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Journal of Immunological Met
Research paper
Silole nanocrystals as novel biolabels
Cangel Pui-yee Chana, Matthias Haeusslerb, Ben Zhong Tangb, Yongqiang Dongb,
King-keung Sina, Wing-cheung Makc, Dieter Traud,
Matthias Seydacke, Reinhard Renneberga,*
aDepartment of Chemistry, Biosensors and Bioelectronics Laboratory, The Hong Kong University of Science and Technology,
Clear Water Bay, Kowloon, Hong Kong, ChinabDepartment of Chemistry, Institute of Nano Materials and Technology, The Hong Kong University of Science and Technology,
Clear Water Bay, Kowloon, Hong Kong, ChinacBioengineering Graduate Program, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China
dDivision of Bioengineering and Department of Chemical and Biomolecular Engineering, National University of Singapore, Singaporee8sens.biognostic AG, Robert-Roessle-Strasse 10, D-13125 Berlin, Germany
Received 11 August 2004; accepted 22 September 2004
Available online 4 November 2004
Abstract
A novel class of biofunctional silole nanocrystals with the potential to create highly sensitive immunoassay was firstly
demonstrated. Biolabels were constructed by encapsulating nanocrystalline hexaphenylsilole [Ph2Si(CPh)4; HPS] within
ultrathin polyelectrolyte layers via the layer-by-layer (LbL) technique that provided an binterfaceQ for the attachment of
antibodies. A high ratio of fluorescent dyes to biomolecules (F/P ratio; 2.4�103) was achieved without self-quenching problem.
The aggregation-induced emission (AIE) feature offered silole biolabels the sensitivity 40- to 140-fold higher than that of a
start-of-the-art immunoassay using directly fluorescent-labeled antibodies.
D 2004 Elsevier B.V. All rights reserved.
Keywords: Aggregation-induced emission (AIE); Biolabel; Fluorescence immunoassay (FIA); Layer-by-layer (LbL) technique; Silole
0022-1759/$ - s
doi:10.1016/j.jim
Abbreviation
diacetate; FIA, f
cellulose; LbL, l
saline; PSS, pol
sodium dodecyl
* Correspon
E-mail addr
hods 295 (2004) 111–118
ee front matter D 2004 Elsevier B.V. All rights reserved.
.2004.09.016
s: AIE, aggregation-induced emission; BSA, bovine serum albumin; FITC, fluorescein isothiocyanate; FDA, fluorescein
luorescence immunoassay; Gt a M IgG, goat antimouse IgG; [Ph2Si(CPh)4; HPS], hexaphenylsilole; HPC-SL, hydroxypropyl
ayer-by-layer; LED, light-emitting diode; M IgG, mouse IgG; PAH, poly(allylamine hydrochloride); PBS, phosphate-buffered
y(sodium 4-styrenesulfonate); F/P ratio, ratio of fluorescent dyes to biomolecules; SEM, scanning electron microscope; SDS,
sulfate; THF, tetrahydrofuran.
ding author. Tel.: +852 2358 7387; fax: +852 2705 9670.
ess: chrenneb@ust.hk (R. Renneberg).
C. Pui-yee Chan et al. / Journal of Immunological Methods 295 (2004) 111–118112
1. Introduction
Ratio of fluorescent dyes to biomolecules (F/P
ratio) is a key parameter that has an impact on
sensitivity and is widely used for the evaluation of a
fluorescent label in biochemical assay technologies—
high number of labeling molecules per biomolecule is
desired. Conventional fluorescence immunoassays
(FIA) often encounter problems associated with self-
quenching effects. If more than 10–15 fluorophores
are attached to one antibody, their distance is close to
or within their Foerster radiuses, resulting in signifi-
cant losses in emission intensities or decreases in
fluorescence quantum yields due to involved energy
transfer. Aggregation of fluorophores often quenches
light emission, which has been a thorny problem in
the development of ultrasensitive FIA. In our previous
studies (Chan et al., 2004; Trau et al., 2002), we
reported on the preparation and utilization of a new
class of particulate labels based on nanoencapsulated
fluorogenic precursor fluorescein diacetate (FDA)
with the potential to create highly amplified bio-
chemical assays. A high molar ratio of fluorescent
molecules present in the crystal core to biomolecules
on the particle surface was achieved. Following the
immunoreaction, the FDA core was dissolved by
exposure to organic solvent/sodium hydroxide mix-
ture, leading to the release of the fluorophores
Fig. 1. Schematic illustration of the preparation of biofunctional sil
experimental procedure (Tang et al., 2001) (a) and was ball-milled into
figure) mixed with SDS (b), followed by encapsulation with polyelectrol
specific immunoreagent (d).
(fluoresceins) into the surrounding medium and thus
suppressing self-quenching effect. Our approach
provides high sensitivity and low limits of detection
without the need for long incubation times, making it
an interesting alternative in biolabel technology.
This is a pilot study to investigate the feasibility of
utilizing siloles as fluorophores in immunoassays.
Siloles are of considerable current interest because of
their unusual electronic and optical properties, and
because of their possible application as electron-
transporting materials in light-emitting devices (Chen
et al., 2003a,b,c; Luo et al., 2001; Tang et al., 2001;
Yamaguchi and Tamao, 1998; Yamaguchi et al.,
2000). Siloles exhibit high electron acceptability
(Yamaguchi and Tamao, 1998; Sadimenko, 2001;
Lee et al., 2000) and fast electron mobility (Murata et
al., 2001), which make them efficient luminophores
for a variety of optoelectronics applications. These
properties arise from the unique low-lying LUMO
level associated with the r*–p* conjugation arising
from the interaction between the r* orbital of two
exocyclic r-bonds on the silicon atom and the p*orbital of the butadiene moiety (Chen et al., 2003a;
Tang et al., 2001). The siloles are a group of novel
molecules that are highly photoluminescent in their
aggregation state. This unique emission feature makes
them promising candidate materials for light-emitting
diode (LED) applications (Chen et al., 2003a; Tang et
ole nanocrystals. HPS was synthesized according to published
nanocrystals in an aqueous surfactant, HPC-SL (not shown in the
yte multilayers of nanometer thickness (c), and the attachment of a
Fig. 2. Principle of a sandwich immunoassay using nanocrystalline silole biolabels. The analyte is first immobilized by the capture antibody
preadsorbed on the solid phase (a) and then exposed to antibody-labeled nanocrystal detectors (b). Fluorescence intensity is proportional to the
analyte concentration.
C. Pui-yee Chan et al. / Journal of Immunological Methods 295 (2004) 111–118 113
al., 2001; Yamaguchi et al., 2000; Murata et al., 2001,
2002; Ohshita et al., 2001; Hay et al., 2001) and as
chemical sensors (Sohn et al., 2001, 2003).
The silole biolabels were constructed by simply
encapsulating hexaphenylsilole [Ph2Si(CPh)4; HPS]
nanocrystals of 99 nm in average size within ultrathin
polyelectrolyte layers of poly(allylamine hydrochlor-
ide) (PAH) and poly(sodium 4-styrenesulfonate)
(PSS) via layer-by-layer (LbL) techniques (Trau et
al., 2002). The polyelectrolyte coating was subse-
quently used as an interface for the attachment of
antibodies through adsorption (Fig. 1). As the entire
nanocrystal core is composed of silole molecules and
the biomolecule forms a layer on the encapsulated
crystal surface, the potentially reachable F/P ratio is
exceptionally high. The general concept of the
application of the nanocrystalline silole biolabels as
fluorescent labels in FIAs is depicted in Fig. 2. The
siloles are nonemissive (boffQ) when molecularly
dissolved in organic solvents at room temperature,
while the silole molecules in poor solvents cluster into
nanoaggregates, which turn the emission bonQ and
boost the photoluminescence quantum yields by up to
two orders of magnitude (Chen et al., 2003a). This
intriguing aggregation-induced emission (AIE) feature
is an invaluable property for the development of
ultrasensitive FIA.
2. Materials and methods
2.1. Synthesis of HPS
This compound was prepared as shown in Fig. 1
according to published experimental procedure (Tang
et al., 2001). Briefly, clean lithium shavings (350 mg,
50 mmol) were added to a solution of diphenylace-
tylene (4.5 g, 25 mmol) in dry tetrahydrofuran (THF;
20 mL). The reaction mixture was stirred at room
temperature for 2 h in a dry nitrogen atmosphere. The
mixture was then diluted with 120 mL of THF,
followed by the addition of 3.2 g (12.5 mmol)
dichloro(diphenyl)silane. After refluxing for 5 h, the
reaction mixture was cooled and filtered, and the
filtrate was washed with water. The organic layer was
extracted with diethyl ether and dried over magnesium
sulfate. The solvent was removed, and the residue was
purified by flash chromatography over silica gel using
10% diethyl ether in petroleum ether as the eluent.
Recrystallization from ethanol gave a faintly greenish-
yellow crystal in 68% yield (4.6 g).
2.2. Preparation of nanocrystalline HPS biolabels
Hexaphenylsilole nanocrystal suspension was
prepared by ball milling using small glass beads
(0.25–0.5 mm, Roth, Karlsruhe, Germany). A
suspension of 20 mg of HPS in 2 mL of 1.0%
(w/v) hydroxypropyl cellulose (HPC-SL, JE-1071;
Nippon Soda, Japan) and 0.05% (w/v) sodium
dodecyl sulfate (SDS) was mixed in a glass tube
with 2 g of glass beads. The mixture was vortexed
for 15 min at room temperature and then exposed to
ultrasonic for 15 min. The milling process was
repeated until desired particle size distribution was
obtained. The colloidal suspension was centrifuged
at 6000�g (8000 rpm) for 10 min, and the collected
supernatant was centrifuged at 16000�g (13,100
rpm) for 15 min. The pellet was then resuspended in
the surfactant solution. The morphology of the
C. Pui-yee Chan et al. / Journal of Immunological Methods 295 (2004) 111–118114
milled HPS nanocrystals was examined with a JEOL
6300F ultrahigh-resolution scanning electron micro-
scope (SEM), operating at 10 kV. Particle size
distribution was measured based on the Fraunhofer
and Mie theories of light scattering using CoulterRLS230 (Beckman Coulter, USA) by polarization
intensity differential scattering technology.
Polyelectrolyte multilayers were assembled onto
the nanocrystals by the sequential deposition of
poly(allylamine hydrochloride) (PAH-polyelectrolyte;
Mw 15,000) and poly(sodium 4-styrenesulfonate)
(PSS-polyelectrolyte; Mw 70,000) as described by
Trau et al. (2002); a small amount (0.1 mL) of the
HPS nanocrystal suspension (1.0%, w/v) was added to
0.5 mL of PAH–polyelectrolyte solution (5 mg/mL,
containing 0.5 M NaCl). The suspension was mixed at
constant intervals for 15 min at room temperature. The
excess polyelectrolyte was removed by three repeated
centrifugation/washing with distilled water and redis-
persion cycles. For subsequent assembly of negatively
charged PSS-polyelectrolyte, 0.5 mL of PSS–poly-
electrolyte solution (5 mg/mL, containing 0.5 M
NaCl) was added. The centrifugation/washing and
polyelectrolyte incubation steps were repeated until
the desired number of layers (typically four) was
assembled. The surface charges of the milled and
encapsulated HPS nanocrystals were examined by
microelectrophoresis using a ZetaPlus potential ana-
lyzer (Brookhaven Instrument, Holtsville, NY) by
taking the average of five measurements at the
stationary level.
The polyelectrolyte-coated HPS nanocrystals with
an outermost layer of PSS-polyelectrolyte were con-
jugated to antibodies as described in our previous
study (Chan et al., 2004). The particle suspension
(0.0626%, w/v) was incubated with 200 Ag/mL of
polyclonal goat antimouse IgG (Gt a M IgG, whole
molecule; Arista Biologicals, USA) in 10 mM
phosphate-buffered saline (PBS, pH 7.4) at 20 8C for
1 h. After centrifugation at 16,000�g (13,100 rpm) for
10 min, the supernatant was removed, and its UV
absorption was measured at 280 nm (Cary 50 Conc
UV–Visible Spectrophotometer, Australia). The anti-
body surface coverage of nanoparticles was deter-
mined by the difference in absorption at 280 nm
between supernatant and the original protein solution.
The IgG-coated particles were then separated from
soluble IgG by three centrifugation/washing cycles.
2.3. Solid-phase sandwich fluorescence immunoassay
Two microgram per milliliter of Gt a M IgG (100
AL/well) was coated on Nunc Maxisorp 96-well
microplates (Nunc International, Rochester, NY) in
0.1 mol/L carbonate buffer (pH 9.6) at 4 8C overnight.
After rinsing three times with washing buffer [10 mM
PBS, 0.1% (w/v) bovine serum albumin (BSA,
fraction V), 0.5% (w/v) Tween-20], the wells were
blocked with 300 AL/well of 1.0% BSA solution for
half an hour at 37 8C. The plate was then washed four
times and incubated with dilutions (100 AL/well) ofmouse IgG (M IgG; Arista Biologicals) as an analyte
at 37 8C for 1 h. After washing five times, antimouse-
coated nanocrystal suspensions (0.0125%, w/v) was
dispensed into the wells (100 AL/well), and the
microplate was incubated again at 37 8C for 1 h.
Soluble fluorescein isothiocyanate (FITC)-labeled Gt
a M IgG dilutions (100 AL/well) of 1:128 (Arista,
protein concentration 1.1 mg/mL, F/P ratio 4.4) was
used for comparison. After incubation, excess detector
antibody conjugates were washed off by five washing
cycles with buffer. The fluorescence intensity was
measured using a FLUOstarOPTIMA multifunctional
microplate reader (BMG Labtechnologies, Germany)
with excitation/emission wavelengths of 380/500 and
485/520 nm for measurements of the nanocrystalline
HPS-labeled and the FITC-labeled antibodies (gain
setting of 1600).
3. Results and discussion
This is a novel study to apply the aggregation-
induced emission principle in immunoassay. Hex-
aphenylsilole was chosen as the fluorophore in this
study due to the impressive performance of its LED
device (Chen et al., 2003a). It was readily turned on
at a low voltage (~4 V), emitted intensely at a
moderate bias (55 880 cd/m2 at 16 V), and showed
very high emission efficiencies (15 cd/A current
efficiency, 10 1m/W power efficiency, and 7%
external quantum efficiency). HPS was synthesized
according to published procedures by ring-closing
reaction of 1,4-dilithio-2,3,4,5-tetraphenyl-1,3-buta-
diene with dichlorodiphenylsilane (Tang et al.,
2001). DMSO solution of HPS (22 AM) was
virtually nonemissive. Only a noisy curve was
Fig. 3. Photoluminescence spectra of 22 AM of HPS in DMSO
(red) and water (blue); excitation wavelength: 380 nm.
C. Pui-yee Chan et al. / Journal of Immunological Methods 295 (2004) 111–118 115
obtained even in a 100 times magnified photo-
luminescent spectrum (Fig. 3). However, when the
same concentration of HPS in a poor solvent (e.g.,
water), an intense signal was recorded under
identical measurement conditions. This observation
fits well to the data obtained from the previous
investigation and confirms that HPS is AIE-active
(Chen et al., 2003a).
Fig. 4. Particle size distribution and SEM m
According to the previous study (Chen et al.,
2003a), the AIE phenomenon is caused by the
restricted intramolecular rotation. Rotational energy
relaxation can nonradiatively deactivate excited spe-
cies (Wise et al., 1998; Wong et al., 1998). In the
solutions at room temperature, the active intramolec-
ular rotations of the peripheral phenyl rings around the
axes of the single bonds linked to the central silole
core may effectively annihilate the excitons, thus
making the silole molecules nonemissive. In the solid
aggregates, the stacking forces involved in the crystal
packing may restrict the intramolecular rotations,
which may block the nonradiative channel and
populate the radiative decay, thus making the silole
molecules luminescent.
The particle size distribution of the HPS nano-
crystals determined by light-scattering measurements
is shown in Fig. 4. A narrow disperse system was
obtained with an average size of 99 nm. Approx-
imately, 90% of the nanocrystals were found to be
smaller than 185 nm, and 100% (or all of them) smaller
than 500 nm. This result is in agreement with SEM
analysis of the nanocrystals as shown in Fig. 4. SEM
shows that the nanocrystals take different shapes.
During the milling process, the hydrophobic sur-
face of the stabilizer is most likely associated with the
hydrophobic surface of the nanocrystal, and the
hydrophilic portion of SDS is oriented towards the
aqueous phase of the suspension (Caruso et al., 2000).
The adsorbed HPC-SL layer makes the HPS nano-
icrograph of milled HPS nanocrystals.
Fig. 6. Sandwich fluorescence immunoassay of M IgG using Gt a
M IgG-HPS nanocrystals ( ) and Gt a M IgG-FITC ( ) as labels.
The reported fluorescence intensities were obtained after the
subtraction of the blank signal. Error bars correspond to standard
deviations (FS.D., n=3).
C. Pui-yee Chan et al. / Journal of Immunological Methods 295 (2004) 111–118116
crystals dispersible in water and prevents their
aggregation and crystal growth following extended
storage periods, hence conferring colloidal stability
(Liversidge et al., 1999). The adsorbed, negatively
charged SDS layer introduces surface charges to the
nanocrystals and also increases colloidal stability.
This was verified by microelectrophoresis measure-
ments. The nanocrystals exhibited a f-potential of
�44.5 mV, indicating a negatively charged, highly
stable particle suspension (Fig. 5). The subsequent
alternate adsorptions of PAH- and PSS-polyelectrolyte
layers onto the SDS-coated HPS nanocrystals yielded
f-potentials of ca. +30 and �40 mV, respectively. The
thickness of the polyelectrolyte coating, four layers
total of PAH- and PSS-polyelectrolytes, assembled
onto the amphiphile-modified HPS nanocrystals is
only about 6–8 nm (Caruso et al., 1999; Caruso and
Mohwald, 1999). This thickness is negligible when
compared to the total size of the HPS nanocrystals.
The polyelectrolyte coatings did not cause any
noticeable change in the morphology and size
distribution of the HPS nanocrystals (data not shown).
The polyelectrolyte coating on the HPS nano-
crystals is to impart sufficient colloidal stability and
to provide a suitable interface for the attachment of
biomolecules to the nanocrystals. Gt a M IgG
adsorption onto polyelectrolyte multilayers assembled
onto polystyrene microparticles has been confirmed by
monitoring the change in f-potentials as a function of
particle suspension pH (Yang et al., 2001) and
quantified by single particle light-scattering experi-
ments (Caruso and Mohwald, 1999). In this study, the
amount of Gt a M IgG adsorbed onto the HPS
nanocrystals was determined spectrophotometrically.
Fig. 5. Microelectrophoresis measurements of the HPS nanocrystals
by taking the average of five measurements at the stationary level.
The protein surface coverage was calculated by
assuming an average size of 99 nm for the nanocrystals.
Incubation of the nanocrystals with Gt a M IgG (200
Ag/mL) in 10 mM PBS buffer for 1 h at 20 8C resulted
in the adsorption of 36.7% of the added protein,
correlating to a surface coverage of 2.37 mg/m2. The
theoretically calculated surface coverage value for a
close-packed IgG monolayer is in the range of 2.0–5.5
mg/m2, depending on the different orientations of the
adsorbed IgG molecules (Davalos-Pantoja et al., 2001;
Caruso et al., 1998). To calculate the F/P ratio for the
nanocrystal biolabel, a cubic crystal morphology with
dimensions of 99�99�99 nm was assumed. The
calculated F/P value for the biolabel is 2.4�103, which
is much higher than the ratios of directly covalent
labeled antibodies (carrying four to eight fluorophores
in general). The F/P ratio of an immune detection
system reflects its potential amplification rate.
Fig. 6 shows calibration curves of the sandwich
fluorescence immunoassay performed with Gt a M
IgG adsorbed nanocrystal labels in comparison with a
C. Pui-yee Chan et al. / Journal of Immunological Methods 295 (2004) 111–118 117
direct FITC-labeled antibody conjugate. The fluores-
cence signal is directly proportional to the M IgG
concentration in the range of 0–100 Ag/L. A 40- to
140-fold higher sensitivity with the assays using the
nanocrystal biolabels was observed compared with the
direct FITC-labeled antibodies, depending on the
analyte concentration. The higher sensitivity of the
nanocrystal biolabels compared with standard FITC
conjugates may be explained by the boosting effect of
the higher ratio of dye molecules to binding mole-
cules, and the suppressed self-quenching by the AIE
feature could also have contributed to the improved
signal. This is the first study to show the successful
application of siloles as fluorophores in immunoassay.
In conclusion, the applicability of a new class of
biolabels for immunoassays was demonstrated.
Chemo- and photostabilities make siloles a useful tool
for labeling purposes. The preparation of nanocrystal-
line HPS biolabel is straightforward to perform and is
controllable. By using a nanoscale polyelectrolyte
coating as an interface for bioconjugation onto the
nanocrystals, the siloles do not need to be water-
soluble or possess groups for bioconjugation, as this
functionality is provided by the polyelectrolytes. The
quenching problem normally arising from F/P ratio
labels can be prevented due to the AIE feature of
siloles. Synthesis of water-soluble amphiphilic siloles
through appropriate chemical modifications is now in
progress. The soluble siloles may be used in homoge-
nous assays, i.e., all reagents present in solution to
achieve the measurement without any separation step.
We believe the use of siloles provides us with a useful
alternative to organic fluorophores after optimization.
Acknowledgements
The work described in this paper was partially
supported by the Hong Kong Research Grant Council
(HKUST6086/02M and HKUST6085/02P) and the
University Grants Committee under an Area of
Excellence (AoE) scheme (AoE/P-10/01-1).
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