Covalent immobilization of aggregation-induced emission luminogens in silica nanoparticles through click reaction

Article abstract of DOI:10.1002/smll.201002195

Fluorescent silica nanoparticles (FSNPs) with efficient light emission, colloidal stability, and size tunability are fabricated by one-pot, two-step Stoeber and reverse microemulsion techniques. Tetraphenylethene (TPE)- and silole-functionalized siloxanes are facilely synthesized by click reactions and their sol-gel reactions followed by reactions with tetraethoxysilane generate FSNPs with core-shell structures. The FSNPs are uniformly sized with smooth surfaces, and show high surface charges and hence excellent colloidal stability. UV irradiation of ethanol solutions of the FSNPs gives strong blue and green light at 474 and 486 nm in high fluorescence quantum yields, thanks to the novel aggregation-induced emission attributes of the TPE and silole aggregates in the hybrid nanoparticles. The FSNPs are benign to living cells and function as fluorescent visualizers for intracellular imaging of HeLa cells. Luminogenic siloxanes with aggregation-induced emission characteristics are synthesized by click chemistry, and their sol-gel reactions followed by reactions with tetraethoxysilane furnish fluorescent silica nanoparticles (FSNPs) with core/shell structures. The FSNPs are monodisperse, colloidally stable, and emit blue and green light with high efficiency upon UV irradiation. The FSNPs are benign to living cells and function as fluorescent visualizers for intracellular imaging.

Full text of DOI:10.1002/smll.201002195

full papers
Fluorescent Nanoparticles
Covalent Immobilization of Aggregation-Induced
Emission Luminogens in Silica Nanoparticles Through
Click Reaction
Faisal Mahtab, Jacky W. Y. Lam, Yong Yu, Jianzhao Liu, Wangzhang Yuan, Ping Lu,
and Ben Zhong Tang*
F luorescent silica nanoparticles (FSNPs) with efficient light emission, colloidal
stability, and size tunability are fabricated by one-pot, two-step Stöber and reverse
microemulsion techniques. Tetraphenylethene (TPE)- and silole-functionalized
siloxanes are facilely synthesized by click reactions and their sol–gel reactions followed
by reactions with tetraethoxysilane generate FSNPs with core–shell structures. The
FSNPs are uniformly sized with smooth surfaces, and show high surface charges and
hence excellent colloidal stability. UV irradiation of ethanol solutions of the FSNPs
gives strong blue and green light at 474 and 486 nm in high fluorescence quantum
yields, thanks to the novel aggregation-induced emission attributes of the TPE and
silole aggregates in the hybrid nanoparticles. The FSNPs are benign to living cells
and function as fluorescent visualizers for intracellular imaging of HeLa cells.
physiological pH.[1] There are numerous features that make
silica nanoparticles superior to their polymeric counterparts,
1. Introduction
such as good hydrophilicity and easy surface modification.[2–6]
They also show high thermal stability and possess a large sur-
face area and excellent dispersion in aqueous media. As silica
nanoparticles are nonfluorescent and chemically inert, they
are environmentally friendly and are an ideal host material
for the inclusion of fluorophores and magnetic clusters.
Both inorganic and organic (fluorescein, rhodamine, etc.)
fluorophores are used for the preparation of FSNPs. Among
them, semiconductor quantum dots (QDs) have attracted
much attention, particularly in the area of cellular marking
or imaging, because of their advantages of size-tunable emis-
sion color, long luminescence lifetime, and high resistance to
photobleaching. Their surface, however, needs to be function-
alized in order to improve their hydrophilicity and reduce
their toxicity as QDs are usually chalcogenides of heavy
metals (e.g., CdSe, ZnSe, CdS, and PdTe), which are well-
known toxicants or carcinogens.[7–11]
The fabrication of fluorescent silica nanoparticles (FSNPs)
has been a fast-growing research area for the last two dec-
ades because of their potential applications in a number of
nanotechnological fields ranging from biological to advanced
engineering. Much interest has been placed on the construc-
tion of FSNPs with controlled size, high monodispersity and
fluorescence quantum yield, and good colloidal stability at
Dr. F. Mahtab, Dr. J. W. Y. Lam, Y. Yu, Dr. J. Liu, Dr. W. Yuan, Dr. P. Lu,
Prof. B. Z. Tang
Department of Chemistry
The Hong Kong University of Science & Technology (HKUST)
Clear Water Bay, Kowloon, Hong Kong
E-mail: tangbenz@ust.hk
Dr. F. Mahtab, Dr. J. W. Y. Lam, Y. Yu, Dr. J. Liu, Dr. W. Yuan, Dr. P. Lu,
Prof. B. Z. Tang
HKUST Fok Ying Tung Research Institute
Nansha, Guangzhou, China
FSNPs can be prepared by incorporating fluorophores
into the silica networks via physical processes or chem-
ical reactions. The silica matrix acts as a protective shield,
which reduces the likelihood of penetration of oxygen and
other harmful species that may cause photobleaching of
the embedded fluorophores.[12–14] FSNPs synthesized by the
Prof. B. Z. Tang
Department of Polymer Science and Engineering
Zhejiang University
Hangzhou 310027, China
DOI: 10.1002/smll.201002195
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Immobilization of Luminogens in Silica Nanoparticles Through Click Reaction
Si(OC2H5)3
(CH2)3
N
N
N
9
)
(
TEOS, C2H5OH, NH4OH
Stöber method
Cl
N3
FSNP-1
NaN3
O
Si
O
O
O
Si
O
8
O
DMF, 90 oC, 5 h
Cu(PPh3)3Br, THF, 60 oC, 24 h
7
TEOS, cyclohexane, NH4OH
Microemulsion
N
N
N
5
2
FSNP-
(CH2)3
Si(OC2H5)3
Scheme 1. Fabrication of TPE-containing fluorescent silica nanoparticles FSNP-1 and FSNP-2.
Stöber method suffer from size limitation to below 100 nm.To a “click” reaction due to its high efficiency and selectivity,
overcome this problem, reverse microemulsion has recently mild reaction conditions, and simple isolation procedure.[27]
gained tremendous attention for the fabrication of nanosized, Organic fluorophores have been incorporated chemically
monodispersed FSNPs.
into the silica matrix through an ester or thiourea linkage,
Development of fluorescent bioprobes with high signal which makes them not leak out from the FSNPs under harsh
changes in the presence of analytes is a hot research topic. conditions.[28–30] Few papers have, however, reported the uti-
However, light emissions from most of the FSNPs prepared lization of click chemistry to covalently link fluorophores to
so far using conventional organic dyes have been rather weak. silica nanoparticles. In this work, we explored the possibility
This is due to the emission quenching caused by the aggrega- of hybridizing silica nanoparticles with AIE luminogens by
tion of the fluorophores in the solid state.[15] A low fluoro- click chemistry. We have developed a facile one-pot, two-
phore loading in the nanoparticle may be free of aggregation step protocol for their preparation. The resultant FSNPs are
but can offer only a weak fluorescence signal. The light emis- monodispersed and show high surface charges and hence
sion can be further weakened, rather than enhanced, if more excellent colloidal stability. Thanks to the AIE effect of the
fluorophores are loaded into the nanoparticles, because of the fluorophores and the inert nature of the silica, the FSNPs
notorious aggregation-caused quenching (ACQ) effect.[16]
emit strong visible light upon photoexcitation and function as
We have discovered an “abnormal” phenomenon of fluorescent visualizers for intracellular imaging of HeLa cells.
aggregation-induced emission (AIE) that is exactly oppo-
site to the ACQ effect: a group of propeller-like, nonemis-
sive molecules is induced to emit efficiently by aggregate
2. Results and Discussion
formation.[17–21] The AIE effect greatly boosts the emission
2.1. Synthesis of FSNPs
efficiency of the luminogens, and turns them from weak lumi-
nophores into strong emitters. An example is represented by
4,4′-bis(1,2,2-triphenylvinyl)biphenyl, of which the quantum
To explore the utility of click chemistry for the fabrica-
yields in the solution (ΦF,S) and aggregate (ΦF,A) states are tion of highly emissive FSNPs, we synthesized tetraphe-
≈0 and 100%, respectively, which results in an infinitely large nylethene (TPE)- and silole-containing diynes (9 and 10, see
AIE effect (αAIE = ΦF,A/ΦF,S → ∞).[17] Mechanistic investiga- Scheme 1 and 2) according to our previously reported pro-
tions reveal that the AIE effect is caused by the restriction of cedures.[31,32] We also prepared azide-functionalized siloxane
the intramolecular rotations of the luminogens in the aggre- 8 in high yield by reaction of 3-chloropropyltriethoxysilane
gate state, which block the nonradiative channels and popu- (7) with sodium azide in dry dimethylformamide (DMF) at
late the radiative decay.[22–25]
90 °C for 4 h. To covalently bind the AIE luminogens to the
1,3-Dipolar cycloaddition was discovered by Huisgen in silica nanoparticles at the molecular level, we employed 8 as
1984.[26] Later, Sharpless and co-workers found that this reac- a chemical linker. We carried out the click reaction of 8 and
tion can be accelerated by Cu(I) species. They coined it as 9 in the presence of Cu(PPh3)3Br in distilled tetrahydrofuran
TEOS, C2H5OH, NH4OH
Stöber method
(CH2)3Si(OC2H5)3
FSNP-3
Cu(PPh3)3Br
N
N
N
N
N
8
+
Si
THF, 60 oC, 24 h
N
Si
(C2H5O)3Si(CH2)3
TEOS, cyclohexane, NH4OH
Microemulsion
10
6
FSNP-4
Scheme 2. Synthesis of silole derivative 6 and fabrication of its fluorescent silica nanoparticles FSNP-3 and FSNP-4.
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(THF) at 60 °C under nitrogen. The reaction product gives
an (M + 1)+ peak at 875.4327 in its high-resolution (HR)
mass spectrum (Figure S1 in the Supporting Information, SI),
thereby confirming the occurrence of the cycloaddition reac-
tion and the formation of expected adduct 5 (M+ = 874.4269).
Employing the same conditions, silole-functionalized siloxane
6 was successfully synthesized from 8 with 10, as confirmed
by the HR mass spectrum given in the SI, Figure S2.
A
B
FSNPs with core–shell structures can be prepared from
5 by using the Stöber and reverse microemulsion techniques.
In the former method, the molecules of 5 are hydrolyzed and
condensed in an ethanol/water mixture containing ammo-
nium hydroxide under stirring at room temperature for
30 min.The resultant TPE–silica nanocores are then subjected
to further sol–gel reaction with tetraethoxysilane (TEOS) to
furnish FSNP-1 (Scheme 1). Microemulsion is a well-known
method to produce nanometer-sized particles with narrow
distribution. The formation of a thermodynamically stable
nanoreactor is facilitated by the introduction of Triton-X as
amphiphilic surfactant. A hydrophilic head region is formed
with numerous nanometer-sized water cores and each with
a hydrophobic tail that extends into an apolar continuous
phase of cyclohexane. The nanodroplets of water in the bulk-
oil phase act as nanoreactor for discrete particle formation.
After addition of adduct 5, the mixture was stirred at room
temperature for 30 min. The synthesized TPE–silica nano-
cores then undergo further sol–gel reaction with TEOS, to
generate small-sized FSNP-2 with core–shell architecture.
Similarly, coupling of 8 with 10 affords adduct 6 and
C
D
4000
3000
2000
1500
1000
500
Wavenumber (cm-1)
Figure 2. IR spectra of A) 7, B) 8, C) 9, and D) 5.
the aqueous mixtures and their shrinkages under the high
electron-beam intensity in the TEM chamber.
2.2. Structural Characterization
Figure 2 shows the IR spectrum of 5; for comparison,
its two-step sol–gel reaction in one-pot Stöber and reverse the spectra of 7–9 are also provided. The azido stretching
microemulsion protocols produces FSNP-3 and FSNP-4, vibration of 8 is observed at 2098 cm−1 , which is absent in 5
respectively.
(Figure 2D). The spectrum of 5 also shows no peaks associ-
Analysis by zeta potential analyzer at room temperature ated with the HC≡ and C≡C stretching vibrations of 9 at 3291
shows that all the FSNPs are monodispersed with low poly- and 2107 cm−1 .These results suggest that the azido groups of 8
dispersities down to 0.005 (Figure 1 and Figure S3). The mean have cyclized with the triple bonds of 9 to form triazole rings
diameters of FSNP-1 and FSNP-3 are 185.7 and 255.6 nm, in 5. Similar observations are also found when comparing the
respectively, which are somewhat larger than those measured spectrum of 6 with those of 8 and 10 (Figure S4).
by transmission electron microscopy (TEM; 143.37
10.5
Analysis by energy-dispersive X-ray (EDX) diffraction of
and 217.26 20.4 nm for FSNP-1 and FSNP-3, respectively) the FSNPs shows that they contain the expected elements of
due to the larger hydrodynamic diameters of the FSNPs in carbon, nitrogen, oxygen, and silicon. Examples of the EDX
spectra of FSNP-1 and FSNP-3 are pro-
vided in Figure S5, and Table S1 (SI) gives
the compositions.
B
A
de = 185.1 nm
dm = 185.7nm
PD = 00.006
de = 255.0 nm
dm = 255.6nm
PD = 00.005
2.3. Size and Morphology
It is important to tune the sizes of nano-
particles to meet the requirements of dif-
ferent technological applications.The Stöber
and reverse microemulsion methods give
large- and small-sized FSNPs, respectively.
Actually, the sizes of the nanoparticles can
also be tuned by varying the reaction para-
meters. Larger nanoparticles are obtained
by using higher concentrations of TEOS
and ammonium hydroxide and vice versa, as
demonstrated by our recent publication.[33]
TEM images show that the large-sized
100
200
300
400
500
100
200
300
400
500
Particle size (nm)
Figure 1. Particle size distributions of A) FSNP-1 and B) FSNP-3. Abbreviations: de = effective
diameter, dm = mean diameter, PD = polydispersity.
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Immobilization of Luminogens in Silica Nanoparticles Through Click Reaction
FSNPs possess smooth surfaces, while the surfaces of the small
nanoparticles (i.e., FSNP-2 and FSNP-4) are somewhat rough
(Figure 3 and Figure S6). Analyses by scanning electron micro-
scopy (SEM) also give similar results (Figure 4 and Figure S7).
The mean diameters of FSNP-2 and FSNP-4 determined by
TEM are ≈37.68 2.7 and 59.82 4.1 nm, respectively, thus
proving that the reverse microemulsion method does indeed
generate FSNPs with much smaller sizes than those prepared
by the Stöber technique in a controlled fashion.
2.4. Light Emission
The AIE luminogens are chemically linked to and cov-
ered by the inert silica shell, which protects them from direct
exposure to the harsh environments as well as their potential
leakage to the media. Even if a small amount of the dye mol-
ecules is leaked out, it will not interfere as the dissolved fluor-
ophores will not emit (hence no background signals). This is a
“bonus” or added advantage of the AIE dyes over their con-
ventional dye counterparts. Figure 5 shows the fluorescence
spectra of 9 and 10 and the suspensions of their core–shell
nanoparticles FSNP-1 and FSNP-3 in ethanol. Nearly no fluo-
rescence signals are recorded when the solutions of 9 and 10
are photoexcited, because the multiple phenyl rings of 9 and
10 undergo active intramolecular rotations in the solutions,
which effectively annihilate their excited states and hence
render them nonemissive.[27–30] When the molecules of 9 and
10 are covalently incorporated into, and aggregated in, the
silica networks, strong fluorescence spectra peaked at 474 and
489 nm are recorded in FSNP-1 and FSNP-3, respectively.The
rigid silica networks largely restrict the intramolecular rota-
tions of the luminogens. This blocks the nonradiative relaxa-
tion channels and populates the radiative decay, thus making
the FSNPs highly luminescent.
Figure 4. SEM images of A,B) FSNP-1 and C,D) FSNP-3 at different
magnifications.
By dissolving 9 and 10 and dispersing FSNP-1 and FSNP-
3 fabricated by using the same molar quantities of lumino-
gens (i.e., 5 and 6) in ethanol, their emission intensities are
compared. The light emission from FSNP-1 and FSNP-3 is
1010- and 916-fold higher than those of 9 and 10, respectively.
The absolute fluorescence quantum yields (Φabs) of FSNP-1
and FSNP-3 determined by integrating spheres are 33.4 and
38.2%, respectively. The light emission is very stable, with no
change in the fluorescence spectra detectable after the FSNPs
were placed on shelves for several months without protection
from light and air. Figure 6 shows photographs of ethanol
solutions of 9 and 10 and the suspensions of FSNP-1 and
FSNP-3 taken under UV irradiation. Whereas the solutions
of 9 and 10 are nonemissive, intense blue and green light is
observed in FSNP-1 and FSNP-3, respectively.
Figure 3. TEM images of A–C) FSNP-1 and D–F) FSNP-3 at different magnifications with particle sizes of ≈143.37 10.5 and 217.26 20.4 nm,
respectively.
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potentials. FSNPs are said to be
colloidally stable if their surface
charges are high at the workable
pH because strong electrostatic
repulsion will exist between the
nanoparticles. The functional
groups play an important role in
determining the surface charges
of the FSNPs. In our previous
work, we reacted brominated
TPE and silole with 3-amino-
A
B
FSNP-1
9
FSNP-3
10
propyltriethoxysilane
(APS)
and used the adducts as fluores-
cent cores for the fabrication of
highly emissive and monodis-
persed FSNPs.[33] Their charges
at neutral pH are, however, not
high enough to impart high col-
loidal stability. This is due to the
400
480
560
640 400
Wavelength (nm)
500
600
700
Figure 5. Fluorescence spectra of ethanol solutions of A) FSNP-1 and 9 and B) FSNP-3 and 10.
Concentration: 200 μg mL−1 ; excitation wavelength: A) 353 and B) 370 nm.
T h e fl uorescence spectra of FSNP-2 and FSNP-4 are given presence of free amine groups on the surface, which partially
in Figure S8 (SI). The emissions of FSNP-2 and FSNP-4 are counteract the negative charge contributed by the silanol
observed at slightly longer wavelengths than those of FSNP- groups. Similarly, FSNPs with thiourea linkages obtained by
1 and FSNP-3. Compared with FSNP-1 and FSNP-3, the Φabs reaction of isothiocyanated dye molecules with APS possess
values of FSNP-2 and FSNP-4 are slightly lower (32.8 and 35.0% even lower colloidal stability and precipitate in ethanol and
for FSNP-2 and FSNP-4, respectively), which is understand- water at pH ≥ 7.
able when taking into account that the dye loadings used for
Adducts 5 and 6 are synthesized from 8 instead of APS.
the construction of FSNP-2 and FSNP-4 are only half of those Thus, FSNPs fabricated from these compounds are antici-
for FSNP-1 and FSNP-3. Since FSNP-2 and FSNP-4 exhibit pated to show high surface charges. This is indeed the case.
much smaller particle sizes, this results in higher dye density As shown in Figure 8, FSNP-1 and FSNP-3 exhibit reason-
and stronger aggregation and thus a red shift in the fluorescence ably high zeta potentials even at pH 3. With an increase in
spectra.The photographs shown in Figure 7 further demonstrate the pH value or the solution basicity, their potentials become
the different emission behaviors between FSNP-1/FSNP-3 and higher or more negative because the dissociation of the sur-
FSNP-2/FSNP-4 pairs. This visual observation also supports the face silanol groups is favorable in such media.
notion that the intramolecular rotations of 5 and 6 are restricted
by their covalent incorporation into the silica matrix.
2.6. Cell Imaging
2.5. Colloidal Stability
One of the important areas in which FSNPs have dem-
onstrated great potential is in cancer cell imaging. The AIE
Colloidal stability is an important parameter for FSNPs luminogens are benign to the growth of living cells.[33] They
and can be reflected by their surface charges or zeta are also nontoxic to HeLa cells and interfere little with the
Figure 6. Photographs of ethanol solutions of A) 9 and FSNP-1 and B) 10
and FSNP-3 taken under the illumination of a UV lamp at 365 nm.
Figure 7. Photographs of ethanol solutions of A) FSNP-1 and FSNP-2 and B)
FSNP-3 and FSNP-4 taken under the illumination of a UV lamp at 365 nm.
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Immobilization of Luminogens in Silica Nanoparticles Through Click Reaction
0
FSNP-1
FSNP-3
-20
-40
-60
-80
Figure 10. A,C) Bright-field and B,D) fluorescence images of HeLa cells
labeled with A,B) FSNP-3 and C,D) FSNP-4.
2
5
8
11
pH
Figure 8. Zeta potentials of FSNP-1 and FSNP-3 at different pH values.
intramolecular rotations are further restricted if some of
them are located on the surface. Although the silica shells are
hydrophilic, no fluorescence is observed in the cell nucleus,
probably due to the “large” particle sizes of the FSNPs.
cytoplasmic activities of the cells. To examine the cell staining
ability of our FSNPs, we cultured HeLa cells in the presence
of these nanoparticles. After 6 h of incubation, the FSNPs
are endocytosed through the cell membrane and efficiently
anchor on the cytoplasmic organelles. To compare the uptake
efficiency of FSNPs with different sizes, we stained the cells
with FSNP-1 and FSNP-2. As depicted in Figure 9, both
3. Conclusion
TPE- and silole-functionalized siloxanes have been fac-
FSNPs work as good fluorescent visualizers for intracellular ilely prepared by Cu(I)-catalyzed alkyne–azide 1,3-dipolar
imaging. On the contrary, the images of HeLa cells stained cycloaddition, and their one-pot, two-step sol–gel reaction
by FSNP-3 and FSNP-4 show different brightness, albeit to a with TEOS furnished FSNPs with uniform sizes and smooth
small extent (Figure 10) .
surfaces. The sizes of the FSNPs are tunable by varying the
During the endocytosis, the FSNPs are enclosed by the cell fabrication method. Upon photoexcitation, the FSNPs emit
membrane to form small vesicles, which are then internalized strong blue and green light in high fluorescence quantum
in the cytoplasmic compartment of the cell. The FSNPs are yields. They possess high surface charges and hence exhibit
further processed in the endosomes and lysosomes containing excellent colloidal stability. The FSNPs are benign to living
numerous digestive enzymes and are eventually released to cells and function as fluorescent visualizers for intracellular
the cytoplasm.[34–36] When bound to the biomacromolecules, imaging of HeLa cells. Such attributes make the nanoparti-
the FSNPs may emit even more intensely because their cles promising for an array of biological applications. Because
of the rich acetylene chemistry, we are currently working on
the incorporation of AIE luminogens with different emission
colors into the silica network using the same strategy. Thanks
to the high functionality tolerance of the click reaction, the
surface of the FSNPs can be conjugated with biomolecules
for specific cancer cell labeling. Details will be published in
due course.
4. Experimental Section
Materials: Tetraethoxysilane (TEOS), 3-chloropropyltriethoxysilane
(7), DMF, THF, and other reagents were purchased from Aldrich and
used as received. Diynes 9 and 10 were prepared according to our
previous publications.[31,32]
1
Instrumentation: H and 13C NMR spectra were recorded on a
Bruker ARX 400 spectrometer with tetramethylsilane (TMS; δ = 0)
as internal standard. HR mass spectra were recorded on a Finnigan
TSQ 7000 triple quadrupole spectrometer operating in the MALDI–
TOF mode. The morphologies of the FSNPs were investigated using
Figure 9. A,C) Bright-field and B,D) fluorescence images of HeLa cells
labeled with A,B) FSNP-1 and C,D) FSNP-2.
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JOEL 2010 TEM and JOEL 6700F SEM instruments at an accelerating are given below. In a typical synthesis, micelles were prepared at
voltage of 200 and 5 kV. Their sizes were measured using TEM soft- room temperature by sonication of a homogeneous mixture of
ware (Digital micrograph 365 Demo), which gave values with two cyclohexane (30 mL), Triton X-100 (7.2 mL), n-heptanol (5.6 mL),
digits after the decimal point. Samples were prepared by drop- and water (600 μL) for 30 min. Ammonia solution (800 μL, 28%) was
casting dilute dispersions of the FSNPs onto copper 400-mesh car- then added. After magnetically stirring for 15 min, 5 (100 μL) was
rier grids covered with carbon-coated Formvar films. The solvent was injected. The solution was stirred for another 15 min. After dropwise
evaporated at room temperature in open air. Fluorescence spectra addition of TEOS (400 μL), the reaction mixture was stirred for 24 h
were recorded on a Perkin–Elmer LS 50B spectrofluorometer with at room temperature. The microemulsion was terminated by adding
xenon discharge lamp excitation. Zeta potentials and particle sizes ethanol and the nanoparticles were isolated by centrifugation and
of the FSNPs were determined at room temperature by a Zeta Plus washed with ethanol and water to remove the surfactant. The nano-
Potential Analyzer (Brookhaven Instruments Corporation, USA). particles were then dried in a vacuum at room temperature.
In these experiments, the silica nanoparticles were dispersed in
Cell Culture: HeLa cells were cultured in minimum essen-
ultrapure water (18 MΩ cm). Therefore, the ionic strength was con- tial medium containing 10% fetal bovine serum and antibiotics
stant for each experiment. The pH of the suspension was controlled (100 units mL−1 penicillin and 100 μg mL−1 streptomycin) in a 5%
by adding 0.1 M ammonium hydroxide and 0.1 M hydrochloric acid.
CO2 humidity incubator at 37 °C.
Synthesis of 3-Azidopropyltriethoxysilane (8) : 3-Chloropropyl-
Cell Imaging: HeLa cells were grown overnight on a plasma-
triethoxysilane (5.0 mL, 20.85 mmol), sodium azide (5 g, 77 mmol), treated 25-mm round cover slip mounted onto a 35-mm petri dish
and dry DMF (50 mL) were added to a 100-mL two-necked round- with an observation window. The living cells were stained with
bottomed flask. The solution was heated to 90 °C under a nitrogen FSNPs (200 μL) and incubated for 6 h. The cells were imaged under
atmosphere for 5 h. The low-boiling compounds were removed by an inverted fluorescence microscope (Nikon Eclipse TE2000-U);
distillation under reduced pressure (ca. 10 mm Hg), after which excitation = 330−380 nm, dichroic mirror = 400 nm. The images of
diethyl ether (100 mL) was added to the mixture. The precipitates the cells were captured using a digital CCD camera.
were removed by filtration and the solvent was removed under
vacuum. Distillation of the residual oil under reduced pressure
(2 mm Hg) at 96 °C gave the desired product as a colorless liquid
1
(3.3 g, 68%). H NMR (400 MHz, CDCl3), δ = 3.81 (q, 6H), 3.24 (t,
2H), 1.66−1.70 (m, 2H), 1.21 (t, 9H), 0.66 ppm (t, 2H); 13C NMR
Supporting Information
(100 MHz, CDCl3), δ = 58.4, 53.8, 22.6, 18.2, 7.5 ppm; IR: 2977,
2927, 2883, 2734, 2098, 1284, 1165, 1084, 960, 779 cm−1 .
Supporting Information is available from the Wiley Online Library
Click Reaction: Cycloaddition reactions of 8 with 9 and 10 were
carried under nitrogen using Schlenk tubes. Typical experimental
or from the author.
procedures for the synthesis of 5 and 6 are given below.
Compound 8 (20.0 mg, 0.081 mmol), 9 (15.4 mg, 0.0405 mmol),
and Cu(PPh3)3Br (4.5 mg, 6 mol%) were placed in a 15 mL Schlenk
Acknowledgements
tube. THF (2 mL) was then injected into the solution. After stirring
at 60 °C for 24 h, the reaction mixture was diluted with THF (3 mL)
and centrifuged at 3000 rpm for 15 min. During the reaction, water
was carefully excluded to avoid the possible hydrolysis of 8. The
supernatant was decanted and concentrated. The adduct (5) was
characterized by HR mass spectrometry.
We thank the Research Grants Council (603509, 601608, CUHK2/
CRF/08, and HKUST2/CRF/10), the Innovation and Technology
Commission (ITP/008/09NP and ITS/168/09), the University Grants
Committee of Hong Kong (AoE/P-03/08), and the National Science
Foundation of China (20974028).
This Full Paper is part of the Special Issue on Nanotechnology
Adduct 6 was synthesized by similar procedures using 8
with Soft Matter.
(20.0 mg, 0.081 mmol), 10 (18.74 mg, 0.0405 mmol), and
Cu(PPh3)3Br (4.5 mg, 6 mol%) in THF (2 mL).
Preparation of FSNP-1 and FSNP-3 by the Stöber Method: FSNP-
1 was prepared from 5 and TEOS by a two-step sol–gel reaction.
Compound 5 (≈15 μmol) was added to a mixture of ethanol (32 mL),
ammonium hydroxide (0.64 mL), and distilled water (3.9 mL). The
solution was stirred at room temperature for 30 min, after which an
ethanol solution (5 mL) of TEOS (1 mL) was added dropwise. The
solution was stirred at room temperature for an additional 24 h to
coat the luminogenic nanocores with silica shells.[37] After incuba-
tion, the mixture was centrifuged and the nanoparticles of FSNP-
1 were redispersed in ethanol under sonication for 5 min. The
process was repeated three times and the FSNPs were dispersed in
water or ethanol for further experiments. Similarly, sol–gel reaction
of 6 with TEOS following the same procedure furnished FSNP-3.
Preparation of FSNP-2 and FSNP-4 by the Microemulsion
Method: FSNP-2 and FSNP-4 were prepared according to the lit-
erature method.[38] Typical procedures for the fabrication of FSNP-2
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Received: December 6, 2010
Revised: January 17, 2011
Published online: April 26, 2011
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