Ultrabright benzoselenadiazole-based semiconducting polymer dots for specific cellular imaging

Article abstract of DOI:10.1039/c3ra42565g

Semiconducting polymer dots (Pdots) have recently emerged as a new class of extraordinarily bright fluorescent probes with promising applications in biological imaging and sensing. Herein we synthesized a novel series of highly emissive orange-fluorescent copolymers, poly[9,9-dioctylfluorenyl-2,7-diyl)-co- 1,4-benzo-{2,1′-3}-selenadiazole)] (PFBS) and tuned the ratio of fluorene to benzoselenadiazole (BS) from 98:2 to 50:50 to investigate the influence of BS molar ratio on the emission properties of the resulting Pdots. An optimal quantum yield of 44% could be obtained for PFBS Pdots at a fluorene to BS ratio of 70:30. These PFBS Pdots also exhibited great photostability and superior single-particle brightness. We next conjugated biomolecules onto the surface of these PFBS Pdots and demonstrated their ability for specific cellular labeling without any noticeable nonspecific binding. We are now working on the synthesis of near-infrared Pdots based on this BS unit and anticipate this series of ultrabright Pdots will be very useful in a variety of in vitro and in vivo bioimaging applications.

Full text of DOI:10.1039/c3ra42565g

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Ultrabright benzoselenadiazole-based semiconducting
polymer dots for specific cellular imaging
Cite this: RSC Advances, 2013, 3,
17507
Chuan-Pin Chen,3 Pei-Jing Wu,3 Sz-Yu Liou and Yang-Hsiang Chan*
Semiconducting polymer dots (Pdots) have recently emerged as a new class of extraordinarily bright
fluorescent probes with promising applications in biological imaging and sensing. Herein we synthesized a
novel series of highly emissive orange-fluorescent copolymers, poly[9,9-dioctylfluorenyl-2,7-diyl)-co-1,4-
benzo-{2,19-3}-selenadiazole)] (PFBS) and tuned the ratio of fluorene to benzoselenadiazole (BS) from
98 : 2 to 50 : 50 to investigate the influence of BS molar ratio on the emission properties of the resulting
Pdots. An optimal quantum yield of 44% could be obtained for PFBS Pdots at a fluorene to BS ratio of
70 : 30. These PFBS Pdots also exhibited great photostability and superior single-particle brightness. We
next conjugated biomolecules onto the surface of these PFBS Pdots and demonstrated their ability for
specific cellular labeling without any noticeable nonspecific binding. We are now working on the synthesis
of near-infrared Pdots based on this BS unit and anticipate this series of ultrabright Pdots will be very
useful in a variety of in vitro and in vivo bioimaging applications.
Received 24th May 2013,
Accepted 15th July 2013
DOI: 10.1039/c3ra42565g
www.rsc.org/advances
(quantum dots), on the other hand, have shown improved
brightness and photostability over conventional organic
Introduction
dyes.6–10 However, the potential release of the inorganic
component (e.g., cadmium and selenium),10,11 the stochastic
blinking phenomenon,12 and the difficulties in tailoring the
surfaces of quantum dots (QDs)9,13 have impeded their use for
in vivo clinical applications.14–16 Therefore, the exploration of
nanoparticle-based probes that can offer high brightness,
good photostability, and biocompatibility is immensely
needed.
Fluorescence imaging techniques have been widely used to
investigate biological processes in various fields, such as
measuring single-cell activity, gene regulation, DNA repair
mechanisms, and diagnosis of disease.1–3 In an effort to
achieve optimal analysis of biomolecular interactions either in
vitro or in vivo at high temporal/spatial resolution,
a
fluorescence probe should possess several properties includ-
ing high brightness, good photostability, low cytotoxicity,
small size (usually sub-30 nm), and facile bioconjugation.
Although small organic dyes are conventionally used for
general biological studies, their intrinsic photoproperties such
as insufficient brightness and low photostability have limited
their applications in high-sensitivity imaging, long-term
imaging, and high-throughput assays. Therefore, there have
been great efforts to exploit brighter and more photostable
fluorescent probes.
Nanoparticle-based fluorescent probes were thus developed
in which numerous dye molecules were encapsulated inside
nanoparticle matrices that usually consisted of latex, polymer,
or silica. These nanoparticle-based assemblies exhibit higher
brightness than single fluorescent dye but the leaching of the
embedded dyes from the matrices has been a concern.4,5
Besides, the use of an optically inert matrix often limits the
potential brightness and optical functionality of the final
nanoparticle probe. Inorganic semiconductor nanocrystals
Recently, there has been rapid progress in the development
of semiconducting polymer dots (Pdots) due to their excep-
tional fluorescence brightness, fast radiative rate, good
photostability, facile surface functionalization, and low cyto-
toxicity.17–23 Moreover, the good biocompatibility and facile
surface functionalization of Pdots have rendered them
extremely useful for a number of applications both in vitro
and in vivo.24–36 Despite this progress, the currently brightest
Pdot developed by Wu et al. is based on poly[9,9-dioctylfluor-
enyl-2,7-diyl)-co-1,4-benzo-{2,19-3}-thiadiazole)] (PFBT), in
which the absorption maximum locates at y450 nm with its
emission maximum at y540 nm. To make Pdots more
applicable to a wide range of biological applications, it is
necessary to develop Pdots with redder or even near-infrared
(NIR) fluorescence. However, the attempt to synthesize Pdots
with longer absorption/emission wavelengths leads to low
quantum yields, partly due to the self-quenching of polymers
upon condensation into a Pdot form.30,37,38 Recently, Chiu’s
group circumvented this problem by blending 1.5% of the
deep-red emitting polymer (PFDBT) into PFBT matrix.26 In this
way PFBT served as a donor to harvest light and then the
Department of Chemistry, National Sun Yat-sen University, 70 Lien Hai Road,
Kaohsiung 80424, Taiwan. E-mail: yhchan@mail.nsysu.edu.tw
Both these authors contributed equally to this work.
3
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groups of Pdots so that they could be tagged with biological
molecules for specific cellular targeting applications.
Design and synthesis of BS-containing copolymer
Scheme 1 illustrates the general synthetic route for the
synthesis of a PFBS polymer. First, a BS monomer (2) was
prepared by cyclization of o-phenylenediamine with SeO2 to
obtain compound 1, following the bromination in concen-
trated sulfuric acid. PFBS conjugated polymer was then
synthesized by copolymerizing BS monomer with fluorene
derivatives (3, 4) via palladium-catalyzed Suzuki coupling
reaction. We also varied the molar ratio of BS monomers (2–
50%) to investigate the effects of various copolymer composi-
tions on the optical properties of the resulting Pdots. The
comonomer molar ratios of fluorene to BS we fed here were
98 : 2, 90 : 10, 70 : 30, 60 : 40, and 50 : 50, respectively; and
the corresponding copolymers were denominated as PFBS2,
PFBS10, PFBS30, PFBS40, and PFBS50, respectively. It should
be noted that the denominations here are directly from the
feed ratios and may not be the true composition of the
polymers. The true incorporation of BS is difficult to be
determined by NMR due to the significant spectral overlap of
Scheme 1 The synthetic procedures for compounds 1–4 and PFBS copolymers.
energy was transferred to PFDBT acceptor. Although the
emission maximum of these Pdots was red-shifted to y650
nm, the absorption profile remained almost identical to bare
PFBT Pdots because only 1.5% of PFDBT was incorporated,
while higher percentages of PFDBT caused the self-quenching
of PFDBT emission. To address this issue, we describe here the
design and synthesis of benzoselenadiazole (BS)-based semi-
conducting polymers39–41 in an effort to redshift both the
absorption and emission of Pdots while keeping their high
brightness.
fluorene with BS (Fig. S4, ESI ).
3
To form Pdots (Scheme 2A), we first blended PFBS with a
polystyrene-poly(ethyleneglycol)–COOH (PS-PEG–COOH) copo-
lymer in tetrahydrofuran (THF) in order to provide the carboxyl
functional groups needed for further bioconjugation reactions.
The mixture was then injected into a water solution using
nanoprecipitation under vigorous sonication. During Pdot
formation, the hydrophobic polystyrene backbones were
embedded inside the hydrophobic PFBS Pdot core, while the
PEG chains offered
a biocompatible layer to minimize
As shown in Scheme 1, we first synthesized BS monomers
and then polymerized with fluorene via palladium-catalyzed
Suzuki coupling to form the copolymer, poly[9,9-dioctylfluor-
enyl-2,7-diyl)-co-1,4-benzo-{2,19-3}-selenadiazole)] (PFBS). We
also varied the concentration of BS in the copolymer and then
investigated the corresponding optical properties of the
resulting Pdots. Furthermore, we conjugated streptavidin onto
the surfaces of Pdots so that we could carry out specific
cellular targeting via biotin-streptavidin interactions for
fluorescence imaging and flow cytometry experiments. We
believe this work should demonstrate the promising applic-
ability of these ultrabright Pdots in a wide range of cellular
and imaging studies.
nonspecific adsorption. The PFBS Pdots were then conjugated
to streptavidin via 1-ethyl-3-[3-dimethylaminopropyl]carbodii-
mide hydrochloride (EDC)-catalyzed coupling for cellular
labeling. The average particle size of PFBS Pdots after
bioconjugation was determined by dynamic light scattering
(DLS) and transmission electron microscopy (TEM) to be y28
nm in diameter (Scheme 2B).
Optical properties of PFBS Pdots
Fig. 1 shows the absorption spectra of PFBS Pdots of different
ratios of fluorene to BS. The absorption spectra consist of two
absorption peaks in which the one at 340–380 nm is attributed
to the fluorene segment in the copolymer and the other one at
450–480 nm is from BS unit. The inset in Fig. 1 represents the
photograph of PFBS Pdot solutions containing different molar
ratios of BS monomers, clearly showing that the color of Pdot
solutions became deeper (higher absorption in the visible
region) with increasing molar ratio of BS in the copolymers.
Their corresponding fluorescence spectra were displayed in
Fig. 2, where a slight red shift (lower inset in Fig. 2, , 10 nm)
was observed as the BS content was raised. The emission of
fluorene (420–480 nm) was completely quenched as the BS
Results and discussion
To date, PFBT Pdot is one of the most well-studied Pdots due
to its extraordinarily high brightness and excellent photo-
stability. Our aim was to replace S-atom in BT unit of PFBT
with Se-atom to minimally affect Pdot folding morphology so
that the high brightness of PFBT Pdots can be maintained
while both their absorption and emission could be effectively
shifted to longer wavelengths. We also blended the carboxyl-
terminated polystyrene polymer, PS-PEG–COOH, with the
resulting PFBS polymer to provide the carboxylic functional
concentration was higher than 10% (Fig. S1, ESI ), indicating
3
an efficient energy transfer from fluorene to BS monomers.
The upper inset in Fig. 2 shows the photograph of Pdot
solutions with different BS concentrations under a UV lamp.
Interestingly, we found that unlike its sulfur analogue, BT, the
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Scheme 2 Schematic illustration of the preparation of carboxyl-functionalized PFBS Pdots and subsequent bioconjugation for specific cellular targeting. (A) PFBS was
mixed with PS-PEG–COOH in THF and then co-precipitated in water under vigorous sonication to form carboxyl-functionalized PFBS Pdots. The PFBS Pdots were
subsequently conjugated to streptavidin via EDC-catalyzed coupling for specific cellular targeting studies. (B) Hydrodynamic diameters of functionalized PFBS30 Pdots
(average diameter was y26 nm) measured by dynamic light scattering. The inset shows the transmission electron microscopy image of PFBS30 Pdots. Scale bar
represents 50 nm.
emission intensity of BS monomer increased as the BS molar
ratio was increased from 2% to 30%, but decreased drastically
for BS molar ratio higher than 30%. This phenomenon was
further confirmed by the measurements of the fluorescence
quantum yields (W) of PFBS Pdots (see Table 1). We speculate
that the increasing W of Pdots from PFBS2 to PFBS 30 was
ascribed to the electron repulsion of Se atom on the
heterocycles that resulted in high dispersion. This greatly
eliminated self-quenching of the BS fluorescence upon
condensation into a Pdot form.42 However, the excessive
amount of BS in the copolymers (i.e. PFBS40 and PFBS50)
inevitably resulted in the aggregation of the BS chromophores
and thus the quenching of the BS fluorescence.
Fig. 1 UV-visible spectra of PFBS2, PFBS10, PFBS30, PFBS40, and PFBS50 Pdots in
water. The inset shows the photograph of these Pdot solutions (from left to
right: PFBS2 to PFBS50 in sequence).
Fig. 2 Fluorescence spectra of PFBS2, PFBS10, PFBS30, PFBS40, and PFBS50
Pdots in water. The inset shows their corresponding Pdot solutions under UV
(365 nm) illumination (from left to right: PFBS2 to PFBS50 in sequence).
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Table 1 Summary of size and photophyscial properties of PFBS copolymer series
in Pdot form in water
labs,max
Copolymers (nm)a
s
Size
lFL,max/nmb (10213 cm2)c W (%)d (nm)e
PFBS6
452
456
460
467
475
570
572
576
578
579
0.60
1.29
1.98
3.44
5.11
16
23
44
17
8
27
27
26
27
29
PFBS16
PFBS30
PFBS40
PFBS50
a
b
c
Absorption maximum. Fluorescence maximum. Absorption
d
cross section per single Pdot at absorption maximum. Quantum
yield. Hydrodynamic size measured by DLS.
e
Using a time-correlated single-photon counting module
system, we determined fluorescence lifetime (t) of PFBS
Pdots to be y1.5 ns (Fig. 3), which is 2–3 times shorter than
conventional dyes that emit in the visible range. The
fluorescence radiative decay rate constant (KR) and nonradia-
tive rate constant (KNR) containing all possible nonradiative
decay pathways can also be estimated from the combination of
the following two equations:
Fig. 4 (A) Single-particle fluorescence image of PFBT Pdots (upper graph) and
the corresponding histograms showing the intensity distributions (bottom
graph). (B) Single-particle fluorescence image of PFBS30 Pdots (upper graph)
and the corresponding histograms showing the intensity distributions (bottom
graph). The insets in the upper images show the photographs of each
corresponding Pdot solution under a 365 nm lamp. The brightness was
measured using imageJ software. All scale bars represent 4 mm.
W = KR/(KR + KNR
)
(1)
(2)
21
from the product of the per-particle absorption cross section
and fluorescence quantum yield. Table 1 summarizes the per-
particle absorption cross section and fluorescence quantum
yields of PFBS Pdots and clearly indicates that PFBS30 Pdots
exhibit the highest fluorescence brightness. In order to
compare the single-particle brightness of PFBS30 Pdots with
the previously reported PFBT Pdots25 (y30 times brighter than
a single quantum dot that emits at 565 nm (QD565)), we also
prepared PFBT Pdots with a similar particle size to PFBS30
Pdots (i.e. y25 nm). As displayed in Fig. 4, we found that
PFBS30 Pdots showed comparable single-particle brightness to
that of PFBT Pdots, which suggests that the single-particle
brightness of PFBS30 Pdots is in turn y30 times brighter than
that of QD565. The calculated brightness of PFBS30 Pdots
based on the photophysical parameters (Table 1) also indicates
that the per-particle brightness of PFBS30 Pdots is almost the
same as that of PFBT Pdots,17,25 which is consistent with the
experimentally measured results.
t = (KR + KNR
)
here we take PFBS30 Pdots as an example, the PFBS30 Pdots
exhibit a fluorescence radiative decay rate of 2.9 6 108 s21
which is comparable to typical organic dyes and other Pdots
reported elsewhere.17
,
Single-particle fluorescence brightness and photostability of
PFBS Pdots
Single-particle brightness is one of the important features of
Pdots when they are applied in fluorescence imaging and
biosensing. The fluorescence brightness can be estimated
Additionally, we also evaluated the photostability of PFBS30
Pdots in comparison with two other fluorescent probes: CdTe
QDs and Au nanoclusters. As shown in Fig. 5 that all the three
probes were photobleached under the same experimental
conditions (380 nm excitation from a 150 W CW Xenon lamp),
the PFBS30 Pdots appeared to be extraordinarily photostable
so that no obvious decrease in emission the intensity was seen
after photobleaching for 15 min. This result is similar to that
of PFBT Pdots characterized by Chiu’s group previously.25
However, the 3-mercaptopropionic acid-coated CdTe QDs
showed slight photobleaching while the lysozyme-stabilized
Au nanoclusters revealed a significant photobleaching decay
during the 15-minute of xenon light irradiation. These results
demonstrate that the PFBS30 Pdots exhibit improved bright-
ness and photostability over other fluorescent probes.
Fig. 3 Fluorescence decay lifetime (y1.5 ns) of PFBS30 Pdots measured by a
time-correlated single-photon counting instrument. The black line represents
experimental data and the red line depicts the decay curve fitted with a double
exponential decay function.
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Fig. 5 Photostability (normalized fluorescence intensity vs. time) of PFBS30
Pdots (orange curve), CdTe QDs (blue curve), and Au nanoclusters (red curve) in
bulk aqueous solutions. Three photobleaching trajectories were obtained under
the same experimental conditions (380 nm excitation from a 150 W CW Xenon
lamp).
Specific labeling of cellular targets with Pdots
We next employed the PFBS Pdots for cellular labeling to
demonstrate their potential in biological applications. First,
the PFBS30 Pdots were bioconjugated to streptavidin via EDC-
catalyzed coupling. We then performed microtubule labeling
inside HeLa cells where the biotinylated monoclonal anti-
a-tubulin antibody and Pdot-streptavidin bioconjugates were
sequentially incubated with the cells. Fig. 6A–C shows
representative confocal images of Pdot-streptavidin-labeled
microtubules in HeLa cells, indicating that PFBS30 Pdots
could be specifically targeted onto subcellular organelles in
the presence of biotinylated antibody. Fig. 6D–F shows the
confocal images of negative control samples (identical experi-
mental conditions to those in Fig. 6A–C but in the absence of
the primary biotinylated antibody) where no observable
fluorescence signal from PFBS30 Pdots was detected. These
results demonstrate that this new type of Pdots possesses
highly specific binding activity with minimal nonspecific
absorption.
Additionally, we also performed flow cytometry experiments
in which PFBS30 Pdots were used to label membrane proteins
on MCF-7 breast cancer cells. The cells were sequentially
incubated with biotinylated primary anti-human CD326
EpCAM antibody (used to label the EpCAM receptors of
MCF-7 cells) and Pdot-streptavidin bioconjugates. Fig. 6G and
H shows the flow cytometry results that an excellent separation
between the Pdot-streptavidin-labeled cells (Fig. 6G, with
primary antibody) and the negative control/background
(Fig. 6H, without primary anti-body) could be clearly observed.
These results demonstrate that Pdot-streptavidin bioconju-
gates can be labeled onto MCF-7 cells effectively and
specifically with ultralow nonspecific binding, consistent with
the results from confocal microscopy in HeLa cells.
Fig. 6 Two-color confocal fluorescence images of microtubules labeled with
PFBS30-streptavidin probes inside HeLa cells. The blue fluorescence is from
nuclear counterstain Hoechst 34580 and the orange fluorescence is from
PFBS30-streptavidin Pdots. (A) Image of nucleus. (B) Image of microtubules. (C)
The overlay of panels (A) and (B). (D–F) Images of negative control samples
where cells were incubated with PFBS30-streptavidin probes but in the absence
of biotinylated primary antibody. The scale bars are 20 mm. (G–H) Flow
cytometry measurements of PFBS30-streptavidin labeled MCF-7 cells. The black
line in (G) shows the fluorescence intensity distribution of negative control
samples where no primary Biotin anti-human CD326 EpCAM antibody was
present. The orange line in (H) represents the result of PFBS30-streptavidin
labeled cells.
groups for the creation of ultrabright Pdots with redder
absorption and emission wavelengths. A significant red shift
of the emission (y40 nm) of Se-containing copolymers as
compared to its sulfur analogue can be attributed to the
narrower p–p* band gap of the BS unit and the stronger
interchain interactions among neighboring BS moieties. The
optical properties of different molar ratios of fluorene to BS
were systemically characterized and the PFBS30 Pdots were
found to possess the highest fluorescence brightness, which is
comparable to that of PFBT Pdots under 488-nm laser
excitation (i.e. y30 times brighter than QD565). We also
demonstrated the bioconjugation of the PFBS Pdots and their
highly specific binding to both cell surfaces and interiors
without any detectable nonspecific binding. We anticipate the
PFBS Pdots to find broad use both in basic biological studies
and in bioimaging and bioanalytical applications. We now
focus on the development of near-infrared fluorescent Pdots
by extending the conjugation of BS unit.
Conclusions
In summary, this work described herein is the first in a series
of benzoselenadiazole-based Pdots being investigated by our
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bromobenzene (1 mL) was added and further stirred for 3 h.
The mixture was poured into methanol (250 mL). The
precipitate was filtered, washed with methanol, water, and
acetone to remove monomers, small oligomers, and inorganic
salts. The crude product was dissolved in CH2Cl2 (15 mL),
filtered through 0.2 mm membrane, and re-precipitated in
methanol (150 mL). The powder was then stirred in acetone
Experimental section
Materials
The following chemicals were purchased from Sigma-Aldrich
and used as received: 2,7-dibromofluorene, sodium hydroxide,
tert-butyl 6-bromohexylcarbamate, tetrahydrofuran (THF;
anhydrous, ¢99.9%, inhibitor-free), toluene, tetrabutylammo-
nium bromide (Bu4NBr), phenylboronic acid, bromobenzene,
methanol, tetrakis(triphenylphosphine)palladium (Pd(PPh3)4),
dichloromethane (CH2Cl2), 4,7-dibromobenzo[c]-1,2,5-thiadia-
zole, 9,9-dihexylfluorene-2,7-diboronic acid bis(1,3-propane-
(200 mL) for 4 h and then collected by filtration, and dried in
1
vacuum. Yield: 62–78%. H NMR (d, CDCl ): See Fig. S4, ESI
3
3
by taking PFBS30 as an example. GPC: PFBS2 Mn: 9794, Mw:
21 183, PDI: 2.16; PFBS10 Mn: 7590, Mw: 12 305, PDI: 1.62;
PFBS30 Mn: 6175, Mw: 11 334, PDI: 1.83; PFBS40 Mn: 3685, Mw:
6229, PDI: 1.57; PFBS50 Mn: 2395, Mw: 3526, PDI: 1.47.
diol)
ester,
sodium
carbonate,
selenium
dioxide,
o-phenylenediamine, silver sulfate, and hydrochloric acid. A
comb-like polymer, polystyrene grafted with ethylene oxide
functionalized with carboxyl groups (PS-PEG–COOH, main
chain MW 6,500, graft chain MW 4,600, total chain MW
30,000, polydispersity 2.9), was purchased from Polymer
Source Inc. (Quebec, Canada) and used as received without
further purification. All other chemical reagents were pur-
chased from Invitrogen (Carlsbad, CA) and used as received.
High purity water (18.2 M 6 cm) was used throughout the
experiment.
Preparation of PFBS Pdots
Typically, 200 mL of PFBT (1 mg mL21 in THF) and 20 mL of PS-
PEG–COOH (2 mg mL21 in THF) were added into 5 mL of THF.
This mixture was then quickly injected into 10 mL of water
under vigorous sonication. THF was then removed by purging
with nitrogen on a 96 uC hotplate for one hour. The resulting
Pdot solution was filtered through a 0.2 mm cellulose acetate
membrane filter to remove any aggregates formed during
preparation.
Synthesis of 2,1,3-benzoselenadiazole (1)
Bioconjugation and characterization of PFBS Pdots
A solution o-phenylenediamine (1.08 g, 10 mmol) in ethanol
(55 mL) was heated to reflux in a round-bottomed single-
necked flask and then SeO2 (1.17 g, 10.5 mmol) in hot water
(30 mL) was added. The reaction mixture was stirred and
refluxed for 2 h. The solvent was removed using a rotary
evaporator and then the residue was dissolved in ethyl acetate
to extract with brine. The organic extract was separated and
dried over MgSO4. The crude product was purified by column
chromatography on silica gel using CH2Cl2 as eluent to afford
Bioconjugation was performed by using the EDC-catalyzed
reaction between carboxylate-functionalized PFBS Pdots and
the respective amine-containing streptavidin. In a typical
bioconjugation reaction, 80 mL of polyethylene glycol (5% w/v
PEG, MW 3350) and 80 mL of concentrated HEPES buffer (1 M)
were added to 4 mL of Pdot solution, resulting in a Pdot
solution in 20 mM HEPES buffer with a pH of 7.3. Then 240 mL
of streptavidin (1 mg mL21) was added to the solution and
mixed well on a vortex. After that, 80 mL of freshly-prepared
EDC solution (5 mg mL21 in MilliQ water) was added to the
solution, and the mixture was stirred for 4 h at room
temperature. After bionconjugation, 80 mL of BSA (10 wt%)
was added to the Pdot solution and the reaction was continued
for another 20 min. A 80 mL aliquot of Triton X-100 in MilliQ
water (2.5 wt%) was added to the Pdot-streptavidin mixture.
The mixture was then transferred to a centrifugal ultrafiltra-
tion tube (Amicon1 Ultra-4, MWCO: 100 kDa), and then
concentrated to 0.5 mL by centrifugation. Finally, the Pdot-
streptavidin bioconjugates were purified by gel filtration using
Sephacryl HR-300 gel media.
The average particle size was determined by dynamic light
scattering and transmission electron microscopy (TEM) to be
26–29 nm in diameter for PFBS-streptavidin Pdots. TEM
images of the synthesized Pdots were acquired using a JEOL
2010 transmission electron microscope at an acceleration
voltage of 200 kV. For TEM, a drop of Pdot aqueous solution
was placed onto a carbon-coated grid and allowed to evaporate
at room temperature. The absorption spectra of Pdots were
measured using UV-visible spectroscopy (Spectra System
UV2000 HR, Thermo Separation Products). For the fluores-
cence measurements, various concentrations of copper ions
were introduced into the Pdot solutions and the fluorescence
spectra were collected using a Hitachi F-7000 fluorometer
(Hitachi, Tokyo, Japan) under 380 nm or 475 nm excitation.
1
1.8 g (99%) of 1. H NMR (d, CDCl3): 7.46–7.50 (m, 2H), 7.84–
7.87 (m, 2H).
Synthesis of 4,7-dibromo-2,1,3-benzothiadiazole (2)
Bromine (5 mL) was added slowly to a solution of compound 1
(7.0 g) and AgSO4 (12.0 g) in concentrated H2SO4 (100 mL). The
reaction mixture was stirred in the dark for 3 h at room
temperature and then poured into ice–water (300 mL). After
that, the precipitate was dissolved in ethyl acetate and then
extracted with brine for 3 times. The organic layer was
separated, dried over MgSO4 and, filtered through a short
silica plug. The crude product was purified by column
chromatography on silica gel using hexane as eluent to yield
0.8 g (6%) of 2 as light yellow needles. 1H NMR (d, CDCl3): 7.64
(s, 2H).
Synthesis of PFBS copolymer series
Polymers were synthesized by copolymerization of monomers
2, 3, and 4 by Suzuki coupling. In a 100 mL flask, monomer 2
(0.04 mmol–1 mmol), 3 (0–0.96 mmol), and 4 (1 mmol) were
dissolved in toluene (20 mL), and then Bu4NBr (0.04 mmol,
12.5 mg) and Na2CO3 (2 M, 12 mL) was added. The mixture
was degassed and refilled with N2 (repeated 4 times) before
and after addition of Pd(PPh3)4 (0.035 mmol, 55 mg). The
reactants were stirred at 90 uC for 40 h and phenylboronic acid
(100 mg) dissolved in THF (1 mL) was added. After two hours,
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Fluorescence quantum yield of Pdots was determined by using
For microtubule-labeling experiments, tens of thousands of
HeLa cells were plated on a 22 6 22-mm glass coverslip and
cultured until the density reached 60–70% confluence. The
cells were fixed with 4% paraformaldehyde for 10 min,
permeabilized with 0.25% Triton-X 100 in PBS buffer for 15
min, and then blocked in BlockAidTM blocking buffer for
another 30 min. The fixed and blocked HeLa cells were
subsequently incubated with 5 mg mL21 biotinylated mono-
clonal anti-a-tubulin antibody (Biolegend, San Diego, CA, USA)
for 60 min, and then Pdot-streptavidin conjugates for 30 min.
The Pdot labeled cells were then counterstained with Hoechst
34580 and imaged immediately on a fluorescence confocal
microscope (Nikon D-Eclipse C1).
a
dilute solution of 4-dicyanomethylene-2-methyl-6-p-
dimethyl-aminostyryl-4H-pyran in methanol as dye standard.
The detailed procedures for the fluorescence quantum yield
measurements of PFBS Pdots were described in the ESI.
3
Cell culture and labeling
The cervical cancer cell line HeLa and breast cancer cell line
MCF-7 were ordered from Food Industry Research and
Development Institute (Taiwan). Primary cultured HeLa cells
were grown in Dulbecco’s Modified Eagle Medium (cat. no.
11 885, Invitrogen) supplemented with 10% Fetal Bovine
Serum and 1% penicillin-streptomycin solution (5000 units
mL21 penicillin G, 50 mg mL21 streptomycin sulfate in 0.85%
NaCl) at 37 uC with 5% CO2 humidified atmosphere. The cells
were pre-cultured in a T-25 flask and allowed to grow for 5–7
days prior to experiments until y80% confluence was
reached. To prepare cell suspensions, the adherent cancer
cells were quickly rinsed with media and then incubated in 5
mL trypsin-ethylenediaminetetraacetic (EDTA) solution (0.25%
w/v trypsin, 0.25 g L21 EDTA) at 37 uC for 5 min. The cell
suspension solution was then centrifuged at 6000 rpm for 10
min to precipitate the cells to the bottom of the tube. After
taking out the upper media, the cells was rinsed and re-
suspended in 5 mL of culture media. Approximately tens of
thousands cells were split onto a glass-bottomed culture dish
and allowed to grow for 12 h before Pdot tagging. Prior to
fluorescence imaging, the cells were rinsed with PBS buffer to
remove any non-specifically bound Pdots on the cell surface.
For cell labeling experiments, BlockAidTM blocking buffer
was purchased from Invitrogen (Eugene, OR, USA). For
labeling cell-surface markers with IgG, a million MCF-7 cells
in 100-mL labeling buffer (16 PBS, 2 mM EDTA, 1% BSA) was
incubated with 0.3 mL of 0.5 mg mL21 primary biotin anti-
human CD326 EpCAM antibody (eBioScience, San Diego, CA,
USA) on a rotary shaker in the dark and at room temperature
for 30 min, followed by a washing step using labeling buffer.
Then the cells were incubated with 1.5 nM Pdot-streptavidin
conjugates in BlockAidTM buffer for 30 min on a shaker in the
dark and at room temperature, followed by two washing steps
with labeling buffer. Prior to cell incubation, Pdot solutions
were sonicated for 3 min in order to disperse any potential
aggregates. Negative controls were obtained by incubating
cells with Pdot-streptavidin conjugates in the absence of
primary biotinylated-antibody. Cell fixation was performed by
dissolving the cell pellet obtained by centrifugation in 500 mL
of fixing buffer (16 PBS, 2 mM EDTA, 1% BSA, 1% PFA).
Flow cytometry measurements were performed on fresh
samples with 106 cells/0.5 mL and prepared following the
procedure previously described. The flow cytometer Cytopeia-
InFlux Mariner (BD Bioscience, San Jose, CA USA) was used:
Cells flowing in the detection chamber were hit with a 488-nm
laser beam, side- and forward-scattered light and fluorescence
emission were collected, the latter was filtered by a 505 long-
pass and a 590/50 band-pass, and all signals were detected by
PMT arrays. Representative populations of cells were chosen
by selection of an appropriate gate. Detection of cell
fluorescence was continued until at least 104 events had been
collected in the active gate.
Cell imaging
The fluorescence spectra of Pdot-tagged cells were acquired
with a fluorescence confocal microscope (Nikon D-Eclipse C1)
under ambient conditions (24
¡ 2 uC). The confocal
fluorescence images were collected using a diode laser at 488
nm (y15 mW) as the excitation source and an integration
time of 1.6 ms/pixel. A Carl Zeiss 606 (‘‘C-Apochromat’’ 606/
1.2 W Corr) objective was utilized for imaging and spectral
data acquisition; the laser was focused to a spot size of y7
mm2.
Acknowledgements
We would like to thank the National Science Council (NSC 101-
2113-M-110-015-MY2) and National Sun Yat-sen University. We
also gratefully acknowledge support for this research from
Prof. Chao-Ming Chiang, Prof. Wei-Lung Tseng, Prof. Chin-
Hsing Chou, Dr Jiun-Yi Shen from National Taiwan University,
and the Instrumentation Center at National Sun Yat-sen
University for use of instruments.
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