Silole nanocrystals as novel biolabels

www.elsevier.com/locate/jim

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: [email protected] (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


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.


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).


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


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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Silole nanocrystals as novel biolabels

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