A cyanine-modified nanosystem for in vivo upconversion luminescence bioimaging of methylmercury

Article abstract of DOI:10.1021/ja403798m

Methylmercury (MeHg⁺) is a strong liposoluble ion, which can be accumulated in the organs of animals and can cause prenatal nervous system and visceral damage. Therefore, the efficient and sensitive monitoring of MeHg ⁺ in organisms is of great importance. Upconversion luminescence (UCL) detection based on rare-earth upconversion nanophosphors (UCNPs) as probes has been proved to exhibit a large anti-Stokes shift, no autofluorescence from biological samples, a remarkably deep penetration depth, and no photobleaching. In this study, a hydrophobic heptamethine cyanine dye (hCy7) modified by two long alkyl moieties and amphiphilic polymer (P-PEG)-modified nanophosphors (hCy7-UCNPs) was fabricated as a highly sensitive water-soluble probe for UCL monitoring and bioimaging of MeHg⁺. Further application of hCy7-UCNPs for sensing MeHg⁺ was confirmed by an optical titration experiment and upconversion luminescence live cell imaging. Using the ratiometric upconversion luminescence as a detection signal, which provides a built-in correction for environmental effects, the detection limit of MeHg⁺ for this nanosystem was as low as 0.18 ppb. Importantly, the hCy7-UCNPs nanosystem was shown to be capable of monitoring MeHg⁺ex vivo and in vivo by upconversion luminescence bioimaging.

Full text of DOI:10.1021/ja403798m

Article
pubs.acs.org/JACS
A Cyanine-Modified Nanosystem for in Vivo Upconversion
Luminescence Bioimaging of Methylmercury
Yi Liu, Min Chen, Tianye Cao, Yun Sun, Chunyan Li, Qian Liu, Tianshe Yang, Liming Yao, Wei Feng,
and Fuyou Li*
Department of Chemistry and The State Key Laboratory of Molecular Engineering of Polymers and Institute of Biomedicine Science,
Fudan University, Shanghai, 200433, P. R. China
*
S Supporting Information
+
ABSTRACT: Methylmercury (MeHg ) is a strong liposoluble ion,
which can be accumulated in the organs of animals and can cause
prenatal nervous system and visceral damage. Therefore, the efficient
and sensitive monitoring of MeHg+ in organisms is of great
importance. Upconversion luminescence (UCL) detection based on
rare-earth upconversion nanophosphors (UCNPs) as probes has
been proved to exhibit a large anti-Stokes shift, no autofluorescence
from biological samples, a remarkably deep penetration depth, and
no photobleaching. In this study, a hydrophobic heptamethine
cyanine dye (hCy7) modified by two long alkyl moieties and amphiphilic polymer (P-PEG)-modified nanophosphors (hCy7-
+
UCNPs) was fabricated as a highly sensitive water-soluble probe for UCL monitoring and bioimaging of MeHg . Further
application of hCy7-UCNPs for sensing MeHg+ was confirmed by an optical titration experiment and upconversion
luminescence live cell imaging. Using the ratiometric upconversion luminescence as a detection signal, which provides a built-in
+
correction for environmental effects, the detection limit of MeHg for this nanosystem was as low as 0.18 ppb. Importantly, the
+
hCy7-UCNPs nanosystem was shown to be capable of monitoring MeHg ex vivo and in vivo by upconversion luminescence
bioimaging.
INTRODUCTION
developed an iridium(III) complex-modified UCNP for
■
−
8c
monitoring intracellular CN . Liu et al. discovered a
combination of UCNPs and manganese dioxide nanosheets
Lanthanide-doped upconversion nanoparticles (UCNPs) can
convert continuous-wave (CW) near-infrared (NIR) excitation
to visible emission with a large anti-Stokes shift of several
9f
for the selective detection of glutathione. Unfortunately, the
above-mentioned UCNPs systems were unsuitable to be
applied in animal bioimaging. Due to the high affinity of
1
−3
hundred nanometers.
Compared to the organic dye and
inorganic semiconductor nanoparticles, UCNPs have been
proved to exhibit superior features, such as no autofluorescence
from biological samples, a remarkable light penetration depth,
lanthanide ions to some anions (such as PO43 and CO3 ),
the chromophore (as energy acceptor) coated on the surface of
UCNPs by coordination interaction will disassociate when the
chromophore-modified UCNPs are injected into the living
animals intravenously. In addition, the responsive UCL
emission of the reported LRET systems belongs to the visible
region, which limits the application of the reported UCL probes
in animal imaging. As a result, no example of UCNPs with both
sensing and imaging function to monitor special ions or small
molecules within living animals has been reported to date.
In light of the fact that the well-known toxic methylmercury
−
2− 10
4
and no photobleaching in bioapplications. As a result, UCNPs
are ideal luminescent probes for visualizing living cells and
5
,6
whole-body animals. To date, several groups have developed
upconversion luminescence (UCL) nanoprobes for small-
animal bioimaging in vivo. However, most of the reported
UCL bioimaging in vivo is focused on biodistribution of the
UCNPs in different organism, because no recognition unit
exists in the UCNPs.
To achieve sensing function, the UCNPs should be
combined with other chromophores with recognition sites,
through the luminescence resonance energy-transfer (LRET)
process. Nowadays, several LRET systems have been developed
+
11,12
+
(
MeHg ) can be accumulated in the organs of animals,
we
are interested in developing UCL bioimaging of MeHg (as an
example of special analyte) in living animals. In this present
study, we demonstrate an upconversion LRET nanosystem
7
8
9
to detect DNA, special ions, and small molecules in solution,
where the UCNPs (donor) transfer energy to the organic
chromophores (acceptor) resulting in changes in UCL
emission. Compared with the rapid development of UCL
solution detection, only three UCL systems combined with
both sensing and imaging function to monitor special analytes
in cell level have been reported. For example, our group
composed of the nanophosphors NaYF : 20% Yb, 1.6% Er,
4
+
0
(
.4% Tm and MeHg -responsible NIR cyanine dye hCy7
hCy7-UCNPs, Scheme 1) for UCL monitoring of MeHg+
Received: April 16, 2013
©
XXXX American Chemical Society
A
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Article
3
+
4
4
Scheme 1. Schematic Illustration of the Synthesis of UCNPs-
hCy7 and Its Sensing to MeHg with a Change in UCL
Emission
= 800 nm) of Tm and F → I transition (λUCL = 660
15/2
9/2
+
3+ 1a
nm) of Er , respectively. Thus, it is reasonable that the
degree of energy transfer could be modulated by the presence
or absence of MeHg+ when Er and Tm -codoped
3+
3+
upconversion nanocrystals (NaYF : Yb, Er, Tm) and hCy7
4
are chosen as the energy donor and acceptor, respectively. To
combine UCNPs and hCy7 together into one nanosystem,
hCy7 was hydrophobized by introducing two long alkyls to
match the hydrophobicity of oleic acid (OA)-coated UCNPs,
and then both were coated with amphiphilic copolymer
through a hydrophobic−hydrophobic interaction. As shown
in Scheme 1, a water-soluble three layer structure nanosystem
(
called hCy7-UCNPs) was composed of an outside hydrophilic
layer of PEG, a middle hydrophobic layer containing hydro
phobic MeHg -sensitive hCy7 (as the energy acceptor), and a
+
core of UCNPs and could display both red and NIR UCL
emission (as the energy donor).
+
Optical Response of hCy7 to MeHg . In previous
12
reports, the thiosemicarbazide subunit could sensitively react
+
with MeHg to form Cy7′, accompanied by significant changes
in photophysical properties. In the present study, the sensing
+
ability of hCy7 for MeHg was investigated in EtOH/H O (4:1,
2
v/v) by both absorption and fluorescence spectroscopy. The
absorption and fluorescence emission spectra of hCy7 in the
absence and presence of MeHg are shown in Figure 1. The
within living small-animal for the first time. The nanoparticles
NaYF : Yb, Er, Tm without and with modification by hCy7
+
4
were here abbreviated to UCNPs and hCy7-UCNPs,
respectively. Using the ratiometric UCL emission at 800 nm
to 660 nm as a detection signal, the detection limit of hCy7-
+
UCNPs for MeHg in aqueous solution was as low as 0.18 ppb.
+
Importantly, the hCy7-UCNPs could monitor MeHg within
the liver and spleen by UCL bioimaging both ex vivo and in
vivo.
RESULTS AND DISCUSSION
■
Design and Principle of the UCL Nanoprobe for
+
MeHg . Our design strategy was based on the fact that the
lanthanide upconversion nanoparticle has no recognition
function but can be used as an energy donor for the LRET
process. To achieve a degree of modulating energy transfer
(
Scheme 2), the emission spectrum of UCNPs and the
+
absorption band of a MeHg -sensitive chromophore should
overlap. As previously reported by Tian et al., the addition of
MeHg induced a significant red shift of the maximum
12
Figure 1. UV−vis absorption (dashes) and photoluminescence (solid)
+
+
spectra of hCy7 and in the absence and presence of MeHg (excitation
3
+
with 730 nm). The range of main UCL emission bands of Er and
absorption peaks of the NIR chemodosimeter Cy7 from 670
to 845 nm. These two absorption band peaks at 845 and 670
Tm3 is also shown with different color.
+
3
3
nm matched the UCL emissions of H → H transition (λ
4
6
UCL
+
addition of increasing amounts of MeHg to a solution of hCy7
Scheme 2. Schematic Illustration of the Upconversion LRET
Process from the Upconversion Emission of UCNPs to the
Absorption of the Dye hCy7 or hCy7′
induced significant changes in its absorption spectrum. The
5
−1
−1
absorbance at 670 nm (ε = 0.9 × 10 M cm ) decreased
5
−1
−1
gradually, and absorbance at 845 nm (ε = 1.5 × 10 M cm )
gradually increased with an isosbestic point at λ =735 nm,
leading to an evident color change from blue to weak green
(
Figure S4), which indicated a reaction between hCy7 and
+
MeHg . The isosbestic point at 735 nm clearly showed that
only two organic species (free hCy7 and cyclized product
hCy7′) coexisted. In the cyclization process, the central
bridging secondary amine (N1 atom in Scheme S2) of hCy7
was transformed into the tertiary amine imidazole of hCy7′.
The product of the mercury-promoted cyclization reaction was
confirmed by MALDI-TOF-MS (Figure S3). The peak at m/z
1
194.77 (calculated as 1194.78) was ascribed to hCy7, and the
peak at m/z 1160.85 (calculated as 1160.79) was assigned to
the cyclized product hCy7′. Furthermore, DFT calculations
12
B
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Journal of the American Chemical Society
Figure S5) indicated that this cyclization reaction resulted in a
Article
(
transition of electron donor from the central bridging
secondary amine group (N1 atom in Scheme S2) to the indole
amine group (N2 atom in Scheme S2), corresponding to the
conjugation enhancement in the cyclization product. As a
result, a large red-shift in the absorption spectrum with visible
+
color change was observed upon addition of MeHg into the
hCy7 solution. In addition, at the concentration of hCy7 used
+
in this study, the detection limit of hCy7 for MeHg was ∼200
ppb.
+
Moreover, the addition of MeHg to hCy7 resulted in a
prominent emission change from 800 to 820 nm (Figure 1).
12
Furthermore, the competition experiment indicated that very
weak variations in absorption and fluorescence emission of
hCy7 were observed after the addition of an excess of other
nonmercuric metal ions (Figure S6). These results indicated
that hCy7 can serve as a sensitive colormetric and fluorescence
+
indicator for MeHg .
Synthesis and Characterization of hCy7-UCNPs. In
order to fabricate a composite material of UCNPs and hCy7, a
polymer-assisted self-assembly method was utilized through a
8
c,13
hydrophobic−hydrophobic interaction,
and the protocol is
represented by Scheme 1. The hexagonal OA-coated UCNPs in
diameter of ∼25 nm were prepared by the solvothermal
3a,6b
method with OA as the surface ligand.
Due to the presence
of OA on the surface of the nanoparticles, the OA-UCNPs were
hydrophobic. The amphiphilic polymer and hydrophobic hCy7
were cooperatively modified on the surface of the UCNPs
(core) to obtain a water-soluble three-layer structure nano-
system of hCy7-UCNPs (Scheme 1). This nanosystem was
composed of an outside hydrophilic layer of PEG, a middle
hydrophobic layer containing hCy7, and a core of UCNPs.
Transmission electron microscopy (TEM) images indicated
no significant changes in size, shape, and crystallinity of UCNPs
after modification with hCy7 dye. As shown in Figure 2, the
TEM images indicated that both OA-UCNPs and hCy7-
UCNPs were uniform with an average diameter of ∼25 nm and
that no significant aggregates were observed for hCy7-UCNPs
after polymer coating. Moreover, the X-ray diffraction peaks of
UCNPs correlated well with the hexagonal structure of NaYF4
Figure 2. TEM (a) and HR-TEM (b) images of OA-UCNPs. TEM
(c) and HR-TEM (d) images of hCy7-UCNPs. (e) X-ray diffraction
pattern of UCNPs (NaYF : 20% Yb, 1.6% Er, 0.4% Tm). The standard
4
pattern of β-NaYF (JCPDS no.16-0334) is also shown.
4
surface of the hCy7-UCNPs (Scheme 1). This indicated that
the PEG layer existed outside the surface and hCy7 was
successfully doped into the hydrophobic inner layer. By
measuring the absorbance at 670 nm with the UV−vis
absorption technique, the loading amount of hCy7 on the
hydrophobic layer of the hCy7-UCNPs was determined to be
∼13.6 wt % (Figure S10), which indicated that hCy7 could be
(JCPDS No.16-0334), which was also confirmed by the high-
resolution transmission electron microscopy (HRTEM) image
(
Figure 2d).
4
The assembly of the organic dye hCy7 and amphiphilic
calculated as ∼1.66 × 10 units per hCy7-UCNP nanoparticle
polymer P-PEG (Scheme 1) on the surface of the UCNPs was
or ∼6.57 units per luminescence center (the total number of
1
3+
3+
8b
investigated by FTIR and H NMR spectra. In the FTIR
Er and Tm ions). Moreover, the hCy7-UCNPs showed
sufficient stability in water and PBS. In contrast, the loading
amount of the two short ethyl chain-modified Cy7 was ∼3.4%
(Figure S11), much lower than that of the long-chained hCy7.
This fact indicated the introduction of the long-chained hCy7
on the surface of UCNPs was important to improve the loading
spectrum of OA-UCNPs (Scheme S8), the bands at 3344 and
1
1
636 cm− were assigned to OH (asymmetric and symmetric)
stretching and δ(CO), respectively. The bands at 2925 and
1
2
854 cm− were attributed to asymmetric and symmetric C−H
stretching, indicating the presence of OA on the surface of the
OA-UCNPs. When compared with the spectrum of OA-
UCNPs, new peaks at 1130, 1703, and 1731 cm− were
attributed to the characteristic features of the amides and
thiourea groups in the P-PEG and hCy7 appearing in the hCy7-
+
efficiency of MeHg -response dye.
1
LRET Efficiency. Under CW excitation at 980 nm, the
hCy7-UCNPs showed four UCL emission bands at 514−534,
2
534−560, 635−680, and 800 nm, attributed to the H11/2
8c
4
4
4
4
4
3+
UCNPs (Figure S8), indicating that the organic hCy7 and P-
PEG were successfully assembled on the surface of the
→ I15/2, S3/2 → I15/2, and F9/2→ I15/2 transitions of Er and
1a
3
3
3+
H → H transition of Tm , respectively, as illustrated in
4 6
1
nanoparticles by self-assembly. In the H NMR measurement
Figure 3. Compared with the UCL emission spectrum of OA-
UCNPs, significant quenching (10-fold) in UCL emission at
635−680 nm was observed for hCy7-UCNPs. The LRET
efficiency was measured to be ∼90.0%, as deduced from the
upconversion emission spectra of hCy7-UCNPs. In contrast,
simple physical mixing of OA-UCNPs and hCy7 led to only a
of hCy7-UCNPs (Figure S9), the appearance of the chemical
shifts both in the low field assigned to the aromatic protons of
hCy7 and in the high field attributed to the protons (−CH −
2
14
O−) in the polymer P-PEG demonstrated that the organic
dye hCy7 and P-PEG were successfully assembled on the
C
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Journal of the American Chemical Society
Article
+
findings indicated that a reaction between MeHg and the
hCy7 in the hydrophobic layer of hCy7-UCNPs existed.
Moreover, this interaction between hCy7-UCNPs and
+
MeHg also affected the upconversion emission signals by the
+
LRET process. Following the addition of MeHg , UCL
4
4
3+
intensity at 660 nm ( F9 → I
transition of Er ) increased
/2
15/2
3
3
significantly, and UCL emission at 800 nm ( H → H
transition of Tm ) decreased for hCy7-UCNPs under
excitation at 980 nm (Figure 5a). The UCL emission change
4
6
3
+
Figure 3. UCL emission spectra of OA-UCNPs, hCy7-UCNPs, and
hCy7′-UCNPs under excitation at 980 nm.
0.05-fold decrease in the red emission at 660 nm. These results
indicated that the quenching effect in hCy7-UCNPs was mainly
ascribed to the LRET process rather than simply absorbed light
process by hCy7. Moreover, in the Cy7-UCNPs nanosystem,
relative weak quenching in red UCL emission was measured
with a LRET efficiency of ∼23% (Figure S11b). These facts
indicated that the long-chained hCy7 was necessary to be
introduced into upconversion nanosystem to enhance the
LRET efficiency.
+
Sensing of hCy7-UCNPs for MeHg . The sensing ability
+
of hCy7-UCNPs for MeHg was investigated by both UV−vis
absorption and UCL emission spectroscopies. In the UV−vis
absorption spectrum, the hCy7-UCNPs showed a broad visible
band with the maximum absorption wavelength at 670 nm
(Figure 4a). Following addition of MeHgCl, this absorption
Figure 5. (a) Upconversion luminescence spectra of 0.05 mg·mL−
1
hCy7-UCNPs in the aqueous solution upon gradual addition of
MeHg (from 0 to 8 equiv). Inset: the photos showing change in the
+
red UCL emissions. (b) The UCL emissions at 660 ± 2 nm and 800 ±
+
2
nm as a function of MeHg concentration. (c) The ratio of the UCL
+
emission at 660 ± 2 nm to 800 ± 2 nm as a function of MeHg
concentration.
+
of hCy7-UCNPs following addition of MeHg was attributed to
the LRET process. It can be seen from Figure 4 that following
+
the addition of MeHg , the spectral overlap between the red
UCL emission (λem = 635−680 nm) of UCNPs and absorption
(
λabs = 670 nm) of hCy7 was reduced, causing a decrease in the
LRET from the red UCL of UCNPs to hCy7. Consequently,
the red UCL emissions of hCy7-UCNPs increased in the
presence of MeHg (Figure 5a). Nevertheless, spectral overlap
+
Figure 4. (a) Changed in the absorption spectra of 0.05 mg·mL−
1
between the NIR UCL emission (λem = 800 nm) of UCNPs
and absorption (λabs = 845 nm) of hCy7′ was increased, leading
to an increase in the LRET from the NIR UCL of UCNPs to
hCy7′. As a result, the NIR UCL emissions of hCy7-UCNPs
hCy7-UCNPs in the aqueous solution upon gradual addition of
MeHg ions (from 0 to 8.0 equiv, 56.0 μmol in total). (b) Color
change of the aqueous solution of hCy7-UCNPs in the presence of
various representative metal ions.
+
+
stopped following the addition of MeHg (Figure 5a). Using
UCL emission at 800 and 660 nm as detection signals (Figure
+
5
b), the detection limits for MeHg in aqueous solution were
peak was gradually red-shifted from 670 to 845 nm,
corresponding to the color change from blue to green (Figure
measured to be ∼0.80 and 0.82 ppb, respectively.
It should be noted that during this interaction process with
+
4
b). Such a significant red-shift (∼175 nm) in the absorption
MeHg , no significant change in the UCL intensity at 534−560
+
band of hCy7-UCNPs following addition of MeHg was in
agreement with the photophysical change in the pure organic
nm was measured for hCy7-UCNPs, thus the green UCL
emission band could be used as the reference standard. Herein,
the ratio of UCL intensities at 540, 660, and 800 nm, that is,
+
dye hCy7 when interacted with MeHg (Figure S4). These
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UCL660 nm/UCL540 nm, UCL800 nm/UCL540 nm, and UCL660 nm
/
UCNPs was investigated by the reduction activity of the methyl
thiazolyl tetrazolium (MTT) assay. Following incubation of
UCL800 nm) was also chosen as the detection signals to improve
the signal stability and the detection limit. The variation in
UCL ratio (UCL660 nm/UCL540 nm, UCL800 nm/UCL540 nm, and
−1
100−600 μg mL hCy7-UCNPs for 48 h, no significant
difference in the proliferation of HeLa and KB cells was
+
−1
UCL660 nm/UCL800 nm) versus the addition of MeHg is shown
observed (Figure S21). After incubation with 500 μg mL
in Figure 5c and Figure S13−S15. Using UCL660 nm/UCL540 nm
,
hCy7-UCNPs for 48 h, the cellular viability of KB and HeLa
cells was >85% or 80%, respectively, indicating that the hCy7-
UCNPs had low cytotoxicity.
UCL8 /UCL , and UCL /UCL800 nm as detection
00 nm
540 nm
660 nm
+
signals, the detection limits of hCy7-UCNPs for MeHg in
aqueous solution were measured to be ∼0.58, 0.25, and 0.18
ppb, respectively, which were significantly lower than those
when using UCL intensity-based detection (Figure S16−S17).
These facts indicated that ratiometric UCL detection is more
favorable.
To demonstrate the applicability of the hCy7-UCNPs in
+
monitoring intracellular MeHg , laser-scanning upconversion
4b
luminescence microscopy (LSUCLM) experiments were
carried out (Figure 7). As determined by LSUCLM, HeLa
For an excellent chemodosimeter nanosystem, high
selectivity and photostability are very important. To validate
the selectivity of hCy7-UCNPs, some other metal ions
+
+
2+
2+
including some alkali (K , Na ), alkaline earth (Ca , Mg ),
and transition-metal ions were tested under the same
conditions as that of MeHg+ by both absorption and
upconversion emission spectroscopy (Figure S18−S19). Only
+
the addition of MeHg resulted in a prominent change in
absorbance and UCL emission, whereas very weak variations
were observed following the addition of an excess of other
metal ions (Figure S19). Moreover, competition experiments
+
Figure 7. Ratiometric UCL images in living HeLa cells (top, a−d) and
were carried out by adding MeHg to solutions of hCy7-
+
12
50 μM MeHg -pretreated Hela Cells (bottom, e−h) incubated with
UCNPs in the presence of other cations. In the absence or
presence of other cations, significant spectral changes were+
observed for hCy7-UCNPs following addition of MeHg
−1
0
.05 mg mL hCy7-UCNPs for 2 h at 37 °C. Emission was collected
by green UCL channel at 500−560 nm (a and e) and red channel at
6
00−700 nm (b and f), under excitation at 980 nm. (c and g) Overlay
(
Figure S20). These results indicated that the sensing of
of bright-field, green and red UCL imagings. (d and h) Ratiometric
+
hCy7-UCNPs for MeHg was not affected by these common
UCL images with ratio of red to green channels.
coexistent ions. Therefore, hCy7-UCNPs can act as a highly
selective and sensitive ratiometric UCL indicator for MeHg .
+
−
1
cells incubated with hCy7-UCNPs (0.05 mg mL ) for 1 h at
7 °C showed only a weak UCL emission at 660 nm. When the
Furthermore, the photostabilities of hCy7-UCNPs under
illumination of 980 and 670 nm light were investigated. Under
continuous illumination using a 980 nm laser or 670 nm lamp
3
+
cells were supplemented with 50 μM MeHg in the growth
medium for 1 h at 37 °C and then incubated with hCy7-
UCNPs under the same conditions, a strong enhancement in
the red UCL emission was observed in the intracellular region.
Bright-field measurements confirmed that the HeLa cells with
−2
with a power density of 12.6 or 0.3 mW cm for 10 h, the
absorbance of hCy7-UCNPs at 670 nm decreased by 8.9% and
79.0%, respectively (Figure 6). Therefore, using UCL emission
as a detection signal, the hCy7-UCNPs nanosystem showed
significant improvement in photostability, which is more useful
in practical applications.
Monitoring MeHg in Living Cells. Before the application
of hCy7-UCNPs in bioimaging, the cytotoxicity of hCy7-
+
or without treatment with MeHg remained viable throughout
the imaging experiments. Furthermore, in light of the rapid
enhancement of red UCL at 660 nm over green UCL at 540
nm of hCy7-UCNPs when interacted with MeHg , the
+
+
ratiometric UCL imaging was further investigated.
The UCL ratio at the red channel (λUCL = 600−700 nm) to
green channel (λUCL = 500−560 nm) was used as the detection
signal. As shown in Figure 7d, HeLa cells incubated with hCy7-
−1
UCNPs (0.05 mg mL ) for 2 h at 37 °C showed a UCL ratio
+
<
0.4. Following incubation of MeHg -pretreated HeLa cells
−1
with hCy7-UCNPs (0.05 mg mL ), the corresponding UCL
ratio was increased to 0.8 (Figure 7h). These results suggested
that hCy7-UCNPs could be used for monitoring intracellular
+
MeHg with the ratiometric UCL method.
+
UCL Monitoring in Vivo of MeHg . The in vivo
accumulation of MeHg+ is related to the development of
visceral injury, especially liver injury in mammals. As previously
reported, lanthanide UCNPs were enriched in the livers of
1f
living mice following intravenous injection. Herein, the hCy7-
UCNPs nanosystem was further investigated to monitor
MeHg in a mouse model. Male Kunming mice (4 weeks
+
old, ∼20 g) were injected intravenously with hCy7-UCNPs
Figure 6. Photostability of hCy7-UCNPs under continuously
−2
−1
illumination by 980 nm laser (12.6 mW cm ) and 670 nm lamp
(0.2 mL, 0.2 mg·mL in physiological saline) and divided into
−2
(
0.3 mW cm ) for 10 h.
two equal groups. As shown in Figure 8, the left group (control
E
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Journal of the American Chemical Society
Article
increasing incubation time, and no significant change in the
control group was observed. These findings indicated that the+
nanosystem (hCy7-UCNPs) could effectively detect MeHg
both ex vivo and in vivo.
CONCLUSION
■
In summary, we have demonstrated a highly sensitive water-
soluble nanosystem based on cyanine dye-assembled nano-
phosphors for UCL sensing and bioimaging of MeHg ions.
+
+
The cyanine dye hCy7, a MeHg -sensitive dye, was successfully
assembled on the surface of lanthanide UCNPs. This dye-
assembled nanosystem could be used for ratiometric UCL
+
detection of MeHg in aqueous solution with high sensitivity
(
detection limit of 0.18 ppb). This detection limit was lower
Figure 8. (a) In vivo UCL images of 40 μg hCy7-UCNPs-pretreated
than the blood level (5.8 ppb) set by the U.S. EPA.
Importantly, the nanosystem was capable of monitoring
MeHg in living cells and small animals by UCL bioimaging.
Our successful ratiometric UCL-based nanosystem for sensing
and bioimaging MeHg provides a new design strategy for
living mice injected intravenously with 0.2 mL normal saline (left
+
mouce) or 0.1 mM MeHg solution (right mouse). (b) The
+
corresponding UCL images of the livers which were isolated from
the above dissected mice. The UCL emission was collected at 800 ±
+
1
2 nm upon irradiation at 980 nm.
further novel probes for highly sensitive in vivo bioimaging
studies.
group) was injected intravenously with physiological saline (0.2
mL), and the right group was injected intravenously with
MeHg (0.2 mL, 0.1 mM) in saline.
MATERIALS AND INSTRUMENTS
■
+
Materials. All reagents and chemicals were purchased from
commercial sources and used without further purification. 2,3,3-
Trimethylbenzoindolenine, 10-bromoundecanoic acid, phenyl-4-iso-
thiocyanate, OA, 1-octadecane (ODE 90%), 1,2-dichlorobenzene,
UCL imaging in vivo indicated that the hCy7-UCNPs mainly
accumulated in the livers of mice (Figure 8). Compared with
control mice treated with normal saline, the UCL at 800 nm
decreased to 50% of the original value after treatment with
POCl , cyclohexanone, n-butanol, ethylenediamine, and benzene were
3
+
obtained from Alfa Aesar Ltd. Rare earth oxides Y
2 3
O (99.999%),
MeHg (Figure 8a). The mice in the two groups were
Yb O (99.999%), Er O (99.999%), and Tm O (99.999%) were
2
3
2
3
2
3
subsequently dissected to isolate the livers. The UCL emission
purchased from Shanghai Yuelong New Materials Co. Ltd. RECl
+
3
in the liver of MeHg -pretreated mice was weaker than that in
3+
3+
3+
3+
3+
(
RE = Y , Yb , Er , and Tm ) were prepared with the literature
15
the control mice (Figure 8b). Furthermore, the livers of normal
mice were divided into small pieces in another experiment,
method. DMF and deionized water was used to prepare all aqueous
solutions. Solutions of MeHg , Cu , Zn , and Fe ions were
+
2+
2+
2+
+
+
2+
2+
which were then immersed in MeHg solution or in saline for
prepared from their chlorate salts, and solutions of Ag , Ni , Cd ,
2
+
2+
different time periods. As shown in Figure 9, the UCL intensity
Pb , and Co were prepared from their nitrate salts. The amphiphilic
block-polymer poly(maleic anhydride-alt-1-octadecene)-PEG (P-
+
at 800 nm in the MeHg -pretreated group decreased with
14
PEG) was prepared according to the previous literatures. The
intermediates, 1H-Benz[e]indolium, 3-(10-carboxydecyl)-1,1,2-tri-
1
6
methyl bromide (2), 2-chilor-1-formy-3-(hydroxymenthylene)-cyclo-
1
7
18
hex-1-ene (4), and subside thiourea derivative (7), were prepared
according to the previous literatures.
Characterization. The 1H NMR spectra were recorded on a
Bruker spectrometer at 400 MHz. All chemical shifts are reported in
the standard δ notation of parts per million. Electrospray ionization
mass spectra were measured on a Micromass LCTTM system. UV−vis
absorption spectra were recorded on a Shimadzu 3000 spectropho-
tometer. UCL emission spectra were measured on an Edinburgh
FLS920 luminescence spectrometer with an external 3 W adjustable
9
80 nm semiconductor laser (Beijing Hi-Tech Optoelectronic Co.,
China). In our case, all power densities of CW 980 nm excitation for
−2
the UCL measurements were fixed at 45 W cm . FT-IR spectra were
measured using an IR Prestige-21 spectrometer (Shimadzu) from
samples in KBr pellets. X-ray powder diffraction measurements were
performed on a Bruker D diffractometer at a scanning rate of 1°
8
−1
min in the 2θ range of 10−90°, with graphite monochromated Cu
K radiation (λ = 1.5406 nm). TEM images were collected on a JEM
α
2
010 operating at an acceleration voltage of 200 kV. The as-prepared
samples were dispersed in cyclohexane and dripped onto a copper grid
for the TEM tests.
Synthesis of OA-UCNPs. OA-UCNPs were prepared by a
3a,6b
modified solvothermal process according to the reported method.
YCl (0.78 mmol), YbCl (0.20 mmol), ErCl (0.016 mmol), and
Figure 9. UCL imaging (a) and the corresponding change in average
3
3
3
UCL intensity (b) of the hCy7-UCNPs-pretreated livers which were
TmCl (0.004 mmol) were mixed with 6 mL OA and 15 mL ODE in a
3
+
immersed with 0.1 mM MeHg solution (Group 1) or saline (Group
50 mL flask. The solution was heated to 160 °C to form a
homogeneous solution and then cooled down to room temperature.
Eight mL methanol solution containing NaOH (2.5 mmol) and NH4F
2
) for different times (5, 15, and 30 min and 1, 2, and 3 h). The UCL
emission was collected at 800 ± 12 nm upon irradiation at 980 nm.
F
dx.doi.org/10.1021/ja403798m | J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Article
(
4 mmol) was slowly added into the flask and stirred for 30 min.
Cytotoxicity of hCy7-UCNPs. In vitro cytotoxicity was measured
Subsequently, the solution was slowly heated and degassed at 120 °C
for 10 min to remove methanol and then heated to 300 °C and
maintained for 1 h under Ar protection. After the solution was cooled
naturally, nanoparticles were precipitated from the solution with
ethanol and washed with ethanol/cyclohexane (9:1, v/v) for three
times.
by performing MTT assays on the HeLa and KB cells. Cells were
4
seeded into a 96-well cell culture plate at 5 × 10 /well, under 100%
humidity, and were cultured at 37 °C and 5% CO for 24 h; different
2
concentrations of hCy7-UCNPs (0, 100, 200, 300, 400, 500, and 600
μg/mL, diluted in RPMI 1640) were then added to the wells. The cells
were subsequently incubated for 24 or 48 h at 37 °C under 5% CO2.
Then, MTT (10 mL; 5 mg/mL) was added to each well, and the plate
Synthesis of hCy7. The synthetic routine of the dye hCy7 is
shown in the Scheme S1.
was incubated for an additional 4 h at 37 °C under 5% CO . After the
2
Compound 5. Into a flask attached with Dean−Stark trap and a
condenser were added freshly prepared 2-chloro-1-formyl-3-
addition of 100 μL DMSO, the assay plate was allowed to stand at
room temperature for 2 h. The optical density OD570 value (Abs) of
each well, with background subtraction at 690 nm, was measured by
means of a Tecan Infinite M200 monochromator-based multifunction
microplate reader. The following formula was used to calculate the
inhibition of cell growth:
(
hydroxymethylene)cyclohex-1-ene 4 (1.7 g, 10 mmol), 1H-Benz[e]-
indolium, 3-(10-carboxydecyl)-1,1,2-trimethyl-, bromide 2 (7.9 g, 20
mmol), n-butanol (200 mL), and benzene (20 mL). The mixture was
heated to 120 °C for 24h, resulting in a green solution. Solvents were
removed on a rotavapor. Residue was washed with hexane/EtOAc and
eluted with DCM/MeOH from a silica gel plug to give the crude
product. Further chromatography on a silica gel column with gradient
1
9
cell viability(%) = (mean Abs value of treatment group
/
mean Abs value of control) × 100%
DCM/MeOH solvent system led to 14.7 g (60%) of 5 as dark-green
1
LSUCLM Imaging. Experiment to assess MeHg+ uptake was
performed over 1 h in the same medium supplemented with 50 μM
solid. H NMR (400 MHz, CDCl ) δ 8.47 (d, J = 14.2 Hz, 2H), 8.15
3
(
1
(
d, J = 8.5 Hz, 2H), 7.99−7.96 (m, 4H), 7.66−7.61 (m, 2H), 7.52−
7.46 (m, 4H), 6.30 (d, J = 14.2 Hz, 2H), 4.35 (t, J = 7.2 Hz, 4H), 4.07
t, J = 6.7 Hz, 4H), 2.79 (t, J = 5.9 Hz, 4H), 2.28 (t, J = 7.5 Hz, 4H),
+
MeHg . Before the experiments, HeLa cells were washed with PBS
buffer for three times, and then the cells were incubated with 5 μM
hCy7-UCNPs in PBS for 1 h at 37 °C. Cell imaging was then carried
out after washing the cells with PBS. LSUCLM imaging was performed
2
4
6
.04 (s, 12H), 1.97−1.88 (m, 4H), 1.64−1.56 (m, 8H), 1.53−1.46 (m,
H), 1.43−1.34 (m, 8H), 1.33−1.21 (m, 18H), 0.93 (t, J = 7.4 Hz,
+
4b
H). MS (MALDI-TOF-MS): calcd for C H ClN O , 1035.67
with an OLYMPUS FV1000 scanning unit. Cells loaded with hCy7-
68
92
2
4
+
+
[
M] ; found, 1035.10 [M] .
Compound hCy7. Subside thiourea derivative 7 (0.39 g, 2.0
UCNPs were excited by a CW laser at 980 nm (Connet Fiber Optics,
China) with the focused power of ∼14 mW. UCL emission was
collected at 500−560 and 600−700 nm.
mmol) and compound 5 (1.11 g, 1.0 mmol) were dissolved in
anhydrous DMF (50 mL). The mixture was stirred at 85 °C for 12 h
under an argon atmosphere. The solvent was removed under reduced
pressure, and then the crude product was purified by silica gel
Upconversion Luminescence In Vivo Imaging. Animal
procedures were in agreement with the guidelines of the Institutional
Animal Care and Use Committee. In vivo and ex vivo UCL imaging
was performed with a modified upconversion luminescence in vivo
chromatography with DCM/MeOH (30:1) to afford the desired
1
4c
product as a deep-blue solid (40%). H NMR (400 MHz, CDCl ) δ
imaging system designed by our group. In this system, two external
3
1
7
7
2
8
1
0.08 (br, 1H), 9.63 (br, 1H), 8.57 (br, 1H), 7.95 (d, J = 8.4 Hz, 2H),
.90−7.80 (m, 3H), 7.78 (d, J = 12.7 Hz, 2H), 7.50 (t, J = 8.0 Hz 2H),
.36 (t, J = 8.0 Hz 2H), 7.32 (t, J = 8.0 Hz 2H), 7.16 (d, J = 8.7 Hz,
H), 7.11 (t, J = 8.0 Hz 1H), 5.62 (d, J = 13.0 Hz, 2H), 4.13−4.05 (m,
H), 3.91−3.85 (m, 4H), 2.52 (t, J = 5.8 Hz, 4H), 1.94 (s, 12H),
0−5 W adjustable CW 980 nm lasers (Connet Fiber Optics, China)
and an Andor DU897 EMCCD were used as the excitation sources
and the signal collector, respectively. Images of luminescent signals
were analyzed with Kodak Molecular Imaging Software. UCL signals
were collected at 800 ± 12 nm with a bandpass filter (Semrock).
.91−1.85 (m, 2H), 1.82−1.75 (m, 4H), 1.44−1.32 (m, 40H), 0.92 (t,
+
J = 7.3 Hz, 6H); MS (MALDI-TOF-MS): calcd for C H N O S ,
77
104
5
4
ASSOCIATED CONTENT
+
+
■
1
194.74 [M] ; found, 1194.77 [M] .
Assembly of P-PEG and hCy7 (denoted as hCy7-UCNPs).
*
S
Supporting Information
1
8
c
The synthetic details, H NMR, MADLI-TOF MS, FT-IR,
EDXA spectra, and cell viability of hCy7-UCNPs. The changes
of UV−vis absorption and UCL emission spectra in various
hCy7-UCNPs were prepared according to the previous method. The
UCNPs (10 mg) and hCy7 (5 mg) were dispersed in the 5 mL
chloroform by ultrasonication, and then the mixture was stirred at
room temperature to obtain a homogeneous phase. Furthermore, the
amphiphilic polymer (P-PEG, 10 mg) was added, and then the mixture
was stirred overnight at room temperature. The mixture was
centrifugated (14000 rpm, 8 min every time in 20 °C), and the
collected solid was repeatedly washed with water. The precipitate
could be redispersed in deionized water.
+
MeHg . This material is available free of charge via the Internet
at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
fyli@fudan.edu.cn
■
Procedures for Cation Titration. Stock solutions of the metal
ions (2.5 mM) were prepared in H O. A stock solution of hCy7-
2
Notes
UCNPs (containing 10 μM hCy7) was prepared in H O. The sensing
2
The authors declare no competing financial interest.
+
+
of hCy7-UCNPs to MeHg was performed by adding the MeHg
stock solution by means of a micropipet to 2 mL solution of hCy7-
UCNPs. Test samples for selectivity experiments were prepared by
adding appropriate amounts of anions stock solution with a similar
ACKNOWLEDGMENTS
■
The authors thank National Science Foundation of China
(21231004 and 91027004), MOST of China (2011AA03A407
and 2012CB932403), Shanghai Sci. Tech. Comm.
11XD1400200 and 12JC1401300), and the innovative team
of Ministry of Education of China (IRT0911) for financial
+
procedure. In competition experiments, MeHg was added to
solutions containing hCy7-UCNPs and the other metal ions of
interest. All test solutions were stirred for 20 min at room temperature.
For all UCL measurements, excitation was fixed at 980 nm, and UCL
emission was collected from 400 to 850 nm.
Cell Culture. The cell lines HeLa and KB were provided by the
Institute of Biochemistry and Cell Biology, SIBS, CAS (China). The
HeLa and KB cells were grown in modified Eagle’s medium
supplemented with 10% fetal bovine serum at 37 °C and 5% CO2.
HeLa and KB cells were planted on 14 mm glass coverslips and
allowed to adhere for 24 h.
(
support.
REFERENCES
■
(1) (a) Auzel, F. Chem. Rev. 2004, 104, 139. (b) Feng, W.; Sun, L. D.;
Zhang, Y. W.; Yan, C. H. Coord. Chem. Rev. 2010, 254, 1038.
(c) Mader, H. S.; Kele, P.; Saleh, S. M.; Wolfbeis, O. S. Curr. Opin.
G
dx.doi.org/10.1021/ja403798m | J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Article
Chem. Biol. 2010, 14, 582. (d) Wang, G. F.; Peng, Q.; Li, Y. D. Acc.
W.; Li, F. Y. Adv. Funct. Mater. 2012, 22, 2667. (d) Xie, L.; Qin, Y.;
Chen, H.-Y. Anal. Chem. 2012, 84, 1969. (e) Li, C. X.; Liu, J. L.;
Alonso, S.; Li, F. Y.; Zhang, Y. Nanoscale 2012, 4, 6065.
Chem. Res. 2011, 44, 322. (e) Schaf
Ed. 2011, 50, 5808. (f) Zhou, J.; Liu, Z.; Li, F. Y. Chem. Soc. Rev. 2012,
1, 1323.
2) (a) Liu, Y. S.; Tu, D. T.; Zhu, H. M.; Li, R. F.; Luo, W. Q.; Chen,
̈
er, H.; Haase, M. Angew. Chem., Int.
4
(
(9) (a) Ali, R.; Saleh, S. M.; Meier, R. J.; Azab, H. A.; Abdelgawad, I.
I.; Wolfbeis, O. S. Sens. Actuators, B 2010, 150, 126. (b) Mader, H. S.;
Wolfbeis, O. S. Anal. Chem. 2010, 82, 5002. (c) Achatz, D. E.; Meier,
R. J.; Fischer, L. H.; Wolfbeis, O. S. Angew. Chem., Int. Ed. 2011, 50,
260. (d) Fischer, L. H.; Harms, G. S.; Wolfbeis, O. S. Angew. Chem.,
Int. Ed. 2011, 50, 4546. (e) Sun, L. N.; Peng, H. S.; Stich, M. I. J.;
Achatz, D.; Wolfbeis, O. S. Chem. Commun. 2009, 5000. (f) Deng, R.
R.; Xie, X. J.; Vendrell, M.; Chang, Y. T.; Liu, X. G. J. Am. Chem. Soc.
2011, 133, 20168.
X. Y. Adv. Mater. 2010, 22, 3266. (b) Wang, F.; Han, Y.; Lim, C. S.;
Lu, Y. H.; Wang, J.; Xu, J.; Chen, H. Y.; Zhang, C.; Hong, M. H.; Liu,
X. G. Nature 2010, 463, 1061. (c) Wang, F.; Deng, R. R.; Wang, J.;
Wang, Q. X.; Han, Y.; Zhu, H. M.; Chen, X. Y.; Liu, X. G. Nat. Mater.
2
011, 10, 968. (d) Su, Q. Q.; Han, S. Y.; Xie, X. J.; Zhu, H. M.; Chen,
H. Y.; Chen, C.-K.; Liu, R.-S.; Chen, X. Y.; Wang, F.; Liu, X. G. J. Am.
Chem. Soc. 2012, 134, 20849. (e) Mai, H. X.; Zhang, Y. W.; Si, R.; Yan,
Z. G.; Sun, L. D.; You, L. P.; Yan, C. H. J. Am. Chem. Soc. 2006, 128,
(10) Coordination Chemistry of Rare Earth Complexes; Huang, C. H.,
Ed.; Science Press: Beijing, 1997.
6
426. (f) Wu, S. W.; Han, G.; Milliron, D. J.; Aloni, S.; Altoe, V.;
Talapin, D. V.; Cohen, B. E.; Schuck, P. J. Proc. Natl. Acad. Sci.U.S.A.
(11) (a) Zalups, R. K.; Lash, L. H. Toxicol. Appl. Pharmacol. 2006,
214, 88. (b) Ando, S.; Koide, K. J. Am. Chem. Soc. 2011, 133, 2556.
(c) Yang, Y. K.; Yook, K. J.; Tae, J. J. Am. Chem. Soc. 2005, 127, 16760.
2
009, 106, 10917. (g) Jalil, R. A.; Zhang, Y. Biomaterials 2008, 29,
4
122. (h) Xu, Z. H.; Ma, P. A.; Li, C. X.; Hou, Z. Y.; Zhai, X. F.;
(d) Yang, Y. M.; Zhao, Q.; Feng, W.; Li, F. Y. Chem. Rev. 2013, 113,
Huang, S. S.; Lin, J. Biomaterials 2011, 32, 4161. (i) Abel, K. A.; Boyer,
J. C.; van Veggel, F. C. J. M. J. Am. Chem. Soc. 2009, 131, 14644.
1
92. (e) Chen, X. Q.; Baek, K. H.; Kim, Y. M.; Kim, S. J.; Shin, I.;
Yoon, J. Y. Tetrahedron 2010, 66, 4016. (f) Chan, D. S.-H.; Lee, H.-M.;
Che, C.-M.; Leung, C.-H.; Ma, D.-L. Chem. Commun. 2009, 7479.
(
j) Yan, B.; Boyer, J. C.; Habault, D.; Branda, N. R.; Zhao, Y. J. Am.
Chem. Soc. 2012, 134, 16558. (k) Jayakumar, M. K. G.; Idris, N. M.;
Zhang, Y. Proc. Natl. Acad. Sci. U.S.A. 2012, 109, 8483.
(
g) He, H.-Z.; Leung, K.-H.; Fu, W.-C.; Chan, D. S.-H.; Leung, C.-H.;
Ma, D.-L. Environ. Res. Lett. 2012, 7, 044032 (7pp).
12) Guo, Z. Q.; Zhu, W. H.; Zhu, M. M.; Wu, X. M.; Tian, H.
(
3) (a) Liu, Q.; Sun, Y.; Li, C. G.; Zhou, J.; Li, C. Y.; Yang, T. S.;
Zhang, X. Z.; Yi, T.; Wu, D. M.; Li, F. Y. ACS Nano 2011, 5, 3146.
b) Xiong, L. Q.; Chen, Z. G.; Yu, M. X.; Li, F. Y.; Liu, C.; Huang, C.
(
Chem.Eur. J. 2010, 16, 14424.
(
(
(
13) Wang, C.; Cheng, L.; Liu, Z. Biomaterials 2011, 32, 1110.
14) Prencipe, G.; Tabakman, S. M.; Welsher, K.; Liu, Z.; Goodwin,
H. Biomaterials 2009, 30, 5592. (c) Liu, Q.; Yin, B. R.; Yang, T. S.;
Yang, Y. C.; Shen, Z.; Yao, P.; Li, F. Y. J. Am. Chem. Soc. 2013, 135,
A. P.; Zhang, L.; Henry, J.; Dai, H. J. J. Am. Chem. Soc. 2009, 131,
783.
15) Li, Z. Q.; Wang, L. M.; Wang, Z. Y.; Liu, X. G.; Xiong, Y. J. J.
Phys. Chem. C 2011, 115, 3291.
5
029. (d) Sun, Y.; Liu, Q.; Peng, J. J.; Zhou, J.; Yang, P. Y.; Zhang, Y.
J.; Li, F. Y. Biomaterials 2013, 34, 2289.
4) (a) Chatteriee, D. K.; Rufalhah, A. J.; Zhang, Y. Biomaterials
008, 29, 937. (b) Yu, M. X.; Li, F. Y.; Chen, Z. G.; Hu, H.; Zhan, C.;
4
(
(
2
(
(
(
16) Zhang, Z.; Achilefu, S. Org. Lett. 2004, 6, 2067.
17) Reynolds, G. A.; Drexhage, K. H. J. Org. Chem. 1977, 42, 885.
18) Yu, M. X.; Shi, M.; Chen, Z. G.; Li, F. Y.; Li, X. X.; Gao, Y. H.;
Yang, H.; Huang, C. H. Anal. Chem. 2009, 81, 930. (c) Xiong, L. Q.;
Chen, Z. G.; Tian, Q. W.; Cao, T. Y.; Xu, C. J.; Li, F. Y. Anal. Chem.
2
009, 81, 8687. (d) Liu, Q.; Sun, Y.; Yang, T. S.; Feng, W.; Li, C. G.;
Xu, J.; Yang, H.; Zhou, Z. G.; Yi, T.; Huang, C. H. Chem.Eur. J.
008, 14, 6892.
19) Li, C. Y.; Yu, M. X.; Sun, Y.; Wu, Y. Q.; Huang, C. H.; Li, F. Y. J.
Am. Chem. Soc. 2011, 133, 11231.
Li, F. Y. J. Am. Chem. Soc. 2011, 133, 17122. (e) Liu, Q.; Yang, T. S.;
Feng, W.; Li, F. Y. J. Am. Chem. Soc. 2012, 134, 5390.
2
(
(
5) (a) Zhou, J. C.; Yang, Z. L.; Dong, W.; Tang, R. J.; Sun, L. D.;
Yan, C. H. Biomaterials 2011, 32, 9059. (b) Xing, H. Y.; Bu, W. B.;
Zhang, S. J.; Zheng, X. P.; Li, M.; Chen, F.; He, Q. J.; Zhou, L. P.;
Peng, W. J.; Hua, Y. Q.; Shi, J. L. Biomaterials 2012, 33, 1079.
(
c) Boyer, J. C.; Manseau, M. P.; Murray, J. I.; van Veggel, F. C. J. M.
Langmuir 2010, 26, 1157. (d) Cheng, L.; Yang, K.; Li, Y. G.; Chen, J.
H.; Wang, C.; Shao, M. W.; Lee, S. T.; Liu, Z. Angew. Chem., Int. Ed.
2
011, 50, 7385. (e) Cheng, L.; Yang, K.; Shao, M. W.; Lee, S. T.; Liu,
Z. J. Phys. Chem. C 2011, 115, 2686. (f) Hilderbrand, S. A.; Shao, F.
W.; Salthouse, C.; Mahmood, U.; Weissleder, R. Chem. Commun.
2
(
009, 4188.
6) (a) Zhou, J.; Sun, Y.; Du, X. X.; Xiong, L. Q.; Hu, H.; Li, F. Y.
Biomaterials 2010, 31, 3287. (b) Liu, Q.; Chen, M.; Sun, Y.; Chen, G.
Y.; Yang, T. S.; Gao, Y.; Zhang, X. Z.; Li, F. Y. Biomaterials 2011, 32,
8
243. (c) Zhou, J.; Yu, M. X.; Sun, Y.; Zhang, X. Z.; Zhu, X. J.; Wu, Z.
H.; Wu, D. M.; Li, F. Y. Biomaterials 2011, 32, 1148. (d) Xia, A.; Chen,
M.; Gao, Y.; Wu, D. M.; Feng, W.; Li, F. Y. Biomaterials 2012, 33,
5
394. (e) Zhu, X. J.; Zhou, J.; Chen, M.; Shi, M.; Feng, W.; Li, F. Y.
Biomaterials 2012, 33, 4618.
7) (a) Chen, Z. G.; Chen, H. L.; Hu, H.; Yu, M. X.; Li, F. Y.; Zhang,
Q.; Zhou, Z. G.; Yi, T.; Huang, C. H. J. Am. Chem. Soc. 2008, 130,
023. (b) Kumar, M.; Zhang, P. Langmuir 2009, 25, 6024. (c) Jiang,
(
3
S.; Zhang, Y. Langmuir 2010, 26, 6689. (d) Zhang, F.; Shi, Q. H.;
Zhang, Y. C.; Shi, Y. F.; Ding, K. L.; Zhao, D. Y.; Stucky, G. D. Adv.
Mater. 2011, 23, 3775. (e) Rantanen, T.; Pakkila, H.; Jamsen, L.;
Kuningas, K.; Ukonaho, T.; Lovgren, T.; Soukka, T. Anal. Chem. 2007,
7
9, 6312. (f) Rantanen, T.; Jarvenpaa, M. L.; Vuojola, J.; Kuningas, K.;
Soukka, T. Angew. Chem., Int. Ed. 2008, 47, 3811. (g) Zhang, P.;
Rogelj, S.; Nguyen, K.; Wheeler, D. J. Am. Chem. Soc. 2006, 128,
1
(
2410.
8) (a) Liu, J. L.; Liu, Y.; Liu, Q.; Li, C. Y.; Sun, L. N.; Li, F. Y. J. Am.
Chem. Soc. 2011, 133, 15276. (b) Liu, Q.; Peng, J. J.; Sun, L. N.; Li, F.
Y. ACS Nano 2011, 5, 8040. (c) Yao, L. M.; Zhou, J.; Liu, J. L.; Feng,
H
dx.doi.org/10.1021/ja403798m | J. Am. Chem. Soc. XXXX, XXX, XXX−XXX