Xiang Zhou et al.
minimal background emission. However, the TP fluores-
cence increased significantly when the cells were exposed to
2
+
5
0 mm Hg for 3 h. The bright-field image confirmed cell vi-
ability during the imaging experiments. Hence, SAN can be
2
+
used as a probe for sensing Hg in live cells.
2
+
In conclusion, we have developed a Hg -promoted desul-
II
furization reaction for Hg detection by employing a turn-
on two-photon fluorescent probe SAN. Our approach is po-
II
tentially suitable for inorganic and organic Hg sensing with
high specificity from the nanomole to the micromole scale
and live cell imaging. Using the advantages of two-photon
fluorescence, it should be possible to detect trace amounts
2
+
Figure 4. Selectivity studies. Fluorescent responses of SAN toward Hg
2
+
+
2+
2+
2+
+
+
II
and 13 different metal ions (Cd , Li , Mg , Mn , Ni , Na , K ,
of Hg in live tissues.
2
+
2+
2+
+
2+
2+
Ca , Co , Cu , Ag , Zn and Pb ) at 503 nm. cACTHUNGTRENNNUG
2
+
+
A
C
H
T
U
N
G
T
R
E
N
N
U
N
G
(Hg )=5 mm,
cACHTUNGTRENNUNG
buffer (pH 7.4).
Experimental Section
formation, an unperturbed fluorescence response was ob-
Optical Properties Study
2
+
served even when Hg was added to the mixture of various
competing ions, each at a concentration of 50 mm. All these
results undisputedly confirmed that SAN demonstrated an
One-photon fluorescent emission spectra were collected from 400–
600 nm on a PerkinElmer LS 55 instrument with an excitation wave-
length of 380 nm; the excitation and emission slit widths were both 4 nm.
The sample were prepared by mixing SAN, buffer, deionized water, and
2
+
excellent specificity and selectivity towards Hg ions.
To demonstrate the formation of AAN and gain further
2 3
HgCl or CH HgCl with given concentrations to final volume 2 mL. Then
the samples were incubation at 378C for 24 h. Quartz cuvettes with 2 mL
volume were used for emission measurements. For two-photon excitation
experiments, all samples were excited at 760 nm by a mode-locked Ti:-
Sapphire femtosecond pulsed laser (Chameleon Ultra I, Coherent Inc.)
with a pulse width of 140 fs at a repetition rate of 80 MHz. Photolumi-
nescence was recorded on a DCS200PC Photon Counting with single-
photon sensitivity through an Omni-l5008 monochromator (Beijing Zolix
Instruments Co., Ltd). UV/Vis absorption spectra were collected on a
SHIMADZU UV-2550 spectrophotometer from 200 to 700 nm with
2
+
insights into the mechanism of Hg sensing, two experi-
2
+
ments were performed. SAN was treated with Hg ions for
.5 h, and the reaction product was purified using column
0
1
chromatography. The H NMR spectrum of isolated product
identified it as AAN (Figure S1b). We also investigated the
UV/Vis absorbance change during the reaction. Addition of
2
+
Hg ions to the SAN solution resulted in a red shift of the
maximum absorption wavelength, from approximately 295
to 355 nm, in accordance with the pull–push ICT effect of
the two-photon product (AAN; Figure S4 in the Supporting
Information). Based on these results, it is conceivable that
6
00 mL quartz cuvettes. Unless otherwise specified, all spectra were taken
at an ambient temperature in 10 mm phosphate-buffered saline (PBS) at
pH 7.4.
Selectivity Experiments
2
+
Hg
converts the protected thioketal group to the acyl
2+
The Hg selectivity of SAN was evaluated in comparison with thirteen
2
+
+
2+
2+
2+
+
+
2+
2+
group, leading to the formation of AAN and fluorescence
turn-on.
other metal ions (Cd , Li , Mg , Mn , Ni , Na , K , Ca , Co ,
+
2
2+
2+
Cu , Pb , and Zn ) under similar conditions. Fifteen samples with or
without different metal ions were incubated at 378C for 24 h. The fluo-
rescent emission intensity at 503 nm was used to plot a histogram. For
mixture samples containing various competing ions, each at a concentra-
tion of 50 mm, fluorescent response was detected in the absence or pres-
2
+
We next sought to utilize SAN as a TP probe for Hg de-
tection in live-cell environments (Figure 5). The TPM image
of the HeLa cells labeled with 5 mm SAN at 378C showed
2
+
ence of Hg
.
Cell Culture and TPM Imaging
HeLa human cervical carcinoma cells (CCTCC, China) were cultured in
Dulbeccoꢁs modified Eagleꢁs medium (DMEM, Hyclone, China) supple-
mented with 10% fetal bovine serum (FBS, Hyclone), penicillin
À1
À1
(
100 unitsmL ), and streptomycin (100 mgmL ). All the cells were
maintained in a humidified atmosphere of 5:95 (v/v) of CO /air at 378C.
2
One day before imaging, the cells were passed and plated on glass-bot-
tomed dishes (Nest). For labeling, the growth medium was removed and
replaced with DMEM without FBS. The cells were incubated with 3 mm
SAN for 1 h at 378C and were washed three times with DMEM without
FBS. Then 50 mm HgCl
2
was added to the cells, which were imaged after
3
h. As a control, another dish of cells was incubated with 3 mm SAN for
Figure 5. Two-photon microscopy. TPM images (a, d), bright-field images
1 h at 378C and then incubated with DMEM for 3 h. Two-photon fluores-
cence microscopy images of SAN-labeled cells and tissues were obtained
with spectral confocal and multiphoton microscopes (Zesis LSM
710NLO) with a ꢂ40 (NA=0.30 DRY) objective lens. To obtain images
at 500–550 nm, internal photomultiplier tubes were used to collect the
signals in 8-bit unsigned 1024ꢂ1024 pixels at 400 Hz scan speed.
(
b, e), overlay of TPM and bright-field images (c, f) of HeLa cells. HeLa
cells were labeled with a) SAN (5 mm) for 4 h, d) SAN (5 mm) for 1 h and
2
+
subsequently treated with Hg (50 mm) for 3 h. The TP fluorescence was
collected at 500–550 nm upon excitation at 780 nm. Cells shown are rep-
resentative images from replicate experiments (n=4).
Chem. Asian J. 2012, 7, 915 – 918
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemasianj.org
917