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T. Zhang et al. / Tetrahedron 69 (2013) 7102e7106
The fluorescence spectra of Z1 upon addition of Hg2þ were
displayed in Fig. 2b. Free Z1 showed a maximum fluorescence
emission at 656 nm upon excitation at 560 nm in buffer solution
(CH3CN and HEPES (v/v¼4:1, pH¼6.86)). After binding with Hg2þ
,
Hg2þ-Z1 showed a large emission blue-shift of 39 nm, indicating
that a disturbed ICT process. The fluorescence intensity gradually
increased at 617 nm, and the ratio of emission intensities (F617/F710
)
varied from 0.8 to 3.5 (Fig. 2c, the ratio was used to eliminate the
disturbance of Agþ, and the explanation were listed below). This
can be explained by utilizing the ICT processdHg2þ would co-
ordinate with the N atom of the thia aza crown ether, which might
restrict the ICT process. With the increase of Hg2þ concentration,
the fluorescence intensity of the solution increased and the color
changed from red to orange under a UV light (Fig. 2b inset).
According to titration experiments, Job’s plot determined that Z1
and Hg2þ was 1/1 complexion (see Supplementary data). Probe Z1
can detect the Hg2þ concentration at 4.36ꢀ10ꢁ9 M (Limit of de-
tection was calculated according to the method described by
Demchemko.27), which is lower than the upper limit (10 nM) that
the EPA had mandated for Hg2þ in drinking water.
Fig. 4. (a) The fluorescence intensity changes of Z1: the curve (Z1þAgþ) represents
10 equiv of Agþ; the curve (Z1þAgþþHg2þ) represents 5 equiv of Hg2þ was added into
the (Z1þAgþ
) solution. (b) The fluorescence intensity changes of Z1: the curve
(Z1þHg2þ) represents 5 equiv of Hg2þ; the curve (Z1þHg2þþAgþ) represents 10 equiv
of Agþ was added into the (Z1þHg2þ)solution. The concentration of Z1 was 1
mM.
Based on these data, we confirmed that Z1 had higher binding
affinity for Hg2þ (3.19ꢀ106 Mꢁ1 obtained by nonlinear regression
analysis, see Supplementary data) than that for Agþ (7.64ꢀ104 Mꢁ1
obtained by nonlinear regression analysis, see Supplementary
data), which indicated the capacity that Hg2þ could displace Agþ
to form more stable complexes and suggested the mechanism of
sensing Agþ and Hg2þ (Fig. 5). Z1 can successfully distinguish Hg2þ
from Agþ via two different sensing mechanisms, i.e., PET for Agþ
and ICT&PET for Hg2þ. The reason might be that the Agþ does not
get involved into the conjugated system while Hg2þ is involved, Agþ
can only coordinate with four sulfur atoms of Z1 and result in
fluorescence intensity enhancement. While, Hg2þ can coordinate
with not only the four sulfur atoms but also the nitrogen atom and
act as an electron-withdrawing group to regulate the electronic
pushepull system, which consequently leads to fluorescence in-
tensity enhancement and wavelength changes. Further in-
vestigation by leaving Z1 in a solution of pH¼1 has showed that the
emission wavelength hypochromatic shifts similar to Hg2þ (see
Supplementary data). This is because Hþ can easily bind to the
nitrogen atom but to the four sulfur atoms. This experiment also
confirms the previous observation of the Agþ titration.
To obtain an insight into the sensing properties of Z1 toward
metal ions, the fluorescence emission of different ions in
CH3CNeHEPES solution (v/v¼4:1, pH¼6.86) was investigated, and
the results were shown in Fig. 2d. No significant ratio (F617/F710
)
,
changes were observed when Cd2þ, Cr3þ, Cu2þ, Fe2þ, Kþ, Liþ, Mg2þ
Naþ, Ni2þ, Pb2þ, Agþ, Co2þ Ba2þ Ca2þ Mn2þ Fe3þ, and Zn2þ were
added in the sensor solution even at high concentration (50 equiv,
black bars). Completive experiments in the addition of Hg2þ
(5 equiv) showed similar ratio (F617/F710) enhancement (Fig. 2d
red bars). The result suggested that Z1 had good selectivity
toward Hg2þ
.
The emission spectrum changes of Z1 upon the addition of an
increasing amount of Agþ were displayed in Fig. 3a. Free Z1 showed
a maximum fluorescence emission at 656 nm upon excitation at
560 nm in buffer solution (CH3CNeHEPES (v/v¼4:1, pH¼6.86)).
Remarkable fluorescence enhancements were detected along the
addition of Agþ while only a smaller wavelength shift (3 nm) ap-
pears. The disturbance of Agþ was eliminated by ratiometric signals.
Fig. 5. The mechanism of Z1 for sensing Hg2þ and Agþ
.
Fig. 3. (a) Emission spectra of Z1 along with the increased concentration of Agþ
(0e10 equiv) in a CH3CNeHEPES solution (v/v¼4/1, pH¼6.86). The excitation wave-
2.5. Cell imaging
length was 560 nm and the concentration of Z1 was 1 mM. (b) Fluorescence intensity of
Z1 probe in CH3CN and HEPES at pH 6.86 upon gradual addition of Agþ at concen-
trations of 0e10 equiv.
Live cell imaging based on Z1 was investigated with confocal
laser scanning microscopy (CLSM). HeLa cells were utilized to
demonstrate the utility of Z1 for intracellular Hg2þ sensing by cell
2.4. Mechanism of the sensing system
imaging. After incubation with the Z1 solution (5 mM) for 15 min,
the cells were washed three times in PBS buffer. The excitation
wavelength was fixed at 561 nm. Fluorescent signals were collected
from 600 nm to 650 nm. Fig. 6aec shows cell images without the
addition of Z1 in fluorescence image, bright field image, and overlay
image, respectively. Fig. 6def shows the CLSM images of the Z1
stained HeLa cells in fluorescence image, bright field image, and
overlay image, respectively. Weak fluorescence is observed in the
cells. Fluorescence is hardly observed in the merged image.
Fig. 6gei shows the increases of the fluorescence intensity in living
The dual titration of Agþ and Hg2þ was also conducted. Z1 in
CH3CN and HEPES was first titrated with 10 equiv Agþ, and the
fluorescence intensity was significantly enhanced without wave-
length changes. Then 5 equiv Hg2þ was added, the wavelength had
a hypochromatic shift about 39 nm (Fig. 4a). In a contrast experi-
ment, 5 equiv Hg2þ was first added, and the fluorescence intensity
enhanced with a blue-shift of 39 nm. Then 10 equiv Agþ was added,
the fluorescence intensity decreased only a little without wave-
length changes (Fig. 4b).
cells after addition of Hg2þ (50
mM) into the medium and incubated