Alkyne-functionalized superstable graphitic silver nanoparticles for raman imaging

Article abstract of DOI:10.1021/ja507368z

Noble metals, especially gold, have been widely used in plasmon resonance applications. Although silver has a larger optical cross section and lower cost than gold, it has attracted much less attention because of its easy corrosion, thereby degrading plas

Full text of DOI:10.1021/ja507368z

Communication
pubs.acs.org/JACS
Alkyne-Functionalized Superstable Graphitic Silver Nanoparticles for
Raman Imaging
Zhi-Ling Song,† Zhuo Chen,*,† Xia Bian,† Li-Yi Zhou,† Ding Ding,† Hao Liang,† Yu-Xiu Zou,†
Shan-Shan Wang,† Long Chen,§ Chao Yang,† Xiao-Bing Zhang,*,† and Weihong Tan*,†,‡
†Molecular Sciences and Biomedicine Laboratory, State Key Laboratory for Chemo/Biosensing and Chemometrics, College of
Chemistry and Chemical Engineering, College of Biology and Collaborative Innovation Center for Molecular Engineering and
Theronastics, Hunan University, Changsha 410082, China
‡Department of Chemistry and Department of Physiology and Functional Genomics, Center for Research at Bio/nano Interface,
Shands Cancer Center, UF Genetics Institute and McKnight Brain Institute, University of Florida, Gainesville, Florida 32611-7200,
United States
§Faculty of Sciences, University of Macau, Av. Padre Tomas
́
Pereira Taipa, Macau, China
S
* Supporting Information
graphene on a quartz substrate could not be penetrated by sulfur
ABSTRACT: Noble metals, especially gold, have been
widely used in plasmon resonance applications. Although
silver has a larger optical cross section and lower cost than
gold, it has attracted much less attention because of its easy
corrosion, thereby degrading plasmonic signals and
limiting its applications. To circumvent this problem, we
report the facile synthesis of superstable AgCu@graphene
(ACG) nanoparticles (NPs). The growth of several layers
of graphene onto the surface of AgCu alloy NPs effectively
protects the Ag surface from contamination, even in the
presence of hydrogen peroxide, hydrogen sulfide, and
nitric acid. The ACG NPs have been utilized to enhance
the unique Raman signals from the graphitic shell, making
ACG an ideal candidate for cell labeling, rapid Raman
imaging, and SERS detection. ACG is further function-
alized with alkyne-polyethylene glycol, which has strong
Raman vibrations in the Raman-silent region of the cell,
leading to more accurate colocalization inside cells. In sum,
this work provides a simple approach to fabricate
corrosion-resistant, water-soluble, and graphene-protected
AgCu NPs having a strong surface plasmon resonance
effect suitable for sensing and imaging.
compounds. Kalyanaraman found that a Ag−Co bimetallic
structure had more stable plasmonic characteristics than pure Ag
on a quartz substrate. Although their approaches retained the
plasmonic properties of Ag, the preparation processes were
complicated, and because of the solid substrate, the Ag
nanostructures were insoluble in water, which also limited their
applications, especially in bioimaging and biosensing.
One possible approach for fabricating superstable and soluble
Ag NPs involves encapsulating them in appropriate shells.
Indeed, graphene could be an ideal shell material based on its
superior chemical stability, mechanical capacity, optical proper-
ties, thermal stability, and electrical conductivity.5 More
importantly, graphene exhibits admirable impermeability for
small molecules, even helium atoms,6 and has emerged as one of
the most extensively studied nanomaterials.7 High-quality
graphene has been grown onto the surfaces of different transition
metal substrates (Cu, Ni, Pd, Pt, and Co)8 by chemical vapor
deposition (CVD). While it is difficult to grow graphene on the
surface of Ag because of its weak catalytic activity, the use of
inexpensive Cu could overcome this problem, since Cu catalyzes
the growth of graphene and Ag and Cu form good alloys. Here
we report the use of CVD to grow a few layers of graphene on the
surface of AgCu NPs to fabricate superstable graphitic Ag NPs.
The formation of few-layer graphene on the surface of Ag NPs
was catalyzed by Cu at high temperature. Sulfur compounds and
oxides could not penetrate the graphene to contaminate the
surface of Ag, and ACGs efficiently maintained the excellent
plasmonic properties of Ag, even in the presence of hydrogen
peroxide, hydrogen sulfide, and nitric acid.
oble metal nanoparticles have gained considerable
Nattention due to their outstanding optical properties. In
particular, gold nanoparticles (Au NPs), which possess strong
plasmonic properties through their long electronic relaxation,
have been applied in surface-enhanced Raman scattering
(SERS).1 Actually, silver has larger optical cross section and
lower cost than gold,2 making it a more suitable material for
plasmon resonance applications. However, Au is often preferred
because its surface is less susceptible to corrosion. More
specifically, Ag is easily affected by such ambient factors as O2
or H2S, forming silver oxide or silver sulfide on the surface,
thereby degrading plasmonic signals and limiting applications.3
To maintain the excellent properties of Ag, many efforts to
reduce corrosion have been explored.4 Cubukcu reported that
the surface of a Ag nanostructure passivated with a monolayer of
Such stable ACGs could be utilized for various plasmon
resonance applications, such as Raman imaging for intracellular
NP localization. SERS Raman imaging as an emerging field has
generated a lot of interest and applications. Raman-based
methods offer a powerful analytical tool that extends the
possibilities of vibrational spectroscopy with extremely high
sensitivity and multiplexing capabilities to solve more chemical
Received: July 23, 2014
Published: September 18, 2014
© 2014 American Chemical Society
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Communication
and biochemical problems.1 Nanoparticle cellular interactions
are increasingly under investigation to support applications such
as targeted imaging and diagnostics, drug delivery, and heat- or
radiation-based therapeutics.9 In these cases, localization of the
NPs within the cell is critical to the intended therapeutic
function. The stable plasmon resonance effect has been utilized
for enhancing the unique Raman signals from the graphitic shell,
making ACG an ideal candidate for cell labeling, rapid Raman
imaging, and SERS detection. However, the Raman signals of
ACGs often overlap with those from cellular components,
making the signals difficult to distinguish. It is well known that
alkynes possess strong vibrations in the Raman-silent region of
the cell.10 To solve this problem, (4-phenylethynyl)benzylamino
polyethylene glycol (alkyne-PEG) was synthesized and con-
jugated to the graphitic surface of ACGs through simple, but
strong, π−π interactions. By combining alkynyl and graphitic
Raman signals, ACGs were accurately colocalized inside the cells.
The ACG structurally consists of a AgCu alloy core
encapsulated in a graphitic shell (Figure 1a). By utilizing
Figure 2. UV/vis spectra of bare Ag NPs (a) and ACGs (b) after adding
220 mM H2O2 for various times. (c) Digital photos of bare Ag NPs and
ACGs suspensions mixed in 220 mM H2O2 for various times. (d) A/A0
(relative absorbance intensity, where A0 and A are the optical absorbance
without and with the presence of 0.15 mM NaHS, 220 mM H2O2, or 75
mM HNO3, respectively) at different time points.
surface plasmon resonance (LSPR) peak around 410 nm of bare
Ag NPs decreased quickly within 20 min, while peaks around 400
and 600 nm of ACGs showed few changes. Figure 2c shows
digital photos of bare Ag NPs and ACGs solution after addition
of 220 mM H2O2 for different incubation times. Bare Ag NPs
produced bubbles and became colorless because of the violent
reaction between Ag and H2O2. In contrast, no obvious changes
were observed in the ACGs, which benefited from the protective
graphitic shells. Different concentrations of H2O2 were also used
to further investigate the stability of bare Ag NPs and ACGs
solution. The minor changes in UV/vis spectra and color of the
ACGs with time indicate the outstanding ability of ACGs to resist
oxidation compared to bare Ag NPs (Figure S3).
Figure 1. Advanced structural analysis of ACGs. (a) Schematic diagram
of ACG, including a AgCu core and a graphitic shell. (b) TEM image of
ACGs around 25 nm. (c) HR-TEM image of ACGs, with the arrows
showing the 0.35 nm lattice constant of the graphitic layer. (d−f) STEM
images of ACG nanostructures and the elemental map. Left, dark field;
middle, Cu; right, Ag.
Apart from oxides, the Ag surface can also be damaged by H2S
and HNO3, dramatically diminishing plasmonic properties and
rendering Ag unreliable for applications. We further investigated
the ability of ACGs to resist H2S and HNO3 corrosion. Figure 2d
shows the temporal changes of the spectra for ACGs and bare Ag
NPs after adding H2O2, NaHS, and HNO3. For bare Ag NPs, the
intensity of the LSPR absorbance peak decreased significantly,
while ACGs showed no changes in position or intensity in LSPR
peaks. ACGs exhibited considerable resistance against corrosion
from H2O2, H2S, and HNO3 as a result of protective graphitic
shells. Other concentrations of NaHS and HNO3 were also
investigated, and ACGs demonstrated superior stabilities
(Figures S4 and S5). Bare AgCu NPs demonstrated lower
stability compared to ACGs (Figure S6).
The cytotoxicity of ACGs was investigated through MTS
assay. ACGs exhibited good biocompatibilities. Negligible
inhibition of proliferation was observed in MCF-7 breast cancer
cells stained with ACGs (Figure S7).
Next, these stable ACGs were utilized to enhance the
distinctive Raman signals from the graphitic shells, demonstrat-
ing that ACG makes a good Raman tag for cell imaging and an
excellent SERS substrate for bioimaging and biosensing. ACG
has two prominent Raman vibration bands, a graphitic carbon
(G) peak around 1590 cm−1 and a disordered (D) peak around
1330 cm−1 (Figure 3a). The relatively high Raman D peak of
ACG is attributed to increasing intensity for smaller crystallite
transmission electron microscopy (TEM, Figure 1b) and high-
resolution TEM (HR-TEM, Figure 1c), the morphology and
composition of ACG were characterized, clearly exhibiting the
formation of the core−shell structure. The ACG demonstrated a
size distribution with an average diameter ∼25 nm. The space
between the shell layers was ∼0.35 nm, consistent with the
interlayer distance in graphite, suggesting that the AgCu core was
encapsulated by a graphitic shell. The existence of AgCu alloy
was further proved by scanning transmission electron micros-
copy (STEM, Figure 1d−f). Formation of the AgCu alloy was
believed to contribute to the growth of the graphitic shell. More
TEM images were obtained and energy dispersive spectrometry
and selected area electron diffraction were performed (Figure
S1); all tests confirmed the core−shell structure of ACGs. The
plasmonic properties of ACGs could be modulated by changing
the Ag/Cu ratio, as verified by inductively coupled plasma mass
spectrometry (Table S1) and UV/vis spectrophotometry
(Figure S2).
We next assessed the corrosion resistance of ACGs. UV/vis
characterization was utilized to monitor the plasmonic properties
and morphological changes under different conditions. For
comparison, bare Ag NPs were synthesized.11 Temporal changes
of aqueous solutions of bare Ag NPs and ACGs (Figure 2a,b)
after adding 220 mM H2O2 were investigated. The localized
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Communication
NPs. The key virtue of using ACGs as a SERS substrate is the
higher detection sensitivity and vast practical applications by the
strongly enhanced Raman signals. On the other hand, the
chemically inert thin shell of ACGs can prevent direct interaction
between adsorbates and bare AgCu cores, which could help to
prevent photocarbonization of signal molecules or nearby
impurity molecules under strong or prolonged laser irradiation14
while retaining the significant Raman enhancement effect.
Rhodamine 6G (R6G) was used as a model molecule for
detection and to determine SERS enhancement (Figure 3e).
When ACGs were added to the R6G solution, the Raman signals
were significantly enhanced. ACGs were also able to quench R6G
background fluorescence through the fluorescence resonance
energy transfer (FRET) process,8 significantly improving the
detection signal-to-noise ratio. The Raman detection with ACGs
also demonstrates superior stability as compared to bare Ag NPs
(Figure S12), which is critical for a SERS detection substrate.
Raman microscopy is capable of detecting specific vibrational
stretches, as well as imaging cells. This property makes possible
the intracellular localization of NPs to realize their intended
therapeutic role. However, numerous Raman signals often
overlap with those from cellular components, making the signals
difficult to distinguish. Alkyne possesses a strong vibration in the
Raman-silent region of the cell (1800−2800 cm−1).10
Figure 3. SERS with the ACGs. (a) Raman spectrum of the ACGs with
the G and D bands of graphitic shell. High-resolution Raman image of
MCF-7 cells treated without (b) and with (c) ACGs using 1 s
integration/pixel. (d) Rapid Raman image of MCF-7 cells treated with
ACGs using 0.1 s integration/pixel. (e) Raman spectra of 20 μM R6G
with (red) and without (blue) ACGs. BF, bright field; scale bar, 10 μm.
Functionalization of ACGs with alkyne molecules could help
to more accurately localize ACGs in the cells. This could be
achieved by conjugating alkyne with PEG molecules. The alkyne
molecule diphenylacetylene, with a high Raman scattering cross
section,10 was efficiently synthesized and conjugated on the PEG
chain (Figure 4a). Alkyne-PEG is also a good surfactant, having a
size.12 The AgCu core significantly enhanced the Raman signal of
the graphitic shell. Compared to other organic Raman-active
molecules, ACG has enormous Raman scattering cross sections
(∼10−21 cm2 sr−1 molecule−1) as a graphitic nanomaterial,13 and
it is more stable because of its inorganic structure. Both the D and
G bands could be utilized to image cells and tissues.
Figure 3b,c shows the Raman images of MCF-7 cells stained
without and with ACGs, respectively (1 s integration time per
pixel). Strong Raman signals were observed in the ACGs stained
cells. All Raman signals in Figure 3c are distributed throughout
the cellular cytoplasm, with no signal emerging from the nucleus,
indicating the cell distribution of ACGs. Strong and rapid
enhancement of Raman signals by the core−shell structure is a
key advantage of using ACGs as Raman probes for cell imaging.
For example, one Raman image could be acquired in several
minutes (Figure 3d, 0.1 s integration/pixel), a remarkable
reduction from the tens of minutes taken by previous methods
(1−2 s integration/pixel). Similar to Figure 3c, Raman signals in
Figure 3d were also localized in the cellular cytoplasm. Based on
strong Raman scattering, only a few seconds were needed to
acquire one Raman image by using the global imaging mode with
an expanding laser beam. Figure S8 shows the global imaging of
the ACGs stained cells, and the entire image was obtained in only
10 s. Compared to fluorescence imaging, the Raman signals in
the cells have good spatial resolution as a result of the narrow full
width at half-maximum of the Raman peak and the unbleached
Raman signals. This represents a potentially useful tool for
monitoring endocytosis processes of ACGs inside cells. Further
utilizing the ACGs as Raman tags for cell and tissue imaging has
also been investigated. To improve the selectivity, cancer cell
specific aptamers are conjugated onto the ACG surface through
the π−π stacking (Figure S9). The aptamer-functionalized ACGs
exhibit good targeting imaging (Figures S10 and S11), which
indicates great potential in clinical applications.
Figure 4. Preparation of alkyne-PEG-modified ACGs. (a) Synthesis
procedure of alkyne-PEG. (b) Schematic illustration of the alkyne-PEG
functionalization of ACGs.
hydrophilic PEG chain and a hydrophobic diphenylacetylene tail,
which could be functionalized on the surface of ACGs through
strong π−π stacking to help solubilize ACGs.
Figure 4b illustrates the alkyne-PEG-functionalized ACG. The
graphitic shell acts as an ideal platform to adsorb alkyne-PEG
molecules. Meanwhile, the AgCu core could simultaneously
enhance the Raman signals from both the graphitic shell and
alkyne-PEG through the SERS effect, which, in turn, could be
utilized to colocalize ACGs inside cells. Figure 5a (red curve)
shows the Raman spectrum of alkyne-PEG, with a strong and
narrow alkyne peak around 2220 cm−1. The efficiency of
adsorbing alkyne-PEG on ACGs was also clearly demonstrated
(Figure 5a, black curve). Three distinct Raman peaks could be
observed: the peak at 1330 cm−1 mainly comes from the graphitic
shell, while the peak at 2220 cm−1 is assigned to CC stretching
of alkyne-PEG, and the peak at 1590 cm−1 is their mutual peak.
ACGs were also excellent substrates for the enhancement of
Raman signals by orders of magnitude of molecules adsorbed on
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AUTHOR INFORMATION
Corresponding Author
zhuochen@hnu.edu.cn; xbzhang@hnu.edu.cn; tan@chem.ufl.
■
edu
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was financially supported by the National Key Basic
Research Program of China (Nos. 2013CB932702,
2011CB911000), the Research Fund for the Program on
National Key Scientific Instruments and Equipment Develop-
ment (No. 2011YQ0301241402), the National Natural Science
Foundation of China (NSFC 21105025, NSFC 21221003, and
NSFC 21327009), the U.S. National Institutes of Health
(GM079359 and CA133086), and the Hunan Innovation and
Entrepreneurship Program.
Figure 5. Cell imaging with alkyne-PEG-functionalized ACGs. (a)
Raman spectra of alkyne-PEG with (black) and without (red) ACGs.
(b−f) Raman image of MCF-7 cells treated with alkyne-PEG-modified
ACGs using 1 s integration/pixel. BF, bright field; scale bar, 10 μm.
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ASSOCIATED CONTENT
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S
* Supporting Information
Procedures and additional data. This material is available free of
charge via the Internet at http://pubs.acs.org.
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