer (HEPES: 2-[4-(2-hydroxyeth-
yl)-1-piperazinyl]ethanesulfonic acid), 0.10m NaCl]. FSe-1 is
nonfluorescent in aqueous solution, however, addition of
Hg2+ ions triggered a 156-fold increase in the fluorescence
intensity as shown in Figure 1a. This obvious intensity
change laid a foundation for the ultrasensitivity of FSe-1.
When FSe-1 (2.5 mm) reacted with different concentrations
of mercury ions (Figure 3), there was a good linearity be-
tween relative fluorescence intensity and concentrations of
Hg2+ ions in the range of 7.0ꢄ10À8 to 3.4ꢄ10À6 m, the regres-
lined in Scheme 1b. It can be seen that the deselenation re-
action produces fluorescein and HgSe; the former was de-
tected by studying its fluorescence behavior (lex/lem =492/
513 nm, Figure 1a). Note that HgSe showed poor crystalliza-
tion when the reaction was carried out at ambient tempera-
ture and atmospheric pressure. After the product was treat-
ed with hydrothermal synthetic conditions, good-quality
crystals were achieved and characterized by powder X-ray
diffraction, the results of which tallied with the reported
spectrogram of HgSe[18] (Figure 1b). We next studied the
sion equation was (FÀF0)=À48.51+{4.201ꢄ103
ACHTUNGTRENNUNG
[Hg2+]-
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Chem. Eur. J. 2009, 15, 3147 – 3151

Sensitivity and Specificity of a Seleno-Fluorescent Probe
FULL PAPER
Figure 2. Stoichiometric response of FSe-1 to mercury ions. Fluorescence
response of FSe-1 (2.5 mM) to HgCl2 (0.05–3.50 equiv) in HEPES
(pH 7.4, 20 mm) with excitation at l=492 nm and a corresponding emis-
sion maximum at l=513 nm.
Figure 4. a) Fluorescence responses of FSe-1 (2.5 mm) to diverse bioana-
lytes in HEPES buffer (pH 7.4, 20 mm). The black bar represents the sub-
sequent addition of 1.0 mm Hg2+ ions. Light gray bars represent the fluo-
rescence spectra on addition of analytes: 100-fold excess of HQ, NaOCl,
Figure 3. Fluorescence responses of FSe-1 (2.5 mm) to different concentra-
tions of Hg2+ ions (final concentration: 0.07, 0.10, 0.20, 0.40, 0.60, 0.80,
1.0, 1.2, 1.5, 2.0, 2.5, 3.0, 3.4 mm). Spectra were acquired at 258C for
20 min in HEPES (pH 7.4, 20 mm) with excitation at l=492 nm and a
corresponding emission maximum at l=513 nm.
1
C
CÀ
OH, O2, tBuOOH, O2 , H2O2; 500-fold excess of dopamine, GSH;
1000-fold excess of DTT, Vc, l-Cys, thioglycol. All data were obtained
after incubation at 258C for 20 min (lex/lem =492/513 nm). b) Fluores-
cence responses of FSe-1 (2.5 mm) to different metal ions in HEPES
(pH 7.4, 20 mm). Black bars represent the subsequent addition of 1.0 mm
Hg2+ to the mixture. Light gray bars represent the addition of an excess
of a metal ion: 3 mm for Na+, K+, Mg2+, Ca2+; 2 mm for Mn2+, Co2+
,
Ni2+, Zn2+, Cd2+; 0.5 mm for Cu2+; 0.1 mm for Fe3+, Fe2+, Pb2+. All data
were obtained after incubation at 258C for 20 min (lex/lem =492/513 nm).
ACHTUNGTRENNUNG
(ꢄ10À6 m)} with a linear coefficient of 0.9936, and the detec-
tion limit was determined to be 1.0 nm based on 3sslopeÀ1,
which was a lower detection limit than previous mercury
probes. From these data, it is clear that the probe could
quantitatively determine Hg2+ ions in the linear range.
Finally, we studied the selectivity of the fluorescent probe.
Considering the antioxidative function of selenium, the in-
terference effects from various bioanalytes, such as reactive
oxygen species (ROS), bioamines, and biological antioxi-
dants, were investigated. Ultimately, we got a satisfactory
conclusion (Figure 4a). An error of Æ5.0% in the relative
fluorescence intensity was considered tolerable (tolerance
ratio of 1.0 mm Hg2+ ions and final concentration): 1 mm for
1,4-dithiothreitol (DTT), ascorbic acid (Vc), l-Cys, and thio-
tains ultrahigh selectivity for Hg2+ over alkali and alkaline-
earth cations, including for Hg2+ in the presence of various
metal ions found in environmental and biological settings
(Figure 4b). Of note are the observed millimolar concentra-
tions of Na+, K+, Mg2+, Ca2+, Mn2+, Co2+, Ni2+, Zn2+
,
Cd2+, and Cu2+ and the 100-fold excesses of Fe3+, Fe2+, and
Pb2+. In a word, only Hg2+ ions could interact with FSe-1
and make the obvious change in fluorescence intensity, even
when in competition with metal ions that possess a strong
affinity for selenium, such as Cd2+ and Pb2+
.
Owing to its chemical and spectroscopic properties, FSe-1
should be ideally suited to detecting mercury ions in living
cells. To test this proposal, in vivo detection of mercury ions
in RAW 264.7 cells (a mouse macrophage cell line) was
evaluated by confocal microscopy. Live-cell-imaging experi-
ments were performed with hexadecyltrimethylammonium
glycol; 0.5 mm for dopamine; 0.1 mm for hydroquinone
1
C
CÀ
,
(HQ), NaOCl, OH, singlet oxygen ( O2), tBuOOH, O2
and H2O2. Furthermore, we investigated potential interfer-
ence from various metal ions. According to the measured
fluorescence intensities, the results showed that FSe-1 re-
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B. Tang et al.
bromide (CTAB) to enhance membrane permeability.[19]
Thus, RAW 264.7 cells loaded with 5.0 mm of FSe-1 and
0.03 mL CTAB (2%, w/v) for 20 min at 378C showed no
fluorescence (Figure 5a). In contrast, FSe-1-stained cells dis-
played a clear increase in intracellular fluorescence intensity
when 10 mM of HgCl2 was added to the medium and the
cells were incubated for another 30 min at 378C (Figure 5b);
bright-field image measurement confirmed that the cells
were viable throughout the imaging studies (Figure 5c). The
results clearly reveal that FSe-1 exhibits a selective response
for mercury ions over other biologically relevant substances.
Conclusion
In summary, we have described the synthesis and properties
of FSe-1, a new organoselenium fluorescent probe for Hg2+
based on a deselenation reaction in aqueous solution. FSe-1
exhibited ultrahigh selectivity for Hg2+ ions over other rele-
vant metal ions and a very low detection limit of 1.0 nm,
which is lower than the EPA limit for Hg2+ ions in drinking
water. The probe was successfully applied to the imaging of
mercury ions in RAW 264.7 cells with high sensitivity and
selectivity, which was valuable for studying the toxicity and
bioactivity of Hg2+ in living organisms. We anticipate that
this work provides a new approach to design metal ions/
ROS/deoxidant probes that take full advantage of the anti-
oxidative and antitoxic properties of selenium and the affini-
ty between Se and O, S, and N.
Experimental Section
Apparatus and reagents: Fluorescence spectra were obtained by using a
FLS-920 Edinburgh Fluorescence Spectrometer equipped with a xenon
lamp, 1.0 cm quartz cells, and slits of 0.60/0.60 nm. XRD patterns were
obtained by using a Rigaku D/Max-rB X-ray powder diffractometer with
CuKa radiation (l=1.5405 ꢅ). The fluorescence images of cells were
taken by using a LTE confocal laser scanning microscope (Leica Co.,
Germany) with an objective lens (ꢄ40) and the excitation wavelength
was l=488 nm. All pH measurements were performed by using a pH-3c
digital pH-meter (Shanghai Lei Ci Device Works, Shanghai, China). All
reagents were of commercial quality and used without further purifica-
tion. Fluorescein (+90%), Chlorodiphenylphosphine (+98%), and sele-
nium metal powder (+99%) were purchased from Alfa Aesar Chemical
Company. Sartorius ultrapure water (18.2mWcm) was used throughout
the analytical experiments.
Synthesis of FSe-1: We synthesized new probe FSe-1 according to a
modified version of the synthesis of alkyl phenylphosphinoselenoic chlor-
ides proposed by T. Murai and T. Kimura.[20] Fluorescein (0.336 g,
1.01 mmol) in THF (20 mL) was added to a suspension of elemental sele-
nium (0.693 g, 8.77 mmol) in THF (5 mL) at RT under argon. PhPCl2
(1.44 mL, 8.02 mmol) in triethylamine (2 mL) and THF (20 mL) was
added dropwise to this mixture over a period of 10 min with vigorous stir-
ring, then the mixture was stirred at RT under argon overnight. After the
solvent was removed, benzene (25 mL) was added to the residue. The
mixture was stirred at reflux in benzene for 4 h, then any insoluble resi-
dues were filtered off. After the solvent was removed, the residue was
purified by using column chromatography on aluminum chloride neutral
with nC6H14/EtOAc as the eluent to give the desired product as colorless
crystals. M.p.: 257–2588C.
Figure 5. Confocal fluorescence images of Hg2+ in live RAW 264.7 cells
(Leica confocal fluorescence microscope, 40ꢄobjective lens). a) Fluores-
cence image of RAW 264.7 cells incubated with FSe-1 (5.0 mm) and
0.03 mL CTAB (CTAB: hexadecyltrimethylammonium bromide; 2%, w/
v) for 20 min. b) RAW 264.7 cells incubated with FSe-1 (5.0 mm) for
20 min, washed three times with PBS (PBS: phosphate buffered saline)
to remove the remaining probe, and then further incubated with Hg2+
(10.0 mm) for 30 min. c) Bright-field image of RAW 264.7 cells shown in
b). The scale bars represent 25 mm.
Characterization of HgSe: A precipitate was obtained when the reaction
of FSe-1 with Hg2+ was carried out at ambient temperature and atmos-
pheric pressure. Owing to its poor crystallization, the precipitate was
treated with filtration and subsequently dispersed in DMF at 1208C
under hydrothermal synthesis conditions. The resulting precipitate was a
brown-black powder that was characterized by powder X-ray diffraction
and confirmed to be HgSe by comparison with the literature spectrogram
of HgSe.[21]
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Sensitivity and Specificity of a Seleno-Fluorescent Probe
FULL PAPER
Sample preparation: A stock solution of FSe-1 (1.00 mm) in DMSO was
prepared. Probe solutions (0.10 mm) were prepared by diluting this stock
solution of FSe-1 with DMSO. Solutions of HgCl2 (0.10 mM), CTAB
(2%, w/v), and HEPES buffer (0.10m HEPES, 0.10m NaCl; pH 7.4) were
prepared in ultrapure water.
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Fluorescence analysis: FSe-1 (0.10 mm; 0.25 mL), HEPES (2.00 mL;
pH 7.4), and different concentrations of HgCl2 were added in turn to a
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to 0.6 nm.
Staining of cell cultures: RAW 264.7 cells were maintained following pro-
tocols provided by the American Type Tissue Culture Collection. Cells
were seeded at a density of 106 cellsmLÀ1 in high glucose Dulbeccoꢂs
Modified Eagle Medium (4.5 g of glucoseLÀ1) supplemented with 10%
fetal bovine serum, NaHCO3 (2 gLÀ1) and 1% antibiotics (penicillin/
streptomycin, 100 UmLÀ1). Cultures were maintained at 378C under a
humidified atmosphere containing 5% CO2.
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Received: October 20, 2008
Published online: February 9, 2009
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ꢀ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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3151