Homogenous, far-reaching tuning and highly emissive QD-silica core-shell nanocomposite synthesized via a delay photoactive procedure; Their applications in two-photon imaging of human mesenchymal stem cells

Abstract

In this article, we present the exploration of a facile synthetic tactic incorporating delay-photo-oxidation to recover the loss in emission frequently encountered after encapsulating quantum dots (QDs) inside a silica shell. This facile synthesis procedure reproducibly increases emissive intensity of QDs (core)-SiO(2) (shell) (60 nm) nanomaterials by >5 fold (QY from 3% to >15%). The resulting QDs (core)-SiO(2) proved to be a single quantum dot in single SiO(2), homogeneous and highly monodispered; their emissions have been successfully fine-tuned from visible to the near infrared region. We then demonstrate their power in biological imaging by labeling human mesenchymal stem cells under two-photon confocal microscopy. The results of low cytotoxicity, efficient labeling, and specific location nearby the nucleus characters of these nanoparticles should spark an intensive relevant research within a living system.

Full text

Homogenous, far-reaching tuning and highly emissive QD–silica core–shell
nanocomposite synthesized via a delay photoactive procedure; their
applications in two-photon imaging of human mesenchymal stem cells†
Chih-Wei Lai,
a
Yu-Hsiu Wang,
a
Yu-Chun Chen,
a
Cheng-Chih Hsieh,
a
Borade Prajakta Uttam,
b
Jong-Kai Hsiao,*
bc
Cheng-Chih Hsu
a
and Pi-Tai Chou*
a
Received 2nd April 2009, Accepted 28th August 2009
First published as an Advance Article on the web 28th September 2009
DOI: 10.1039/b906575j
In this article, we present the exploration of a facile synthetic tactic incorporating delay-photo-
oxidation to recover the loss in emission frequently encountered after encapsulating quantum
dots (QDs) inside a silica shell. This facile synthesis procedure reproducibly increases emissive
intensity of QDs (core)–SiO
2
(shell) (60 nm) nanomaterials by >5 fold (QY from 3% to >15%). The
resulting QDs (core)–SiO
2
proved to be a single quantum dot in single SiO
2
, homogeneous and highly
monodispered; their emissions have been successfully fine-tuned from visible to the near infrared
region. We then demonstrate their power in biological imaging by labeling human mesenchymal stem
cells under two-photon confocal microscopy. The results of low cytotoxicity, efficient labeling, and
specific location nearby the nucleus characters of these nanoparticles should spark an intensive relevant
research within a living system.
Introduction
Quantum dots (QDs), synonym of semiconducting nano-
particles, possess a number of advantageous optical properties
such as high extinction absorption coefficient, size-tunable
wavelength, sharp emission bandwidth, and superior photo-
stability, etc.
1,2
Structurally, composition of QDs has evolved
from basic homo-QDs to versatile hetero-QDs with core–shell
configuration. The addition of inorganic shell growth on the
central core expands the possible interplay of energy levels in
QDs.
3
Depending on the energetic relationship between the core
and shell, hetero-QDs frequently encountered can be classified as
type-I and type-II.
4
In type-I QDs, the conduction band of the
shell is higher in energy than that of the core, while the relative
energy level is opposite in the valence band. In contrast, the
energy levels of both valence and conduction bands in type-II
QDs’ cores are either lower or higher than that of the shells. Such
a configuration permits spatially separated interband transitions.
Having a direct band-gap transition, type-I QDs, usually have
high quantum yield (QY) in visible domain, while type II QDs
can be largely tuned to bear emissions in far visible to near-
infrared (NIR) region by utilizing the indirect interband transi-
tion character. Regardless of the difference in their emissive
mechanisms, however, both types of QDs have demonstrated
great potential in bio-imaging.
5
Unless the excitons are trapped
in the surface defect sites, in theory, QDs should exhibit intense
emission due to the lack of high-frequency vibrational quench-
ing. This is particularly important regarding the imaging appli-
cation toward NIR since in this region classical organic dyes
greatly suffer from high frequency vibrational (overtone) deac-
tivation; a universal quenching mechanism for excited molecules
dubbed as the energy gap law.
6
Despite their superiority, a major obstacle for QDs in bio-
imaging is known to be their cytotoxicity. Among numerous
attempts to circumvent this disadvantage, QDs coated with silica
shell, QDs–SiO
2
, have proven to possess less cytotoxicity and
versatile functionalities towards practical applications, allowing
biological molecules to form covalent bonding with the silica
material surface.
7,8
However, QDs–SiO
2
core–shell materials
prepared via this prevailing microemulsion method suffer
considerable loss in emission QY.
9
Recently, several synthetic
approaches have been proposed to achieve high QY QDs–SiO
2
,
including layer-by-layer assembly and outer inorganic shell
thickening, etc.
10,11
One convenient approach should be credited
to photo-oxidation of QDs, which involves light exposure to
QDs after surface modification (prior to silica shell coating).
Although this protocol seems to alleviate the QY deprivation, the
performance of the resulting QDs remains sceptical due to the
lack of bio-compatible evidence, inferior homogeneity, and poor
dispersibility.
12
In an aim to improve the QDs–SiO
2
quality, using cysteine
(–Cy) as a capping ligand in phase transfer, we then proposed an
alternative, facile protocol, in which the as prepared QDs-Cy
surface was subject to a photo-oxidation process only when QDs-
Cy had been located inside the SiO
2
shell. As a result, highly
emissive QDs-Cy–SiO
2
for both type I and type II QDs were
prepared, and the emission wavelength could be fine-tuned from
the visible to NIR region of 1000 nm. It is noteworthy that to
a
Department of Chemistry, National Taiwan University, Taipei 106,
Taiwan. E-mail: chop@ntu.edu.tw
b
Department of Medical Imaging, National Taiwan University Hospital
and College of Medicine, Taipei, Taiwan. E-mail: jongkai@gmail.com
c
Institute of Biomedical Engineering, National Taiwan University, Taipei,
106, Taiwan
† Electronic supplementary information (ESI) available: Experimental
section, including further experimental detail procedure, TEM picture,
emission spectra, instrument information, EDX and MTT data. See
DOI: 10.1039/b906575j/
8314 | J. Mater. Chem., 2009, 19, 8314–8319 This journal is ªThe Royal Society of Chemistry 2009
PAPER www.rsc.org/materials | Journal of Materials Chemistry

our knowledge, QDs–SiO
2
with NIR emission has not yet been
reported.
13
The resulting QDs-Cy–SiO
2
are homogeneous with
great dispersibility. We then investigated their two photon
absorption (TPA) effect as well as in-vitro bio-viability. Using
stem cells as a cellular model, their capability for two-photon cell
imaging is successfully demonstrated.
Experimental
Materials
Tri-n-octylphosphine oxide (TOPO, 99%, Aldrich), tri-n-butyl-
phosphine (TBP, technical grade 98%, SHOWA), di-n-octyl-
amine (DOA, 98%, ACROS), hexadecylamine (HDA, 90%,
TCI), CdO (99.99%, Strem), selenium (Se) powder (99.5%,
200 mesh, Aldrich), CdCl
2
(99.99%, Aldrich), tellurium (Te)
powder (99.8%, 200 mesh, Aldrich), and zinc stearate
(RiedeldeHa€
en), hexanol (98%, ACROS), Triton X-100
(ACROS), tetraethyl orthosilicate (98%, ACROS), and ammo-
nium hydroxide (28–30 wt%, Fluka) were used without further
purification.
Phase transfer
According to our previous synthetic protocol, CdSe/ZnS and
CdTe/CdSe/ZnS were prepared for further surface modification.
4
(see ESI†). The water-soluble CdSe/ZnS and CdTe/CdSe/ZnS
QDs were made using a stepwise procedure reported by
Chen et al. and Mattoussi et al.
1d,14
Briefly, TBP/TOPO-capped
CdSe-ZnS core–shell particles were prepared from the growth
and annealing of CdO. 20 mg of the resulting TBP/TOPO-cap-
ped CdSe-ZnS and 80 mg of cysteine were then placed in
a reaction vessel containing 15 mL of methanol, the pH of which
was adjusted to >10 with tetramethylammonium hydroxide
pentahydrate to aid the dissolution process. Under dark condi-
tions and regular airflow, the mixture was heated under reflux at
65 C overnight. After cooling to room temperature, the cysteine-
capped CdSe/ZnS (QDs-Cy) nanocrystals were then precipitated
with ether. For further purification, methanol was added to
dissolve the precipitate, followed by the addition of ether to
re-precipitate the nanocrystals. The vacuum dried QDs-Cy were
diluted in 10 ml DI water.
Synthesis of QDs-Cy–SiO
2
QDs-Cy–SiO
2
with different CdSe/ZnS core–shell sized nano-
particles were prepared from reverse micelles.
15
The as-prepared
QDs-Cy nanoparticles (see above) were used as seeds for growth
of the SiO
2
shell. Briefly, TEOS (100 mL), and various amounts of
QDs-Cy (3 5.5 10
5
M) within water (520 mL) were added to
a solution containing cyclohexane (7.4 mL), hexanol (1.8 mL),
and Triton X-100 (1.8 mL), which the QDs concentration was
measured with an UV-Vis absorption instrument.
16
In the
microemulsion system, a mixture of silica precursor, TEOS, and
the CdSe/ZnS core–shell nanoparticles of capped water-soluble
ligand was stirred over 6 h. NH
4
OH was then added in the dark.
After 24 h polymerization process, CdSe/ZnS-Cy were coated
with silica, extracted by centrifugation, and re-precipitated twice
with ethanol. The QDs-Cy–SiO
2
was then suspended in deion-
ized water. Under oxygen gas, the reaction solution was
illuminated by a 366 nm UV lamp (0.72 mW/cm) light source for
several hours (1–6 h).
Cytotoxicity examination
Human mesenchymal stem cells (hMSC) were cultured in
Dulbecco’s modified Eagle’s medium (DMEM) (Cellgro, Hern-
don, VA, USA), supplemented with 10% heat-inactivated fetal
bovine serum (FBS), penicillin (50 U/ml), and streptomycin
(0.05 mg/ml). All cultures were kept in moist atmosphere of 5%
CO
2
, 95% air at 37 C. Cells were passaged through trypsiniza-
tion, and nucleated cells were centrifuged at 100 g for harvesting.
Phosphate buffer saline (PBS) used for washing and rinsing were
composed of 137 mM NaCl, 2.68 mM KCl, 10 mM Na
2
HPO
4
,
1.76 mM KH
2
PO
4
, and tuned to pH 7.4. For cytotoxicity eval-
uation, cells were seeded in a 24 well plate at 5 10
3
cells/well
density in 500 mL culturing medium 24 h prior to the feeding of
the particles. Including the control, six different dosages of
particles were given to each well and the incubation concentra-
tion was confirmed with inductively coupled plasma-mass spec-
trometry (ICP-MS): 0, 0.19, 0.37, 0.75, 1.49, and 2.98 mM Cd.
After 24 h incubation, each well was washed with phosphate
buffer saline (PBS) twice, and replenished with 500 mL culturing
medium with 10% of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide, Sigma) agent. After 1 h incubation
and medium removal, the newly formed purple MTT-formazan
was dissolved in 200 mL dimethyl sulfoxide (DMSO, Sigma) and
the absorbance was measured at 570 nm by a multi-plate spec-
trophotometer.
In vitro Imaging
hMSCs were seeded in a 6 well plate at 5 10
3
cell/well density in
2 mL of culturing medium 24 h prior to particle feeding. After
24 h incubation with 2.98 mM of QD-Cy–SiO
2
, cells were washed
three times with PBS and then fixed in a 3.7% paraformaldehyde
solution in PBS at room temperature for 5 min. The cells were
then washed twice with PBS and incubated with 0.5% Triton
X-100 (Sigma-Aldrich) plus 1% bovine serum albumin (BSA;
Sigma-Aldrich) in PBS for 5 min. Then, fixed cells were incu-
bated with 5 mg/mL 40,6-diamidino-2-phenylindole (DAPI,
molecular probe) in PBS for nucleus staining for 10 min at room
temperature. Afterwards, 7.5 mL methanolic stock solution of
rhodamine phalloidin (Invitrogen) was diluted into 1 mL PBS
and used for actin staining for 20 to 30 min. The sample slide was
examined by a TCS SP2 confocal spectral microscope and MP
two-photon imaging system with a X40 water immersion objec-
tive, and using 800 nm two-photon diode laser and 543 nm
He–Ne laser as the excitation source. Since DAPI and CdSe/Zn-
Cy–SiO
2
with emission wavelengths of 530 nm could both be
excited through an 800 nm two-photon laser as DAPI features
a broad emission spectrum, an appropriate dye separation
procedure was performed.
Results and discussion
Freshly prepared type-I CdSe/ZnS-Cy and type-II CdTe/CdSe/
ZnS-Cy nanocrystals, made by phase-transfer procedures with
a cysteine (Cy) water-soluble ligand, commonly suffer from
rather weak emission intensity (QY #3%). To circumvent this, it
This journal is ªThe Royal Society of Chemistry 2009 J. Mater. Chem., 2009, 19, 8314–8319 | 8315

has been known that photo-oxidation of QDs to reduce the
surface defects plays a key role in enhancing the luminescence of
QDs, particularly in the aqueous solution.
12
Although this
workup procedure is rather succinct, the emphasis of this tactic
lies in the selection of the occasion. For example, applying photo-
oxidation at this stage could successfully regain the QY;
however, the effort would be devastated as significant quenching
re-occurred during silica encapsulation under a microemulsion
system. Therefore, in our viewpoint, a more appropriate
proposal to initiate photo induced oxidation reaction would be
after silane polymerization, referred here as delay photo-oxida-
tion. The advantage mainly lies in the fact that the cysteine
ligands, having been locked inside SiO
2
, remain unperturbed
during photo-oxidation. We then expect the as prepared
QDs-Cy–SiO
2
to be homogeneous for QDs (inside SiO
2
), well
dispersive and highly emissive.
As a result, the delay photo-oxidation strategy did effectively
preserve the emission intensity for both type I and type II
QDs-Cy–SiO
2
, and the degree of emission preservation depended
on the time of illumination. As shown in Fig. 1A, the immediate
photoluminescence spectra of freshly prepared silica (60 nm)
coated CdSe/ZnS (4.9 nm/0.7 nm) core–shell nanoparticles under
oxygen atmosphere, water as the solvent, and increasing period
of UV-366 nm (0.72 mW/cm) exposure demonstrates positive
enhancement in QY recoveries at 610 nm. The optimum expo-
sure period appeared to be 6 h as 5-fold recovery (QY 15%)
was achieved in comparison to the control. In addition to
emission gain, a blue shift of about 3 nm/h was also observed
within the photo-oxidation period of 6 h and then remained
unchanged after prolonged illumination. The shifting in emission
wavelength was the consequence of size reduction, as the rough
surface of the nanoparticles was etched after photo-oxidation.
12
To verify the origin of the emission as well as to demonstrate the
wavelength tunability of our synthetic approach, photo-
luminescence (PL) spectra of various sizes of silica coated QDs
(type I CdSe/ZnS-Cy, type II CdTe/CdSe/ZnS-Cy) subject to
photo-oxidation were measured. From the PL spectra shown in
Fig. 1B, several important remarks can be pointed out: first of all,
in response to the increase in size of QDs (core, vide infra), the
band-edge emission peaks systematically shift to the lower band
gap energy. As shown in Fig. 1B, emissions from different sizes
(measured by transmission electron microscopy (TEM), vide
infra) of CdSe/ZnS-Cy–SiO
2
at 530, 545, 580, 610 nm and
different sizes of CdTe/CdSe/ZnS-Cy–SiO
2
at 780 nm and
1027 nm demonstrate that the synthetic protocol employed is
capable of generating QDs with emissions ranging from visible to
the NIR region. This size dependent PL emission for both
CdSe/ZnS-Cy–SiO
2
and CdTe/CdSe/ZnS-Cy–SiO
2
, together
with similar emission spectral features of the bare QDs-Cy
(without SiO
2
, not shown here), warrants the origin of the
emission from the band gap. Note that each peak exists as a clean
single shape indicating that CdSe/ZnS-Cy–SiO
2
and CdTe/CdSe/
ZnS-Cy–SiO
2
nanocrystals maintained their unique optical
properties and dispersed homogeneously. More importantly, the
quantum yields of all QDs–SiO
2
prepared via delay photo-
oxidation are higher than 15% versus those of <0.05 prepared by
either non-photo-oxidation or photo-oxidation on non-SiO
2
coated QDs-Cy. Finally, this protocol is facile and reproducible,
producing monodispersity and high emission intensity of single
QDs at the center of smooth silica nanoparticles.
In addition to the exploitation of the cysteine ligand, 3-mer-
captopropionic acid (3-MPA) was also selected as a candidate for
QD surface modification to study the influence of the surface
ligand and the effectiveness of delay photo-oxidation. As both
cysteine and 3-MPA contain reducing functional groups, they
are expected to enhance the QY of the conjugated QDs.
17
Nonetheless, under identical treatment of 6 h illumination in an
oxygen atmosphere, the QDs-3-MPA–SiO
2
nanomaterials did
not receive comparable emission recovery as obtained by
QDs-Cy–SiO
2
(Fig. S1, ESI†). The difference in QY enhance-
ment may be rationalized by the stronger reducing power of the
amine functionality compared to that of the carboxylic group.
This finding reinforces the importance of amine functional
groups on the surface, and is in agreement with pervious reports,
stating that the amine functional ligand modification on e.g. ZnO
nanoparticles and alkyl-amine modification on Zn
x
Cd
1x
Se alloy
nanoparticles augments emission intensity much more efficiently
compared to 3-MPA or thiolglycolic acid species. It is also noted
that SiO
2
–QDs modified with 3-amino- propyltrimethoxysilane
carry a stronger emission intensity than uncoated SiO
2
–QDs.
9a,18,19
In brief, amino functional ligands seem to serve as an indis-
pensable capping agent in respect to delay photo-oxidation
enhancing emission.
As for the morphology, the TEM images of cysteine ligand
capped QDs, including CdSe/ZnS and CdTe/CdSe/ZnS nano-
crystals are shown in Fig. S2. (ESI†). Compared to the TEM
pictures of CdSe/ZnS with TOPO and HDA, QDs-Cy was less
clear in imaging because of the water-soluble capping ligand on
the QDs surface. The use of water-soluble QDs-Cy assured that
the nanoparticles were located in the water pool before
polymerization, which is an important criterion for excellent
silica encapsulation. As one can perceive in Fig. 2A, the size of
the core (3.5 nm) in the silica shell provides positive identification
of QDs-Cy. Insert of Fig. 2 clearly indicates that CdSe/ZnS
(3.5–5.8 nm) and CdTe/CdSe/ZnS (7.1 nm) nanocrystals
(Fig. 2A–2D) are successfully encapsulated within the silica
shells. Although QDs embedded in silica appeared as an amor-
phous structure, a more detailed examination by high resolution
TEM (HRTEM) revealed the crystal lattice of QDs nanocrystals
Fig. 1 (A) Photoluminescence spectra of freshly prepared CdSe/ZnS-
Cy–SiO
2
(4.9 nm/0.7 nm) core–shell nanoparticles with 610 nm emission
after illumination. Increases in luminescence were obtained with
increasing period of UV-366 nm (0.72 mW/cm) exposure. (B) The QDs-
Cy–SiO
2
PL peaks resulted from 530, 545, 580, 610, 780, (3.5–5.8 nm in
diameter) and 1027 nm (7.1 nm in diameter) correlating to the CdSe/ZnS,
CdTe/CdSe/ZnS cores.
8316 | J. Mater. Chem., 2009, 19, 8314–8319 This journal is ªThe Royal Society of Chemistry 2009

(see insert of Fig. 2C). The monodisperisty and the overall size of
the nanomaterials of 60 nm measured by TEM show that the
shape of nanocrystals were retained after the polymerization and
photo-oxidation process. The advanced verification of the
composition of QDs-Cy–SiO
2
nanoparticles was then analyzed by
energy dispersive X-ray spectroscopy (EDX). The results shown
in Fig. S3A† (CdSe/ZnS-Cy) and S3B† (CdTe/CdSe/ZnS-Cy)
unveil the elemental characteristic peaks and the corresponding
atomic percentage ratios, which render sufficient evidence for
successful encapsulation of the CdSe/ZnS-Cy and CdTe/CdSe/
ZnS-Cy in the SiO
2
shell.
Previously, our group has demonstrated that QDs such as
CdSe, and CdTe possessed outstanding two-photon absorption
cross-section values (>10 000 GM), and it would be eligible to
assume that QDs-Cy–SiO
2
is applicable in two-photon imaging
as well.
20
As for the naked eye view, the QDs-Cy–SiO
2
two-
photon visible imaging pictures (e.g. 530 nm) excited by NIR
laser (800 nm) in aqueous solution were taken and depicted in the
insert (lower right corner) of Fig. 2A. Also, the insert (lower right
corner) of Fig. 2D shows the two-photon emission of
CdTe/CdSe/ZnS-Cy–SiO
2
(l
ex
1200 nm) captured by
a conventional hand-held infrared viewer coupled with a CCD
detector. The results affirm the potential of QDs-Cy–SiO
2
in two-
photon bio-imaging.
We then applied the above silica nanoparticle embedded QDs
with in vitro optical imaging; particular focus is on two-photon
imaging. Prior to the experiment, a cytotoxicity test of the QDs-Cy–
SiO
2
nanocrystals is necessary, in which human mesenchymal stem
cells (hMSCs) were adopted as a target. Stem cells are self-renewabl e
and capable of differentiating into different kinds of cells that are
essential for tissue transplantation. Such unidirectional
differentiation forbids the reversible process once the cells are in the
designated locations. Consequently, labeling of stem cells with
QDs-Cy–SiO
2
is biomedically important and will facilitate the
understanding of stem cell biology. The hMSCs were immortalized
by the gene transfer of a combination of human telomerase reverse
transcriptase (hTERT) with human papillomavirus. The differen-
tiation ability of these hMSCs has been previously demonstrated.
21
To examine the cytotoxicity of the as prepared particles, cells
were incubated with six different dosages of particles for 24 h, the
concentration of which was confirmed with inductively coupled
plasma-mass spectrometry (ICP-MS): 0, 0.19, 0.37, 0.75, 1.49,
and 2.98 mM Cd. The feedback suggested that bare CdSe/ZnS-Cy
are detrimental to cells as the cellular viability dropped down to
70% at higher incubation concentrations (2.98 mM, see ESI†
for QD/cell calculation). In contrast, both CdSe/ZnS-Cy–SiO
2
and CdTe/CdSe/ZnS-Cy-SiO
2
were harmless toward cells as the
viabilities of the cells within the prescribed dosages remained well
above 90% even after 24 h incubation (Fig. S4, ESI†).The uptake
efficiency of the nanoparticles, using CdSe/ZnS-Cy–SiO
2
as the
representing QDs, was found to be 13%, in equivalence of
1.40 10
7
QDs-Cy–SiO
2
per cell. Note that a harsh condition of
1:1 HNO
3
/H
2
O acid digestion was performed on a hot plate for
6 h to digest the silica protected core before ICP-MS measurement.
To ensure the event of internalization and the location of the
particles inside the cell, confocal microscopy with two-photon
laser and fluorescence staining has been performed. In this
experiment, hMSCs incubated with 2.98 mM of QD-Cy–SiO
2
for
24 h were fixed with paraformaldehyde solution and stained with
molecular dyes DAPI and rhodamine phalloidin for nucleus and
cytoskeleton labeling, respectively. The confocal image shown in
Fig. 3 clearly indicates that QDs-Cy–SiO
2
of green fluorescence
was internalized into the cells and mainly resided in the
Fig. 2 TEM pictures of core–shell–shell nanomaterials (CdSe/ZnS-Cy–
SiO
2
) (2A–C) and core–shell–shell–shell (CdTe/CdSe/ZnS-Cy–SiO
2
) (2D)
nanoparticles after illumination. Upper right insets: emission of the
corresponding nanoparticles under UV lamp. Lower right insets in (A)
and (D): emission of the corresponding nanoparticles with two-photon
laser excitation. Lower right inset in (C): HRTEM picture of CdSe/ZnS-
Cy–SiO
2
.
Fig. 3 Microscopic observation of the QDs-Cy–SiO
2
internalization.
hMSCs were treated with silica coated QDs for 24 h and then processed
for two photon and confocal microscopic examination. Cell nuclei were
stained with 40,6-diamidino-2-phenylindole (blue color). Actin fibers were
stained with rhodamine phalloidin to confirm the cell boundary
(red color). QDs-Cy–SiO
2
exhibit a two photon emission (green fluo-
rescence, 530 nm) for in vitro bio-applications. Fluorescence overlay
image demonstrates the internalization of QDs-Cy–SiO
2
which resided
near the nucleus.
This journal is ªThe Royal Society of Chemistry 2009 J. Mater. Chem., 2009, 19, 8314–8319 | 8317

cytoplasm nearby the nucleus. Although the exact location of the
particle confinement was not fully determined, collective images
from Z-stack scanning (see Fig. 4) advised that no particles were
located on the cell membrane or inside the nucleus. The confocal
imaging illustrated efficient labeling and specific location char-
acters of the CdSe/ZnS-Cy–SiO
2
. The biocompatibility of
CdSe/ZnS-Cy–SiO
2
with 530 nm was ensured in two ways:
(1) negligible cytotoxicity from cell proliferation test; and
(2) total preservation of two-photon signal after internalization
into the cell as observed under the confocal microscopy. It should
be noted here that although Prasad and co-workers have
reported that QD nanocomposites after silica shell modification
could be uptaken by cancer cells for two-photon imaging, the
structural robustness of the core–shell to avoid heavy element
release remains sceptical.
22
Our findings provide solid and strong
evidence that QD nanocomposites after silica shell modification
are very practical and versatile in biological two-photon imaging.
Conclusions
By using a bottom-up synthesis approach, this article illustrates
a synthetic strategy to prepare QDs–SiO
2
nanocomposites and
their potential applications for bio-imaging. This facile and
simple method incorporating a delay photoactive procedure not
only embeds core–shell nanoparticles, such as CdSe/ZnS and
CdTe/CdSe/ZnS, into SiO
2
for reducing its cytotoxicity, but also
makes semiconductor nanocrystals retaining their unique and
excellent optical properties. We then successfully demonstrate
their power in two-photon imaging of human mesenchymal stem
cells. For future work, QDs-Cy–SiO
2
(shell) featuring both NIR
emission and two photon absorption effects simultaneously will
be explored. We also believe that this facile synthetic protocol
can be exploited in other QD species with unique optical
properties to preserve their own emission wavelength and
intensity after being embedded in silica nanoparticles. This
protocol should be vital for the synthesized nanomaterials to be
non-toxic with minimal dosage required to generate effective
fluorescent signal within a living system.
Acknowledgements
This work was funded by the National Science Council of
Taiwan, R.O.C. We thank the excellent technical assistance of
Technology Commons, College of Life Science, NTU (Taiwan)
with the confocal laser scanning microscopy (CLSM).
Notes and references
1(a) A. P. Alivisatos, Science, 1996, 271, 933; (b) M. Danek,
K. F. Jensen, C. B. Murray and M. G. Bawendi, Chem. Mater.,
1996, 8, 173; (c) M. Nirmal and L. Brus, Acc. Chem. Res., 1999, 32,
407; (d) H. Mattoussi, J. M. Mauro, E. R. Goodman,
G. P. Anderson, V. C. Sundar, F. V. Mikulec and M. G. Bawendi,
J. Am. Chem. Soc., 2000, 122, 12142.
2(a) B. Murray, D. J. Norris and M. G. Bawendi, J. Am. Chem. Soc.,
1993, 115, 8706; (b) J. K. Jaiswal, H. Mattoussi, J. M. Mauro and
S. M. Simon, Nat. Biotechnol., 2003, 21, 47; (c) A. P. Alivisatos,
J. Phys. Chem., 1996, 100, 13226; (d) C. B. Murray, C. R. Kagan
and M. G. Bawendi, Annu. Rev. Mater. Sci., 2000, 30, 545;
(e) H. Weller, Angew. Chem., Int. Ed. Engl., 1993, 32, 41;
(f) J. M. Klostranec and W. C. W. Chan, Adv. Mater., 2006, 18, 1953.
3(a) S. Kim, B. Fisher, H. J. Eisler and M. G. Bawendi, J. Am. Chem.
Soc., 2003, 125, 11466; (b) C.-Y. Chen, C.-T. Cheng, J.-K. Yu,
S.-C. Pu, Y.-M. Cheng, P.-T. Chou, Y.-H. Chou and H.-T. Chiu,
J. Phys. Chem. B, 2004, 108, 10687; (c) P.-T. Chou, C.-Y. Chen,
C. T. Cheng, S.-C. Pu, K.-C. Wu, Y.-M. Cheng, C.-W. Lai,
Y.-H. Chou and H.-T. Chiu, ChemPhysChem, 2006, 7, 222;
(d) B. Blackman, D. M. Battaglia, T. D. Mishima, M. B. Johnson
and X. G. Peng, Chem. Mater., 2007, 19, 3815; (e) C.-Y. Chen,
C.-T. Cheng, C.-W. Lai, Y.-H. Hu, P.-T. Chou, Y.-H. Chou and
H.-T. Chiu, Small, 2005, 1, 1215; (f) C.-T. Cheng, C.-Y. Chen,
C.-W. Lai, W.-H. Liu, S.-C. Pu, P.-T. Chou, Y.-H. Chou and
H.-T. Chiu, J. Mater. Chem., 2005, 15, 3409.
4(a) B. O. Dabbousi, J. Rodriguez-Viejo, F. V. Mikulec, J. R. Heine,
H. Mattoussi, R. Ober, K. F. Jensen and M. G. Bawendi, J. Phys.
Chem. B, 1997, 101, 9463; (b) D. V. Talapin, A. L. Rogach,
A. Kornowski, M. Haase and H. Weller, Nano Lett., 2001, 1, 207;
(c) P. Reiss, J. Bleuse and A. Pron, Nano Lett., 2002, 2, 781.
5(a) S. Kim, Y. T. Lim, E. G. Soltesz, A. M. De Grand, J. Lee,
A. Nakayama, J. A. Parker, T. Mihalijevic, R. G. Laurence,
D. M. Dor, L. H. Cohn, M. G. Bawendi and J. V. Frangioni, Nat.
Biotechnol., 2004, 22, 93; (b) L. Bakueva, I. Gorelikov, S. Musikhin,
X. S. Zhao, E. H. Sargent and E. Kumacheva, Adv. Mater., 2004, 16,
926; (c) W. Jiang, S. Mardyani, H. Fischer and W. C. W. Chan,
Chem. Mater., 2006, 18, 872; (d) Y. Lei, H. Y. Tang, L. D. Yao,
R. C. Yu, M. F. Feng and B. S. Zou, Bioconjugate Chem., 2008, 19, 421.
6(a) W. Siebrand, J. Chem. Phys., 1967, 47, 2411; (b) K.-Y. Chen,
C.-C. Hsieh, Y.-M. Cheng, C.-H. Lai and P.-T. Chou, Chem.
Commun., 2006, 4395.
7(a) Jr. M. Bruchez, M. Moronne, P. Gin, S. Weiss and
A. P. Alivisatos, Science, 1998, 281, 2013; (b) N. Gaponik,
D. V. Talapin, A. L. Rogach, K. Hoppe, E. V. Shevchenko,
A. Kornowski, A. Eychmu1ller and H. Weller, J. Phys. Chem. B,
2002, 106, 7177; (c) T. Zhang, J. L. Stilwell, D. Gerion, L. Ding,
O. Elboudwarej, P. A. Cooke, J. W. Gray, A. P. Alivisatos and
F. F. Chen, Nano Lett., 2006, 6, 800; (d) A. Derfus, W. Chan and
S. Bhatia, Nano Lett., 2004, 4, 11; (e) C. Kirchner, T. Liedl,
S. Kudera, T. Pellegrino, A. Munoz Javier, H. E. Gaub, S. Stolzle,
N. Fertig and W. J. Parak, Nano Lett., 2005, 5, 331.
8 S. Santra, P. Zhang, K. Wang, R. Tapec and W. H. Tan, Anal. Chem.,
2001, 73, 4988.
9(a) S. Selvan and T. Tan, Adv. Mater., 2005, 17, 1620; (b) M. Darbandi,
R. Thomann and T. Nann, Chem. Mater., 2005, 17, 5720.
10 Z. Zhelev, H. Ohba and R. Bakalova, J. Am. Chem. Soc., 2006, 128,
6324.
Fig. 4 Confocal microscopy examination of QDs-Cy–SiO
2
labeled
hMSCs. For confirmation of the exact intracellular location of QDs-Cy–
SiO
2
, hMSCs treated with QDs-Cy–SiO
2
for 48 h were stained with
40,6-diamidino-2-phenylindole (DAPI) for visualization of the cell
nucleus and the actin fibers were stained with rhodamine phalloidin. The
QDs-Cy–SiO
2
was located at the cytoplasm near the nucleus. No
QDs-Cy/SiO
2
was detected at the cell membrane or nucleus. The finding
indicates efficient labeling and specific location characters of the
QDs-Cy/SiO
2
.
8318 | J. Mater. Chem., 2009, 19, 8314–8319 This journal is ªThe Royal Society of Chemistry 2009

11 R. Koole, M. M. van Schooneveld, J. Hilhorst, C. D. Doneg
a, C. D. ’t
Hart, A. van Blaaderen and D. Vanmaekelbergh, Chem. Mater., 2008,
20, 2503.
12 (a) A. A. Bol and A. Meijerink, J. Phys. Chem. B, 2001, 105, 10203;
(b) L. Manna, E. C. Scher, L.-S. Li and A. P. Alivisatos, J. Am.
Chem. Soc., 2002, 124, 7136; (c) Y. Wang, Z. Tang, M. A. Correa-
Duarte, I. Pastoriza-Santos, M. Giersig, N. A. Kotov and
L. M. Liz-Marz
an, J. Phys. Chem. B, 2004, 108, 15461–15469.
13 (a) M. Darbandi, W. Lu, J. Fang and T. Nann, Langmuir, 2006, 22,
4371; (b) T. T. Tan, S. T. Selvan, L. Zhao, S. Gao and J. Y. Ying,
Chem. Mater., 2007, 19, 3112.
14 C.-Y. Chen, C.-T. Cheng, C.-W. Lai, P.-W. Wu, K.-C. Wu, P.-
T. Chou, Y.-H. Chou and H.-T. Chiu, Chem. Commun., 2006, 263.
15 (a) S. Santra, R. P. Bagwe, D. Dutta, J. T. Stanley, G. A. Walter,
W. Tan, B. M. Moudgil and R. A. Mericle, Adv. Mater., 2005, 17,
2165; (b) C.-W. Lai, Y.-H. Wang, C.-H. Lai, M.-J. Yang,
C.-Y. Chen, P.-T. Chou, C.-S. Chan, Y. Chi, Y.-C. Chen and
J.-K. Hsiao, Small, 2008, 4, 218.
16 (a) W. W. Yu, L. Qu, W. Guo and X. Peng, Chem. Mater., 2003, 15,
2854; (b) B. O. Dabbousi, J. Rodrigez-Viejo, F. V. Mikulec,
J. R. Heine, H. Mattoussi, R. Ober, K. J. Jensen and
M. G. Bawendi, J. Phys. Chem., 1997, 101, 9463.
17 Y.-J. Chen and X.-P. Tan, Small, 2009, 5, 2012.
18 F.-C. Liu, T.-L. Cheng, C.-C. Shen, W.-L. Tseng and M. Y. Chiang,
Langmuir, 2008, 24, 2162.
19 M. L. Kahn, T. Cardinal, B. Bousquet, M. Monge, V. Jubera and
B. Chaudret, ChemPhysChem, 2006, 7, 2392.
20 S.-C. Pu, M.-J. Yang, C.-C. Hsu, C.-W. Lai, C.-C. Hsieh, S. H. Lin,
Y.-M. Cheng and P.-T. Chou, Small, 2006, 2, 1308.
21 (a) J.-K. Hsiao, M.-F. Tai, H.-H. Chu, S.-T. Chen, H. Li, D.-M. Lai,
S.-T. Hsieh, J.-L. Wang and H.-M. Liu, Magn. Reson. Med., 2007, 58,
717; (b) T. Okamoto, T. Aoyama, T. Nakayama, T. Nakamata,
T. Hosaka, K. Nishijo, T. Nakamura, T. Kiyono and J. Toguchida,
Biochem. Biophys. Res. Commun., 2002, 295, 354.
22 W.-C. Law, K.-T. Yong, I. Roy, G. X. Xu, H. Ding, E. J. Bergey,
H. Zeng and P. N. Prasad, J. Phys. Chem. C, 2008, 112, 7972.
This journal is ªThe Royal Society of Chemistry 2009 J. Mater. Chem., 2009, 19, 8314–8319 | 8319