Imaging different interactions of mercury and silver with live cells by a designed fluorescence probe rhodamine b selenolactone

Article abstract of DOI:10.1021/ic902192a

Rhodamine B selenolactone has been designed, synthesized, and characterized as a new fluorescent probe for imaging both Hg²⁺ and Ag⁺ in live cells to better understand their distinct toxicities to organisms. The probe is designed based on the fact that selenium has a strong affinity for mercury and silver, and is constructed by incorporating a Se atom into the spirocyclic structure of rhodamine. It exhibits a rapid and specific spectroscopic off-on response to Hg²⁺ and Ag⁺ instead of other species, with detection limits of 23 nM Hg²⁺ and 52 nM Ag ⁺. Moreover, the probe is membrane-permeable, and can react with Ag⁺ even in the presence of Cl^- because of the higher affinity of Se than Cl^- for Ag⁺, which makes it of potential use for imaging not only Hg²⁺ but also Ag⁺ in live cells. This applicability has been demonstrated by imaging Hg²⁺ and Ag⁺ in Hela cells. It is observed that the reaction of Ag ⁺ with the probe inside the cells occurs much slower than that of Hg²⁺, which is ascribed to the high concentration of cellular chloride ions inhibiting the formation of sufficient free Ag⁺. The present finding is helpful to get an insight into the different interaction mechanism of Hg²⁺ and Ag⁺ with cells, and more applications of the probe may be expected for studying the behaviors of Hg ²⁺ and Ag⁺ in various biosystems.

Full text of DOI:10.1021/ic902192a

1
206 Inorg. Chem. 2010, 49, 1206–1210
DOI: 10.1021/ic902192a
Imaging Different Interactions of Mercury and Silver with Live Cells by a Designed
Fluorescence Probe Rhodamine B Selenolactone
Wen Shi, Shuna Sun, Xiaohua Li, and Huimin Ma*
Beijing National Laboratory for Molecular Sciences, Institute of Chemistry, Chinese Academy of Sciences,
Beijing 100190, China
Received November 5, 2009
Rhodamine B selenolactone has been designed, synthesized, and characterized as a new fluorescent probe for
2
þ
þ
imaging both Hg and Ag in live cells to better understand their distinct toxicities to organisms. The probe is
designed based on the fact that selenium has a strong affinity for mercury and silver, and is constructed by
incorporating a Se atom into the spirocyclic structure of rhodamine. It exhibits a rapid and specific spectroscopic off-on
2
þ
þ
2þ
þ
response to Hg and Ag instead of other species, with detection limits of 23 nM Hg and 52 nM Ag . Moreover, the
probe is membrane-permeable, and can react with Ag even in the presence of Cl because of the higher affinity of Se
þ
-
-
þ
2þ
þ
than Cl for Ag , which makes it of potential use for imaging not only Hg but also Ag in live cells. This applicability
has been demonstrated by imaging Hg and Ag in Hela cells. It is observed that the reaction of Ag with the probe
2
þ
þ
þ
2þ
inside the cells occurs much slower than that of Hg , which is ascribed to the high concentration of cellular chloride
ions inhibiting the formation of sufficient free Ag . The present finding is helpful to get an insight into the different
þ
2
interaction mechanism of Hg and Ag with cells, and more applications of the probe may be expected for studying
þ
þ
2þ
þ
the behaviors of Hg and Ag in various biosystems.
2
þ
þ
Introduction
fluorescent probe capable of imaging both Hg and Ag in
live cells, so as to eliminate the effect of different probes. So
far, many excellent fluorescent probes have been proposed
Mercury has long been known to be a highly toxic metal,
which may cause prenatal brain damage, cognitive and
motion disorders, vision and hearing loss, and even death;
whereas silver (a similar ds-block element) hardly exhibits
toxicity to humans, and has been widely used as a broad-
2þ
þ 3
1
for the detection of Hg or Ag , but very few are available
for cell imaging. In particular, to the best of our knowledge,
none of them is suited for imaging Ag in live cells. The main
difficulty of this is that high concentration (more than mM
levels) of cellular chloride ions may lead to the precipitation
of Ag as AgCl, thereby hindering the reaction of Ag with a
fluorescent probe.
4
þ
2
spectrum antimicrobial agent. To better understand their
distinct toxicities to organisms, it may be useful to develop a
5
þ
þ
*
To whom correspondence should be addressed. E-mail: mahm@
iccas.ac.cn. Fax: þ86-10-62559373.
1) (a) Nolan, E. M.; Lippard, S. J. Chem. Rev. 2008, 108, 3443–3480.
b) Boening, D. W. Chemosphere 2000, 40, 1335–1351.
2) (a) Klasen, H. J. Burns 2000, 26, 117–130. (b) Berger, T. J.; Spadaro,
J. A.; Chapin, S. E.; Becker, R. O. Antimicrob. Agents Chemother. 1976, 9, 357–
58.
3) (a) Huang, W.; Song, C. X.; He, C.; Lv, G. J.; Hu, X. Y.; Zhu, X.;
The combination of fluorochromes with cleavable active
bonds has opened a novel alternate route to developing
specific spectroscopic probes. Recently, rhodamine as an
excellent fluorochrome has attracted considerable interest
to prepare various spectroscopic off-on-type probes by virtue
of its easy formation of a colorless and non-fluorescent
(
6
(
(
3
(
Duan, C. Y. Inorg. Chem. 2009, 48, 5061–5072. (b) Lyoshi, S.; Taki, M.;
Yamamoto, Y. Inorg. Chem. 2008, 47, 3946–3948. (c) Lee, M. H.; Lee, S. W.;
Kim, S. H.; Kang, C.; Kim, J. S. Org. Lett. 2009, 11, 2101–2104. (d) Choi, M. G.;
Kim, Y. H.; Namgoong, J. E.; Chang, S. K. Chem. Commun. 2009, 3560–3562.
(
4) (a) Ko, S. K.; Yang, Y. K.; Tae, J.; Shin, I. J. Am. Chem. Soc. 2006,
1
28, 14150–14155. (b) Suresh, M.; Shrivastav, A.; Mishra, S.; Suresh, E.; Das, A.
Org. Lett. 2008, 10, 3013–3016. (c) Zhang, X.; Xiao, Y.; Qian, X. Angew.
Chem., Int. Ed. 2008, 47, 8025–8029. (d) Yoon, S.; Miller, E. W.; He, Q.; Do,
P. H.; Chang, C. J. Angew. Chem., Int. Ed. 2007, 46, 6658–6661.
(5) Incharoensakdi, A.; Takabe, T. Plant Cell Physiol. 1988, 29, 1073–
1075.
(6) (a) Richard, J. A.; Meyer, Y.; Jolivel, V.; Massonneau, M.; Dumeunier,
R.; Vaudry, D.; Vaudry, H.; Renard, P. Y.; Romieu, A. Bioconjugate Chem.
2008, 19, 1707–1718. (b) Li, X. H.; Zhang, G. X.; Ma, H. M.; Zhang, D. Q.; Li, J.;
Zhu, D. B. J. Am. Chem. Soc. 2004, 126, 11543–11548. (c) Chen, X. Q.; Sun, M.;
Ma, H. M. Curr. Org. Chem. 2006, 10, 477–489.
(e) Suresh, M.; Mishra, S.; Mishra, S. K.; Suresh, E.; Mandal, A. K.; Shrivastav,
A.; Das, A. Org. Lett. 2009, 11, 2101–2104. (f) Li, K. H.; Xu, Z. T.; Fettinger,
J. C. Inorg. Chem. 2004, 43, 8018–8022. (g) Chatterjee, A.; Santra, M.; Won, N.;
Kim, S.; Kim, J. K.; Kim, S. B.; Ahn, K. H. J. Am. Chem. Soc. 2009, 131, 2040–
2
041. (h) Coskun, A.; Akkaya, E. U. J. Am. Chem. Soc. 2005, 127, 10464–1046.
(i) Swamy, K. M. K.; Kim, H. N.; Soh, J. H.; Kim, Y.; Kima, S. J.; Yoon, J. Chem.
Commun. 2009, 1234–1236. (j) Ho, M. L.; Chen, K. Y.; Lee, G. H.; Chen, Y. C.;
Wang, C. C.; Lee, J. F.; Chung, W. C.; Chou, P. T. Inorg. Chem. 2009, 48, 10304–
1
0311.
pubs.acs.org/IC
Published on Web 12/30/2009
r 2009 American Chemical Society

Article
Inorganic Chemistry, Vol. 49, No. 3, 2010 1207
Scheme 1. Synthesis of RBSe
China] was used for pH measurements. Fluorescence imaging
experiments were performed on a FV 1000-IX81 confocal laser
scanning microscope (Olympus, Japan) with FV5-LAMAR for
excitation at 515 nm and a variable bandpass emission filter
set to 530-630 nm through a 40 ꢀ 0.9 NA objective. Optical
sections were acquired at 0.8 μm.
Rhodamine B, selenium powder and AgNO were purchased
3
from Beijing Chemical Company. Mercuric choride was ob-
tained from Tianjin Jingjin Chemical Reagent Plant. All other
chemicals used were local products of analytical grade. Distil-
led-deionized water was used throughout. The stock solution
7
,8
spirocyclic structure. However, most of the rhodamine
derivatives are spirolactams (constructed through a pH-
sensitive N atom), which readily suffer from influence of
(
1.0 mM) of RBSe was prepared by dissolving the requisite
amount of it in 1,4-dioxane. Stock solutions (1-100 mM) of
other species were prepared by dissolving their compounds in
water or acidic solutions.
7c
acidity. To overcome the problem, we previously synthe-
sized rhodamine B thiolactone by replacing the N atom with
þ
2þ
Synthesis of RBSe. To a stirred solution of rhodamine B
a S atom (a weak H -receptor), which can serve as a Hg
-
(
239 mg, 0.5 mmol) in 1,2-dichloroethane (5 mL) was added
phosphorus oxychloride (0.3 mL) dropwise. After refluxing for
h, the reaction solution was cooled and evaporated in vacuo to
selective fluorescent probe with higher tolerance against pH
changes in aqueous media. Nevertheless, the probe is
unsuitable to be used for comparing the interacting behaviors
of Hg and Ag with live cells by fluorescence imaging
because of its insensitivity to Ag .
8a
4
give rhodamine B acid chloride as violet-red oil, which was used
directly in the next step. Separately, a suspension of selenium
powder (79 mg, 1.0 mmol) in dry tetrahydrofuran (THF, 10 mL)
2þ
þ
þ
þ
On the basis of the fact that selenium is a weaker H -
was prepared, and then LiAlH (38 mg, 1.0 mmol) was added
4
receptor and has a strong affinity for both mercury and
at 0 °C under an argon atmosphere. The mixture was stirred at
9
0
°C under an argon atmosphere for 30 min, yielding a hetero-
silver, herein we design rhodamine B selenolactone (RBSe;
2þ
þ
geneous grayish suspension. A solution containing crude rho-
damine B acid chloride in 6 mL of THF was added dropwise to
the selenium powder suspension. After stirring overnight at
room temperature, the solvent was removed under reduced
pressure to give a violet-red oil. Then, 5 mL of water was added
to the oil, and the formed precipitate was filtered. The precipi-
tate was washed several times with water and dried in air to give
a violet-red powder. The crude product was purified by silica-gel
column chromatography with petroleum ether (60-90 °C)/ethyl
Scheme 1) as a Hg - and Ag -specific fluorescent probe by
incorporating a Se atom into the spirocyclic structure of
rhodamine. The probe itselfis nearly non-fluorescent because
of its selenospirocyclic character. Upon addition of various
species, however, the probe produces a rapid and specific
2
þ
þ
fluorescence-on reaction with Hg or Ag only. Most
þ
notably, the probe is capable of reacting with Ag in the
presence of a high concentration of Cl because Se has a
-
þ
- 10
acetate (25:1, v/v) as eluent, affording 101 mg of RBSe (yield
stronger binding ability toward Ag than does Cl .
Furthermore, the probe has been successfully used to com-
1
4
0%). Mp 173-174 °C. H NMR (300 MHz, CDCl
3
, 298 K)
2
þ
þ
δ 7.85 (dd, J = 7.5 Hz, 0.7 Hz, 1H), 7.52-7.41 (m, 2H), 7.26 (d,
pare the imaging behaviors of Hg and Ag in Hela cells to
gain a better understanding of their different toxicities.
J = 7.5 Hz, 1H), 6.70 (d, J = 8.7 Hz, 2H), 6.32-6.27 (m, 4H),
13
.32 (q, J = 6.9 Hz, 8H), 1.16 (t, J = 6.6 Hz, 12H); C NMR
3
(
1
1
3
300 MHz, CDCl , 297 K) δ 201.2, 158.6, 152.3, 148.3, 140.9,
Experimental Section
33.9, 130.0, 128.4, 128.0, 122.5, 111.2, 108.3, 97.7, 62.6, 44.3,
2.6; ESI-MS m/z 507.2 [M þ H] . Elemental analysis, calcd. for
þ
Apparatus and Reagents. A Hitachi F-2500 spectrofluori-
meter was used for fluorescence measurements. The absorption
spectra were recorded with a TU-1900 spectrophotometer
30 2 2
RBSe (C28H N O Se), C 66.53, H 5.98, N 5.54%; found, C
6
a = 16.1745(4), b = 12.1930(2), c = 12.1745(3) A, R = 90°, β =
9
6.14, H 5.98, N 5.71%. Crystal data for RBSe: orthorhombic,
˚
(
Beijing Purkinje General Instrument Co. LTD). NMR spectra
were measured on a Bruker DMX-300 spectrometer at 300 MHz
in CDCl with tetramethylsilane as the internal standard. Elec-
3
˚
0°, γ = 90°, V = 2401.00(9) A , T = 173(2) K, space group
-3
c
Pna2(1), Z = 4, D = 1.398 g cm , 5287 reflections measured,
3
R1 [I > σ(I)] = 0.0373, wR2 (all data) = 0.0900, GOF = 0.513.
CCDC no. 737187. These data can be obtained free of charge
from The Cambridge Crystallographic Data Centre via www.
ccdc.cam.ac.uk/data_request/cif.
trospray ionization (ESI) mass spectra were measured with an
LC-MS 2010A (Shimadzu) instrument. Elemental analyses were
carried out with a Flash EA 1112 instrument. A single crystal
was characterized on an R-AXIS Rapid IP (Rigaku). A Delta
3
20 pH-meter [Mettler-Toledo Instruments (Shanghai) Co.,
General Procedure for Detection of Metal Ions. Unless other-
wise noted, all the measurements were made according to the
following procedure. In a 10 mL glass tube, 5 mL of 20 mM
HEPES buffer (pH 7.2) and 50 μL of the stock solution of RBSe
were mixed, followed by addition of an appropriate volume of
(
7) (a) Dujols, V.; Ford, F.; Czarnik, A. W. J. Am. Chem. Soc. 1997, 119,
7
386–7387. (b) Kim, H. N.; Lee, M. H.; Kim, H. J.; Kim, J. S.; Yoon, J. Chem.
2þ
þ
Soc. Rev. 2008, 37, 1465-1472, and references therein. (c) Beija, M.; Afonso,
C. A. M.; Martinho, J. M. G. Chem. Soc. Rev. 2009, 38, 2410-2433, and
references therein. (d) Zhao, Y.; Zhang, X. B.; Han, Z. X.; Qiao, L.; Li, C. Y.; Jian,
L. X.; Shen, G. L.; Yu, R. Q. Anal. Chem. 2009, 81, 7022–7030.
Hg or Ag sample solution. The final volume was adjusted to
0 mL with the HEPES buffer, and the reaction solution was
1
mixed well. After 5 min at room temperature, a 3 mL portion of
the reaction solution was transferred to a quartz cell of 1 cm
optical length to measure absorbance or fluorescence intensity/
spectrum with λex/em = 520/580 nm and both excitation and
emission slit widths of 10 nm. In the meantime, a blank solution
(
8) (a) Shi, W.; Ma, H. M. Chem. Commun. 2008, 1856–1858. (b) Chen,
X. Q.; Nam, S. W.; Jou, M. J.; Kim, Y.; Kim, S. J.; Park, S.; Yoon, J. Org. Lett.
008, 10, 5235–5238. (c) Zhan, X. Q.; Qian, Z. H.; Zheng, H.; Su, B. Y.; Lan, Z.;
Xu, J. G. Chem. Commun. 2008, 1859–1861.
9) (a) Yoneda, S. J.; Kazuo, T. S. Toxicol. Appl. Pharmacol. 1997, 143,
74–280. (b) Tran, D.; Moody, A. J.; Fisher, A. S.; Foulkes, M. E.; Jha, A. N.
Aquat. Toxicol. 2007, 84, 11–18.
10) (a) Mehra, M. C.; Gubeli, A. O. Can. J. Chem. 1970, 48, 3491–3497.
b) F. S ꢀe by, F.; Potin-Gautier, M.; Giffaut, E.; Borge, G.; Donard, O. F. X. Chem.
Geol. 2001, 171, 173–194. (c) Cypionka, H. Arch. Microbiol. 1989, 152, 237–
43. (d) Tsipouras, N.; Rix, C. J.; Brady, P. H. Clin. Chem. 1995, 41, 87–91.
2
2þ
þ
(
containing no Hg or Ag was prepared and measured under
the same conditions for comparison.
2
General Procedure for Cell Imaging. The Hela cells were grown
on glass-bottom culture dishes (MatTek Co.) in Dulbecco’s
modified eagle media (DMEM) supplemented with 10% (v/v)
fetal bovine serum, penicillin (100 μg/mL), and streptomycin
(
(
2

1
208 Inorganic Chemistry, Vol. 49, No. 3, 2010
Shi et al.
Figure 2. Fluorescence spectra (λex = 520 nm) of RBSe (5 μM) in 20 mM
HEPES buffer (pH 7.2) containing 0.5% (v/v) 1,4-dioxane in the presence
þ
3þ
2þ
2þ
3þ
2þ
2þ
of various species (50 μM of Ag , Al , Cd , Cu , Fe , Hg , Pb ,
2þ
-
þ 2þ
Zn , H O , or ClO ; 0.2 M of NaCl; 0.15 M of K ; 2 mM of Ca ; 1 mM
; 1.5 mM of
2 2
-
3-
2-
2-
of NO
3
; 50 mM of PO
4
; 30 mM of CO
3
; 10 mM of SO
4
2
þ
Mg or reduced glutathione).
Figure 1. View of the structure of RBSe with displacement atomic
ellipsoids drawn at the 20% probability level, excluding H atoms.
(
100 μg/mL) at 37 °C. Before use, the adherent cells were washed
three times with phenol red-free DMEM. For mercury imaging,
cells were first loaded with 10 μM RBSe in DMEM at 37 °C for 20
min, washed three times with phosphate buffered saline solution
(pH 7.4) to remove the free RBSe, and then incubated in phos-
phate buffered saline media with 10 μM HgCl for 10 or 20 min.
2
-
For silver imaging, unless otherwise stated, a Cl -free buffer of 20
mM HEPES (pH 7.4) containing 0.14 M NaNO was used to
3
avoid the formation of insoluble AgCl precipitates. Hela cells were
prepared in a similar manner as for mercury imaging, except that
the Cl -free buffer was employed for the last cell washing and
-
Figure 3. Change of fluorescence intensity at 580 nm of RBSe (5 μM)
with time in the presence of 10 μM of Hg or Ag in 20 mM HEPES
buffer (pH 7.2) at room temperature.
2þ
þ
AgNO
3
loading.
Results and Discussion
Synthesis and Characterization of RBSe. The probe
RBSe was prepared by treating rhodamine B with phos-
phorus oxychloride, and then letting the formed rhoda-
1
1
mine B acid chloride react with selenium powder in the
presence of LiAlH . The structure of RBSe was con-
4
1
13
firmed by H NMR, C NMR, MS, and X-ray analyses
Figures S1 and S2 in the Supporting Information). The
(
single crystal of the probe was well grown as a yellow
needle from the ethyl acetate/petroleum ether (1:25, v/v)
solution, which has a unique selenospirocyclic structure
(
Figure 1).
Spectroscopic Properties of RBSe. A detailed investiga-
tion was conducted on spectroscopic properties of RBSe
and their variations upon addition of different sub-
stances. Slightly different from rhodamine B thiolactone,
Figure 4. Effect of pH on fluorescence intensities (λex/em = 520/580 nm)
2
þ þ
of RBSe (5 μM) and its reaction solution with 50 μM of Hg or Ag . The
2
12
RBSe has a very weak fluorescence (Φ ≈ 0.01) with a
þ
4
-1
-1
lower fluorescence intensity in the case of Hg may result from its
stronger quenching effect.
molar absorptivity of about 1 ꢀ 10 M cm at 560 nm,
which might result from the different property between Se
and S. Various metal ions, oxidizing and reducing agents
were examined under the same conditions to assess the
specificity of the fluorescence-on reaction. As shown in
fluorescence enhancement (∼10 fold) at about 580 nm
and the retrievement of magenta color characteristic of
rhodamine B. Time course studies revealed that the
reaction was fast and nearly complete in 30 s in both
2
þ
þ
Figure 2, RBSe reacts with only Hg and Ag instead of
the other common species tested, accompanying a great
2
þ
þ
cases of 2 equiv. Hg and Ag (Figure 3); the resulting
absorbance and fluorescence signals remained constant
for at least 24 h. The pH of reaction media varying from 4
to 9 causes negligible changes in the fluorescence inten-
sity, and the probe itself is rather stable over a wider range
of pH (Figure 4). Then, we examined the sensitivity of
(
11) Ishihara, H.; Koketsu, M.; Fukuta, Y.; Nada, F. J. Am. Chem. Soc.
001, 123, 8408–8409.
12) Fluorescence quantum yield Φ was determined according to the
literature: Karstens, T.; Kobs, K. J. Phys. Chem. 1980, 84, 1871–1872.
2
(

Article
Inorganic Chemistry, Vol. 49, No. 3, 2010 1209
Figure 5. Changeinfluorescence intensity(ΔF) ofRBSe(5 μM) reacting
þ
2þ
þ
with Ag and Hg for 5 min at varied concentrations. ΔF = F - F
where F and F are the fluorescence intensity at 580 nm before and after a
species was added to the probe solution, respectively.
0
,
Figure 6. Fluorescence spectra of RBSe (5 μM) reacting with Ag for
5 min in the environment of high Cl concentration. (a) RBSe in 0.15 M
NaCl aqueous media without Ag (control experiment). (b) RBSe was
-
0
þ
3 3
added tothe mixture of0.15MNaCl and10μMAgNO . (c) AgNO (final
concentration 10 μM) was added to the mixture of 0.15 M NaCl and 5 μM
RBSe. (d) The spectrum of the sample (b) was recorded after 4 h. (e) The
spectrum of the sample (c) was recorded after 4 h. (f) RBSe in the presence
2
Scheme 2. Possible Reaction Mechanism of RBSe with Hg or Ag
þ
þ
3
of 10 μM AgNO without NaCl.
that of sample (f). This is due to the higher affinity of
Se than Cl for Ag , since the solubility product
-
þ
-
56
of Ag Se (K = 1.6 ꢀ 10 ) is much lower than that
2
sp
-
10 10
of AgCl (K = 1.56 ꢀ 10 ).
sp
2
RBSe to Hg and Ag . The change in fluorescence
þ
þ
Cell Imaging. The probe RBSe is a less polar lactone
derivative, which is favorable for penetrating the cell
membrane; while its reaction product, rhodamine B, is a
more polar zwitterion, which would facilitate its stay in
cells. Together with the above observations, it can be thus
concluded that RBSe not only displays a highly selective
intensity of RBSe (5 μM) was found to be directly
2
þ
proportional to the concentration of 0.1-5 μM Hg
þ
2þ
and 0.1-10 μM Ag , with detection limits of 23 nM Hg
þ
and 52 nM Ag (S/N = 3), respectively. Although the
corresponding stability constants can not be obtained
because of the irreversibility of the reactions (vide infra),
the observed apparent stoichiometric ratios of RBSe to
Hg and Ag were 1:1 and 1:2 (Figure 5), respectively,
which are in agreement with those in their typical com-
plexes HgSe and Ag Se.
2
þ
and sensitive fluorescence response to both Hg and
Ag in water but also may be suitable for imaging these
þ
2
þ
þ
two ions in cells. Figure 7 shows fluorescence images of
RBSe-loaded Hela cells in the absence and presence of
þ
þ
Ag . Surprisingly, after treatment for 10 min, Ag only
switches on fluorescence of the cell surface. A parallel
experiment for Hg with the same incubation time of 10
2
Reaction Mechanism. To study the reaction mechanism
of the present system, the reaction products of RBSe with
2
þ
2þ
2
þ
þ
Hg and Ag were subjected to electrospray ionization
mass spectral analysis, confirming the generation of
min reveals that Hg can turn on the fluorescence of the
whole cell (Figure S4 in the Supporting Information).
Moreover, in the case of Hg , we cannot capture the
þ
2þ
rhodamine B (m/z = 443 [MþH] ) as a major final
product (Figure S3 in the Supporting Information); the
introduction of KI into the system can not produce a
colorless solution. This indicates that the reactions of
similar image that fluoresces only on the surface but
not inside, even if image time interval was decreased to
0.5 min. Nevertheless, a longer incubation time of 20 min
leads to a brighter and broader fluorescence image in both
8
2
þ
þ
RBSe with Hg and Ag are irreversible, and may
proceed through the following way: the selenium atom
of RBSe recognizes and binds Hg and Ag , and the
2
þ
þ
cases of Hg and Ag (Figure S4 in the Supporting
Information). The above results indicate that the reaction
rate of Ag with RBSe is noticeably slower than that of
2
þ
þ
2
þ
þ
þ
subsequent complexation of Hg
or Ag promotes
2
þ
hydrolytic cleavage of the selenolactone bond, causing
the release of the rhodamine B and thereby the retrieval of
the fluorescence (Scheme 2).
Hg in intracellular environments. The possible reason
for this phenomenon is that the high concentrations of
cellular chloride ions inhibit the fast formation of suffi-
þ
-
þ
Reaction of RBSe with Ag in the Presence of Cl . We
investigated the reaction behavior of RBSe with Ag in
the environment of high Cl concentration. As can be
cient free Ag , which is consistent with our observation in
the above in vitro experiments (Figure 6). Moreover,
þ
-
-
higher extracellular Cl concentrations (above 100 mM)
in intact multicellular organisms even prevent free Ag
þ
seen from Figure 6, the spectrum of sample (b) is nearly
identical to that of sample (c), which indicates that, in
the presence of 0.15 M Cl , RBSe can still react with
from accessing cells effectively (Figure S5 in the Support-
ing Information), thus scarcely showing fluorescence
enhancement. However, it is understandable that, in
-
þ
trace free Ag dissociated from the AgCl precipitate,
-
and the reaction is not affected by their adding order.
The results from samples (d) and (e) show that, with
the increase of reaction time, RBSe can gradually strip
common aqueous environments containing lower Cl
concentration, Ag may enter unicellular organisms in
þ
a higher concentration, exerting the well-known antimi-
crobial effect.
þ
Ag from AgCl, though the reaction rate is slower than

1
210 Inorganic Chemistry, Vol. 49, No. 3, 2010
Shi et al.
þ
Figure 7. Confocal fluorescence images of Ag in Hela cells. (A) Hela cells loaded with 10 μM RBSe for 20 min. (B) RBSe-loaded cells incubated with
1
þ
0 μM Ag for 10 min. (C) The corresponding differential interference contrast (DIC) image of sample B. Scale bars, 50 μm.
Conclusion
evidence to evaluate the claimed safety of silver as a disin-
fectant and medicine.
In summary, RBSe with a unique selenospirocyclic struc-
ture has been developed as a new fluorescent probe, which
Acknowledgment. We thank Prof. X. H. Fang and Prof.
X. Y. Jiang for culturing cells. This work is supported by
grants from the NSF of China (Nos. 20935005, 90813032,
20875092), the 973 Program (2010CB933502), and the
Chinese Academy of Sciences.
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can produce a fluorescence-on reaction with both Hg and
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Ag through deselenation. Fluorescent confocal imaging
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reveals for the first time that the reaction of Ag with RBSe
in live cells is much slower than that of Hg , which is
ascribed to the high concentration of cellular chloride ions
prohibiting the presence of sufficient free Ag . These differ-
ent interactions in cells may be responsible for the very
2
Supporting Information Available: X-ray structural data of
RBSe in CIF format, NMR spectra of RBSe, electrospray
ionization mass spectral analysis, and other supplementary
figures. This material is available free of charge via the Internet
at http://pubs.acs.org.
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different toxicities of Hg and Ag . Further applications
of the probe to various biosystems may provide more direct