High-intensity near-IR fluorescence in semiconducting polymer dots achieved by cascade FRET strategy

Article abstract of DOI:10.1039/c3sc50222h

Near-IR (NIR) emitting semiconducting polymer dots (Pdots) with ultrabright fluorescence have been prepared for specific cellular targeting. A series of π-conjugated polymers were synthesized to form water dispersible multicomponent Pdots by an ultrasonic

Full text of DOI:10.1039/c3sc50222h

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High-intensity near-IR fluorescence in semiconducting
polymer dots achieved by cascade FRET strategy†
Cite this: DOI: 10.1039/c3sc50222h
a
b
b
b
b
*
Xuanjun Zhang,‡ Jiangbo Yu,‡ Yu Rong, Fangmao Ye, Daniel T. Chiu
and Kajsa Uvdal
a
*
Near-IR (NIR) emitting semiconducting polymer dots (Pdots) with ultrabright fluorescence have been
prepared for specific cellular targeting. A series of p-conjugated polymers were synthesized to form
water dispersible multicomponent Pdots by an ultrasonication-assisted co-precipitation method. By
optimizing cascade energy transfer in Pdots, high-intensity NIR fluorescence (F ¼ 0.32) with tunable
excitations, large absorption–emission separation (up to 330 nm), and narrow emission bands (FWHM ¼
44 nm) have been achieved. Single-particle fluorescence imaging show that the as-prepared NIR Pdots
were more than three times brighter than the commercially available Qdot705 with comparable sizes
under identical conditions of excitation and detection. Because of the covalent introduction of
carboxylic acid groups into polymer side chains, the bioconjugation between NIR-emitting Pdots and
streptavidins can be readily completed via these functional groups on the surface of Pdots. Furthermore,
through flow cytometry and confocal fluorescence microscopy the NIR-emitting Pdot–streptavidin
conjugates proved that they could effectively label EpCAM receptors on the surface of MCF-7 cells, via
specific binding between streptavidin and biotin.
Received 25th January 2013
Accepted 27th February 2013
DOI: 10.1039/c3sc50222h
www.rsc.org/chemicalscience
non-blinking property, and fast emission rates make them well-
suited to bioimaging and biosensing applications.6–21
Introduction
Optical imaging with uorescence microscopy is a vital tool in
the study of living systems owing to its attractive features, such
as high sensitivity and spatiotemporal resolution.1 Among
different uorescent labels, water-soluble uorescent dyes tend
to suffer from rapid photobleaching, which limits their broad
applicability in long-term monitoring of live cells with high
sensitivity. Fluorescent inorganic nanoparticles, particularly,
quantum dots (Qdots) have gained much attention in the past
decades owing to their unique optical features such as higher
photostability and brightness, tunable emission wavelengths,
narrow emission bands, as well as lower susceptibility to
cellular efflux mechanisms when compared with small-mole-
cule labels.2–5 Recently, semiconducting polymer nanoparticles
(Pdots) have emerged as a new class of promising uorescent
nanoprobes. Their superior characteristics such as non-toxic
features, ultrabright photoluminescence, high photostability,
When biological applications are targeted, uorescence in
the deep red and near-infrared (NIR) is highly desirable,
because this wavelength region offers maximal penetration into
biological tissues, good spectral separation from auto-
uorescence, and less scattering in turbid media.22,23 However,
because most of the NIR emitters are at molecules with
extended p-conjugations or strong donor–acceptor charge
transfer chromophores, self-quenching is oen a serious issue
when they are condensed into a nanoparticle or solid form.24–28
As self-quenching is difficult to overcome, the development of
bright NIR-emitting Pdots is a signicant challenge. Recently
some strategies, such as Pdots doped with NIR dyes14,21 and
hybrid Pdots–Qdots,29 have been demonstrated to realize NIR-
emitting Pdots, which is usually applied to improve the sensi-
tivity and specicity of bio-imaging and tracking, since the
strong autouorescence of living tissues, as well as the scat-
tering and the absorption of short-wavelength light in tissues
can greatly decrease sensitivity. However, the leakage of the dyes
from Pdots13,14,19 and the toxicity of the Qdots29 are important
limitations for using dye-doped NIR-emitting Pdots or hybrids
as uorescent probes. In particular, the leakage of NIR dyes
from Pdots results in poor optical properties, such as reduced
emission color purity and quantum yields, because the donor–
acceptor ratio in Pdots signicantly affects the energy transfer
efficiency. Another drawback of current Pdots is a broad uo-
rescence emission band, which can lead to constraints in
aDivision of Molecular Surface Physics
& Nanoscience, Department of Physics,
¨
¨
Chemistry, and Biology, Linkoping University, Linkoping 58183, Sweden. E-mail:
kajsa@ifm.liu.se; Fax: +46 13 28 8969; Tel: +46 13 281208
bDepartment of Chemistry, University of Washington, Seattle, Washington 98195, USA.
E-mail: chiu@chem.washington.edu; Fax: +1-206-685-8665; Tel: +1-206-5431655
† Electronic supplementary information (ESI) available: DLS, ow cytometry
result of 4-NIR Pdots and Qdot705, other characterization and measurement
methods. See DOI: 10.1039/c3sc50222h
‡ X. Zhang and J. Yu contributed equally to this work.
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multiplex applications because of spectral interference with triuoroacetic acid. Boron dipyrrin (BODIPY)-containing poly-
other uorophores, especially for Pdots with high brightness, mer P4 was designed as terminal acceptor because BODIPY
where leakage of emitted photons into other detection channels derivatives exhibit favorable optical properties, such as high
can easily overwhelm the uorescence from other non-Pdot extinction coefficients and uorescence quantum yields, negli-
probes. To address these problems, we report here the prepa- gible photobleaching, and narrow emission peaks.30 Our
ration and application of bright NIR-emitting Pdots with narrow purpose is to combine the benecial signature properties of
emission band and large absorption–emission separation both donor and acceptor in light-harvesting system, such as the
¨
(Scheme 1) via cascade Forster resonance energy transfer strong absorption of primary donor and narrow emission band
(FRET).
and high uorescence quantum yield of terminal acceptor.
Here, P4 can be doped into P1 or P2 Pdots by co-precipita-
tion. However, the absorption of P4 (peak at 690 nm) does not
overlap well with the emission spectrum of P1 (peak at 440 nm)
or P2 (peak at 540 nm), which leads to poor energy transfer from
primary donors to P4. This issue was also observed in our recent
work, where we achieved NIR uorescence by encapsulation of
NIR emitting dye silicon 2,3-naphthalocyanine bis(trihexylsilyl
oxide) into Pdots.14 Therefore, in this work we introduced
polymer P3 to facilitate efficient energy transfer from the
primary donor to the terminal acceptor P4. P1 exhibits an
absorption peak at 380 nm and uorescence peak at 440 nm.
Results and discussion
Polyuorene (P1 in Scheme 1) and poly[(9,9-dioctyluorenyl-2,7-
diyl)-co-(1,4-benzo{2,10,3}thiadiazole)] (P2) were used as primary
donors in cascade FRET systems because of their high bright-
ness and absorption proles. In the synthesis, monomer 2,7-
dibromo-9,9-bis(3-(tert-butyl propanoate))uorene was co-poly-
merized (molar ratios of 5 and 9%, respectively, for P1 and P2),
which enabled further covalent functionalization and bio-
conjugation aer removal of protecting tert-butyl groups by
Scheme 1 Schematic illustration of NIR fluorescence in Pdots obtained by cascade FRET.
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Fig. 1 (a and b) TEM images and size distribution measured by DLS of 3-NIR Pdots; (c and d) TEM images and size distribution measured by DLS of 4-NIR Pdots; (e)
absorbance (solid line) and fluorescence spectra (dashed line) of 4-NIR (blue, lexc ¼ 380 nm) and 3-NIR Pdots (red, lexc ¼ 450 nm); (f) time-resolved fluorescence decay
of P1 Pdots, and 4-NIR Pdots; (g) time-resolved fluorescence decay of P2 Pdots, and 3-NIR Pdots.
(peak at 690 nm) overlaps well with the emissions of P3 (peak at
680 nm). These optimized spectral overlaps (Scheme 1) ensure
¨
an efficient Forster-type cascade energy transfer, which is
further established by the time-resolved uorescence results
(Fig. 1f). Owing to these perfect spectral overlaps, a minimum
amount of P4 is needed to quench the donor emissions, and
thus greatly reduced self-quenching of the nal NIR uores-
cence from P4.
We have also found an interesting result. As polymer P3 is
an intermediary in the cascade FRET system described above,
the molar ratio between monomer uorene and 2,3-
bisphenylthieno[3,4-b]pyrazine in the P3 polymer chain signif-
icantly inuenced the nal optical properties. As we know, the
thieno[3,4-b]pyrazine (TP) unit has a high electron affinity, and
the degree of intramolecular charge transfer from uorene
monomer to TP varies with TP molar ratio in the polymer
chain.31 Therefore, by optimizing the optical property and
energy transfer, a ratio of 13% of monomer TP in P3 polymer
was found as optimal for P3 as an effective intermediary for
cascade FRET. We also discovered that the molar ratio of
BODIPY monomer in P4 is also crucial to the nal NIR-emis-
sion brightness of the NIR Pdots. Experimental results showed
that in the prepared Pdots, even though the doping ratio of
polymer P4 (doped into P1 or P2) is very low (weight percentage
< 1%), the high molar ratio of BODIPY units in the P4 polymer
chain induced signicant self-quenching of nal NIR emission
due to the high local concentration of BODIPY monomers
contained in a single polymer chain. In this work, 8% molar
ratio of BODIPY monomer contained in P4 was determined to
ensure the best emission performance in the cascade FRET
system of Pdots.
Fig. 2 (a, b) Single-particle fluorescence images of Qdots and 3-NIR Pdots
obtained under identical excitation and detection conditions. (c) Histograms of
intensity distribution of single-particle fluorescence for Qdots and 3-NIR Pdots
(log-normal fitting on the histograms resulted in the black and blue fitting curves,
as well as mean values of 1200 and 3900 CCD counts for Qdots and 3-NIR Pdots,
respectively). (d) Flow-cytometry measurements of the intensity distributions of
MCF-7 cells labeled with Qdot705–streptavidin (negative labeling, cyan curve;
positive labeling, orange curve) and 3-NIR Pdot–streptavidin (negative labeling,
green curve; positive labeling, red curve). All the positive and negative labeling
was completed and measured under identical experimental conditions. In the
negative labeling, primary biotinylated antibodies were absent.
The absorption spectrum of P2 (peak at 460 nm) overlaps well
with the emission spectrum of P1. The absorption spectrum of
P3 (peak at 540 nm) also perfectly overlaps with the emission
spectrum of P2 (peak at 540 nm). Likewise, the absorption of P4
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Fig. 3 Confocal fluorescence images of MCF-7 cells labeled with 3-NIR Pdot–SA probes. (a) Positive labeling using 3-NIR Pdot–SA probe. (b) Negative labeling carried
out under the same condition as (a) but in the absence of biotinylated antibody on the surface of the MCF-7 cells. Images from left to right: blue fluorescence from the
nuclear stain Hoechst 34580; deep red fluorescence images from 3-NIR Pdot–SA probes; Nomarski (DIC) images; combined fluorescence images. Scale bars: 20 mm.
Pdots composed of different polymers described above could the nal 4-NIR Pdots exhibited a high-intensity uorescence
be readily prepared by a co-precipitation method (details shown peak at 710 nm with very large absorption-uorescence sepa-
in Experimental section). The prepared Pdots were water ration (330 nm) and a high quantum yield of 32% (Fig. 1e). The
dispersible and were stable for several months. Generally, the emission peak is very narrow with a full width at half
prepared Pdots had an average size of about 16 nm, which are maximum (FWHM) of 44 nm, which is even narrower than that
comparable to commercial Qdots705 (ESI, Fig. S1†), as reported of commercially available Qdots705. Similarly efficient FRET
by TEM analysis and dynamic light scattering (DLS) (Fig. 1a–d). was also achieved in 3-NIR Pdots with optimized weight ratio
Here, multicomponent Pdots formed from P1, P2, P3 and P4 are of P2 : P3 : P4 ¼ 100 : 50 : 6.2 and the nal Pdots gave an
denoted as 4-NIR Pdots and those formed from P2, P3 and P4 intense uorescence peaking at 720 nm (FWHM ¼ 44 nm) with
polymers are denoted as 3-NIR Pdots.
a quantum yield of 30%. Because NIR emission was derived
By careful optimization of the weight ratios of premixed from semiconducting polymers that comprised the Pdots,
polymers P1, P2, P3 and P4 (50 : 50 : 70 : 8) in THF solution, there was no leakage issue, which oen plagues dye-doped
the emissions of P1, P2 and P3 in Pdots were quenched and Pdots.
Scheme 2 Synthesis route of monomer F. Reagents and conditions: (i) 1-Bromooctane, Na2CO3/DMF, 80 ꢀC, 48 h; (ii) hydrazine hydrate, reflux in ethanol overnight;
(iii) 2-hydroxy-40-iodoacetophenone, reflux in ethanol for 4 h; (iv) lead tetraacetate, THF, r.t. for 2 h; (v) NH3 (aq)–acetic acid, PEG300, 100 ꢀC, 2 h; (vi) Et3N/BF3$OEt2,
CHCl3, 50 ꢀC, 12 h.
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Time-resolved uorescence decay was measured by time- receptors on the MCF-7 cell surface. In the control experiment,
correlated single-photon counting (TCSPC) to study the FRET where the biotinylated antibody on the surface of the MCF-7
process. As shown in Fig. 1f, the lifetime of P1 Pdots at 440 nm cells was absent, no uorescence on the cell surface was
is 0.65 ns, but the lifetime of P1 in 4-NIR Pdots was much too detected (Fig. 3b), which further conrmed that the covalent
fast to be measured because the decay curve was comparable bioconjugation of streptavidin to Pdots was successful and the
with the instrument response function (IRF) (black curve in cellular targeting was specic through biotin–streptavidin
Fig. 1f). The lifetime of P4 in 4-NIR Pdots was 3.48 ns. Similarly interaction.
efficient energy transfer and very short lifetime of P2 was also
observed in 3-NIR Pdots (Fig. 1g). These results agree well with
the total quenching of P1 or P2 emission by energy transfer (in
Conclusions
4-NIR and 3-NIR Pdots, respectively), which were also proved by
the emission spectra of these Pdots in Fig. 1e showing efficient
energy transfer in both 4-NIR and 3-NIR Pdots.
To sum up, we demonstrated a simple approach for the design
and preparation of ultrabright NIR-emitting Pdots with narrow
uorescence band and large absorption–emission separation
with the aid of cascade FRET and careful design of polymers.
Single particle uorescence brightness measurements and ow-
cytometric analysis of Pdots-labeled MCF-7 cells showed that
these NIR emitting dots are >3 times brighter than Qdot705.
This work has successfully addressed the self-quenching issue
of NIR-emitting Pdots by employing the cascade FRET strategy,
which combined the benecial signature properties of both
donor and acceptor in a light-harvesting system. The prepara-
tion method presented here is general and can be extended to
the preparation of other NIR-emitting nanoparticles for
different biological applications.
To further develop and test ultrabright Pdots more suitable
for bioimaging, we decided to focus on 3-NIR Pdots because
their absorbance (peak at 460 nm) conveniently matches the
commonly available laser wavelengths at 473 and 488 nm for
uorescence microscopy and detection. A useful estimate of
uorescence brightness is given by the product of the absorp-
tion cross-section and the uorescence quantum yield. We
carried out single-particle imaging to experimentally evaluate
and compare the brightness of 3-NIR Pdots and commercially
available Qdot705. Fig. 2a–c showed typical single-particle
images of Qdot705 and 3-NIR Pdots, respectively, which were
obtained under identical excitation and acquisition conditions.
With a low excitation power from the 488 nm laser, very bright,
near-diffraction-limited spots were clearly observed for single 3-
NIR Pdots (Fig. 2b). In addition, 3-NIR Pdots were much
brighter than Qdot705 under uorescence microscopy. From
the distribution curves of single-particle brightness of several
thousand Qdot705 and 3-NIR Pdots, we determined 3-NIR Pdots
were >3 times brighter than Qdot705 (Fig. 2c). Such a prominent
contrast is primarily due to the high per-particle absorption
cross-section of Pdots, which would be particularly useful for
long-term uorescence detection or tracking requiring low
excitation powers.
Experimental section
Materials
All of the chemicals and solvents were purchased from
Sigma-Aldrich unless indicated elsewhere. Monomer C13 and
monomer E,32,33 and 2-hydroxy-40-iodoacetophenone34 were
synthesized by the methods reported before.
Synthesis
Polymers were synthesized by Suzuki coupling using a series of
To evaluate the NIR-emitting Pdots for bioimaging, we per- monomers shown in Chart 1.
formed bioconjugation between the Pdots and streptavidin (SA)
Synthesis of polymer P1. To a 100 mL ask was added
for specic targeting towards cell surface receptors. Streptavidin monomer A (0.9 mmol, 493 mg), monomer B (1.02 mmol,
was used ubiquitously because of its remarkable binding 569 mg), monomer C (0.1 mmol, 58 mg), Bu4NBr (15 mg),
affinity to biotin. The nal streptavidin-conjugated Pdots were toluene (20 mL) and Na2CO3 (2 M, 10 mL). The mixture was
used to label the specic cell-surface receptor, EpCAM, which is stirred at room temperature and the ask was degassed and
widely used for the detection of circulating tumor cells. MCF-7 recharged with N2, which was repeated four times before and
cells were sequentially incubated with biotinylated primary aer addition of Pd(PPh3)4 (0.02 mmol, 23 mg). The reactants
anti-EpCAM antibody and 3-NIR Pdot–SA. Flow-cytometry were stirred at 90 ꢀC for 48 h and then phenylboronic acid
measurements of negative and positive labeling (Fig. 2d and S2, (100 mg) dissolved in THF (1 mL) was added. Aer 2 h, bro-
ESI†) revealed that both 3-NIR Pdot–SA and 4-NIR Pdot–SA mobenzene (1 mL) was added and further stirred for 3 h. The
could specically bind to the biotinylated antibody on the mixture was poured into methanol (200 mL). The precipitate
surface of the MCF-7 cells like commercial Qdot705–SA. was ltered off, washed with methanol, water and acetone to
However, the uorescence intensity of cells labeled with 3-NIR remove monomers, small oligomers and inorganic salts. The
Pdots and 4-NIR Pdots are >3 times higher than that labeled crude product was dissolved in DCM (15 mL), ltered through a
with Qdot705. This ensemble result of 3-NIR Pdots agrees well 0.2 mm membrane and re-precipitated in methanol (150 mL).
with that based on single-particle brightness measurements.
The powder was then stirred in acetone (200 mL) overnight.
To further evaluate the NIR emitting Pdots for potential then collected by ltration, and dried in vacuum. Yield: 580 mg
1
bioimaging, MCF-7 cells labeled with 3-NIR Pdots–SA conjugate (74%). H NMR (500 MHz, CDCl3): d 7.84 (d, J ¼ 7.8 Hz, 2H),
were studied using confocal uorescence microscopy. As shown 7.63–7.73 (m, 4H), 2.12 (br, 4H), 1.32 (s, 1H), 1.10–1.25 (m, 20H),
in Fig. 3a, the 3-NIR Pdot–SA probes effectively labeled EpCAM 0.68–0.92 (m, 10H). GPC: Mn 5400, Mw 11 400, PDI 2.11. The
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aer addition of Pd(PPh3)4 (0.02 mmol, 23 mg), respectively.
ꢀ
The reactants were stirred at 90 C for 48 h and then phenyl-
boronic acid (100 mg) dissolved in THF (1 mL) was added. Aer
2 h, bromobenzene (1 mL) was added and further stirred for 3 h.
The mixture was poured into methanol (200 mL). The precipi-
tate was ltered off, washed with methanol, water and acetone
to remove monomers, small oligomers and inorganic salts. The
crude product was dissolved in DCM (15 mL), ltered through a
0.2 mm membrane and re-precipitated in methanol (150 mL).
The powder was then stirred in acetone (200 mL) overnight and
collected by ltration, and dried in vacuum. Yield: 532 mg
1
(71%). H NMR (300 MHz, CDCl3): d 7.84 (d, J ¼ 8.1 Hz, 2H),
7.53–7.62 (br, 4H), 7.48 (t, J ¼ 7.2 Hz, 0.28H), 7.36–7.43 (br,
1.07H), 2.12 (br, 4H), 1.10–1.22 (m, 20H), 0.76–0.89 (m, 10H).
The molar fractions of uorene and thieno[3,4-b]pyrazine (TP)
units were estimated by the elemental analysis data for [uo-
rene]–[TP] ¼ 0.87 : 0.13. Calc. (%) C, 88.3, H, 9.78, N, 0.88;
found, C, 88.5, H, 9.9, N, 0.89. GPC: Mn 6200, Mw 14 200, PDI
2.29.
The synthesis route of monomer F is shown in Scheme 2.
Synthesis of 1. Methyl 4-hydroxybenzoate (0.1 mol, 15.2 g), 1-
bromooctane (0.13 mol, 25 g) and Na2CO3 (0.28 mol, 30 g) were
stirred in DMF (100 mL) at 80 ꢀC for 48 h under N2. Aer cooling
to room temperature, water 100 mL and DCM (100 mL) were
added and stirred for 5 min. The DCM phase was washed with
water (100 mL) and Na2CO3 solution (1 M, 100 mL), and dried
over MgSO4. Aer removal of the solvent, the residue was
puried by column chromatography (CHCl3–hexane ¼ 1 : 1) to
gave colorless crystals. Yield: 76%. 1H NMR (500 MHz, CDCl3): d
7.98 (d, J ¼ 8.5 Hz, 2H), 6.90 (d, J ¼ 8.5 Hz, 2H), 4.00 (t, 2H), 3.88
(s, 3H), 1.79 (m, 2H), 1.24–1.40 (m, 10H), 0.89 (t, 3H). 13C NMR
(500 MHz, CDCl3): d 167.12, 163.15, 131.75, 122.50, 114.25,
68.41, 52.03, 32.01, 29.54, 29.43, 29.37, 29.33, 26.20, 22.87, and
14.31. ESI-MS: m/z 264.40. Calc. for C16H24O3 (%): C, 72.69; H,
9.15. Found: C, 72.82; H, 8.99.
Synthesis of 2. Methyl 4-(octyloxy)benzoate (1) (0.1 mol,
26.4 g) and hydrazine hydrate (30 mL, excess) were reuxed in
ethanol (80 mL) overnight. Aer cooling to room temperature,
the crystals were ltered off, washed with ethanol, and dried in
vacuum. The ltrate was concentrated to 50 mL and water
(60 mL) was added under vigorous stirring. The white crystals
were ltered off and washed with ethanol–H2O (v/v ¼ 1 : 1) and
dried in a vacuum desiccator. Overall yield: 91%. 1H NMR (500
MHz, DMSO-d6): d 9.61 (s, 1H), 7.79 (d, J ¼ 8.5 Hz, 2H), 6.96 (d,
J ¼ 8.5 Hz, 2H), 4.42 (s, 2H), 4.01 (t, 2H), 1.72 (m, 2H), 1.22–1.46
(m, 10H), 0.87 (t, 3H). 13C NMR (500 MHz, DMSO-d6): d 165.76,
161.04, 128.86, 125.48, 114.09, 67.76, 31.42, 28.90, 28.84, 28.76,
25.66, 22.27, 14.14. ESI-MS: m/z 265.01. Calc. for C15H24N2O2
(%): C, 68.15; H, 9.15; 10.60. Found: C, 68.22; H, 9.04; N, 10.48.
Synthesis of 3. 2-Hydroxy-40-iodoacetophenone (10 mmol,
2.62 g) and 2 (10 mmol, 2.64 g) were dissolved in ethanol and
heated to reux for 4 h. Aer cooling to r.t. the powder was
ltered off and washed with ethanol and dried in vacuum. Yield:
4.72 g, 93%. 1H NMR (500 MHz, DMSO-d6): d 13.63 (s, 1H), 11.20
(s, 1H), 7.92 (d, J ¼ 8.5 Hz, 2H), 7.39 (d, J ¼ 8.5 Hz, 1H), 7.29 (s,
1H), 7.25 (d, J ¼ 8.5 Hz, 1H), 7.06 (d, J ¼ 8.5 Hz, 2H), 4.06 (t, 2H),
2.45 (s, 3H), 1.74 (m, 2H), 1.42 (m, 2H), 1.26–1.33 (m, 8H), 0.87
Chart 1 Molecular structure of the monomers used in polymer synthesis.
tert-butyl protecting group was removed by TFA in DCM at room
temperature.13
Synthesis of polymer P2. To a 100 mL ask was added
monomer B (1.02 mmol, 569 mg), monomer C (0.18 mmol,
104 mg), monomer D (0.82 mmol, 241 mg), Bu4NBr (15 mg),
toluene (20 mL) and Na2CO3 (2 M, 10 mL). The mixture was
stirred at room temperature and the ask was degassed and
recharged with N2, which was repeated four times before and
aer addition of Pd(PPh3)4 (0.02 mmol, 23 mg). The reactants
were stirred at 90 ꢀC for 48 h and then phenylboronic acid
(100 mg) dissolved in THF (1 mL) was added. Aer 2 h, bro-
mobenzene (1 mL) was added and further stirred for 3 h. The
mixture was poured into methanol (200 mL). The precipitate
was ltered off, washed with methanol, water and acetone to
remove monomers, small oligomers and inorganic salts. The
crude product was dissolved in DCM (15 mL), ltered through a
0.2 mm membrane and re-precipitated in methanol (150 mL).
The powder was then stirred in acetone (200 mL) overnight and
collected by ltration, and dried in vacuum. Yield: 436 mg
(75%). 1H NMR (500 MHz, CDCl3): d 7.94–8.12 (m, 7.08 H), 7.73
(d, J ¼ 6 Hz, 1.64H), 2.40–2.60 (m, 0.72H), 2.04–2.22 (br, 4H),
1.678 (br, 0.72H), 1.33 (s, 3.24H), 1.45–1.21 (m, 20H), 0.96 (br,
4H), 0.81 (t, J ¼ 4.2 Hz, 6H). GPC: Mn 6000, Mw 13 200, PDI 2.2.
The tert-butyl protecting group was removed by TFA in DCM at
room temperature.13
Synthesis of polymer P3. To a 100 mL ask was added
monomer A (0.7 mmol, 384 mg), monomer B (1.02 mmol,
569 mg), monomer E (0.3 mmol, 134 mg), Bu4NBr (15 mg),
toluene (20 mL) and Na2CO3 (2 M, 10 mL). The mixture was
stirred at room temperature and the ask was degassed and
recharged with N2, which was repeated four times before and
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(t, 3H). 13C NMR (500 MHz, DMSO-d6): d 163.58, 161.60, 159.01, (2 M, 3 mL) and ethanol (0.7 mL). The mixture was stirred at
156.36, 129.98, 129.76, 127.15, 125.59, 124.33, 119.06, 113.92, room temperature and the ask was degassed and recharged
97.12, 67.61, 31.09, 28.58, 28.51, 28.40, 21.93, 13.81, 13.77. ESI- with N2, which was repeated four times before and aer addi-
MS: m/z 508.84. Calc. for C23H29IN2O3 (%): C, 54.34; H, 5.75; N, tion of Pd(PPh3)4 (0.004 mmol, 4.6 mg), respectively. The reac-
5.51. Found: C, 54.08; H, 5.63; N, 5.72.
tants were stirred at 83 ꢀC for 30 h and then phenylboronic acid
Synthesis of 4. Intermediate 4 was synthesized by a previ- (20 mg) dissolved in THF (0.5 mL) was added. Aer two hours,
ously reported method.35 Lead tetraacetate (5.38 g, 12.13 mmol) bromobenzene (0.5 mL) was added and further stirred for 2 h.
was added to a suspension of 3 (5.08 g, 10 mmol) in dry THF The mixture was poured into methanol (100 mL). The precipi-
(150 mL) in small portions over a period of 5 min. Aer stirring tate was ltered off, washed with methanol, water and acetone
at room temperature for 2 h, the resulting solid was removed by to remove monomers, small oligomers and inorganic salts. The
ltration. The ltrate was concentrated by rotary evaporator and crude product was dissolved in DCM (5 mL), ltered through a
puried by silica gel chromatography (hexane–ethyl acetate ¼ 0.2 mm membrane and re-precipitated in methanol (60 mL). The
4 : 1). 1H NMR (500 MHz, CDCl3): d 7.94 (d, J ¼ 8.5 Hz, 1H), 7.74 powder was collected by ltration, and dried in vacuum. Yield:
(s, 1H), 7.71 (d, J ¼ 9.0 Hz, 2H), 7.59 (d, J ¼ 8.5 Hz, 1H), 6.92 (d, 64.8 mg (77%). 1H NMR (300 MHz, CDCl3): d 7.55–7.95 (m,
J ¼ 8.5 Hz, 2H), 4.03 (t, 2H), 2.50 (s, 3H), 1.81 (m, 2H), 1.47 (m, 6.72H), 4.06 (t, J ¼ 6.9 Hz, 0.32H), 2.09 (br, 3.66 H), 1.06–1.40
2H), 1.26–1.39 (m, 8H), 0.91 (t, 3H). 13C NMR (500 MHz, CDCl3): (m, 12H), 0.61–0.95 (m, 18H). GPC: Mn 9200, Mw 26 300, PDI
d 197.76, 194.48, 163.42, 142.66, 138.53, 136.93, 136.71, 131.70, 2.86.
130.50, 129.43, 114.34, 99.48, 68.34, 31.82, 29.32, 29.23, 29.10,
27.53, 26.00, 22.67, 14.15. ESI-MS: m/z 478.24. Calc. for Preparation of Pdots
C
23H27IO3 (%): C, 57.75; H, 5.69. Found: C, 57.88; H, 5.54.
Synthesis of 5. Synthesis of derivatives of 5 have been
The semiconducting polymer dots were prepared by co-precip-
itation method. All polymers were dissolved into anhydrous
THF, respectively, to form a 1 mg mLꢁ1 THF solution, then as
the volume ratio of 50 : 50 : 70 : 8 to mix polymers P1, P2, P3, P4
(for 4-NIR Pdots), and ratio of 100 : 50 : 6.2 to mix P2, P3, P4 (for
3-NIR Pdots) in THF. Then 0.2 mL mixed solution was added
into 1.8 mL anhydrous THF, which was further injected directly
into 10 mL DI water under ultrasonication. THF was removed by
N2 ow at room temperature. The sizes of these two kinds of
particles (4-NIR Pdots and 3-NIR Pdots) were characterized as
15.7 nm by DLS analysis.
reported before.35 In this work, we used a modied method with
much shorter reaction time using low molecular-weight poly-
(ethylene glycol) (PEG) as solvent. Concentrated aqueous
ammonia (NH3 content 28–30%, 9 mL) was added to a solution
of 4 (2 mmol, 0.96 g) in acetic acid (15 mL) and PEG300 (25 mL).
The mixture was stirred at 100 ꢀC for 2 h. The resulting solid was
ltered off and washed thoroughly with ethanol. The crude
product was puried by silica gel chromatography (DCM). Yield:
0.43 g, 47.6%. 1H NMR (500 MHz, CDCl3): d 8.30 (s, 2H), 7.87 (d,
J ¼ 8.5 Hz, 4H), 7.55–7.62 (m, 4H), 7.37 (s, 1H), 7.07 (d, J ¼ 8.5
Hz, 4H), 4.07 (t, 4H), 1.86 (m, 4H), 1.25–1.56 (m, 20H), 0.91 (t,
6H). 13C NMR (500 MHz, CDCl3): d 159.99, 145.28, 134.63,
134.18, 132.64, 131.03, 130.68, 128.75, 125.62, 120.65, 115.19,
110.80, 100.14, 89.31, 68.42, 32.05, 29.64, 29.54, 29.48, 26.31,
22.87, 14.30. ESI-MS: m/z 905.36. Calc. for C45H50I2N2O2 (%): C,
59.74; H, 5.57; N, 3.10. Found: C, 59.57; H, 5.64; N, 3.02.
Synthesis of monomer F. Dry triethylamine (1 mL, 7.2 mmol)
was added to a solution of 5 (0.90 g, 1 mmol) in chloroform
(250 mL), followed by addition of BF3$OEt2 (2.51 mL, 20.3
mmol). Aer the reaction mixture was stirred at 50 ꢀC for 12 h, it
is washed with water. The organic layer is separated, dried over
anhydrous magnesium sulfate, and concentrated by a rotary
evaporator to give a blue solid. The crude product was puried
by silica gel chromatography (hexane–chloroform ¼ 1 : 1) to
give F as a deep blue solid. Yield: 0.7 g, 73.5%. 1H NMR
(500 MHz, CDCl3): d 7.94 (s, 2H), 7.74 (d, J ¼ 8.5 Hz, 4H), 7.64 (s,
1H), 7.61 (d, J ¼ 8.5 Hz, 2H), 7.55 (d, J ¼ 8.5 Hz, 2H), 7.02 (d, J ¼
8.5 Hz, 4H), 4.03 (t, 4H), 1.83 (m, 4H), 1.49 (m, 4H), 1.29–1.41
(m, 16H), 0.91 (t, 6H). 13C NMR (500 MHz, CDCl3): d 160.47,
150.78, 137.00, 132.51, 132.37, 132.18, 131.59, 127.30, 122.33,
120.32, 114.36, 113.91, 89.51, 67.92, 31.70, 29.23, 29.12, 25.95,
22.54, 13.99. ESI-MS: m/z 952.43. Calc. for C45H49BF2I2N2O2 (%):
C, 56.74; H, 5.19; N, 2.94. Found: C, 57.01; H, 4.96; N, 3.05.
Bioconjugation
We performed bioconjugation by utilizing the EDC-catalyzed
reaction between carboxyl groups on Pdots surface and amine
groups on biomolecules. 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 func-
tionalized Pdot solution (50 mg mLꢁ1 in MilliQ water), resulting
in a Pdot solution in 20 mM HEPES buffer with a pH of 7.4.
Then, 240 mL of mg mLꢁ1 streptavidin (purchased from Invi-
trogen (Eugene, OR, USA)) was added to the solution and mixed
well on a vortex. 80 mL of freshly prepared EDC solution (5 mg
mLꢁ1 in MilliQ water) was added to the solution, and the above
mixture was kept under stirring. Aer 3 h at room temperature,
80 mL 10 wt% BSA solution was added, the mixed solution was
stirred for 20 min, then 80 mL 2.5 wt% Triton-X 100 (0.25% (w/v),
20 mL) were added. Finally, the resulting Pdot bioconjugates
were transferred to a centrifugal ultraltration tube (Amiconꢀ
Ultra-4, MWCO: 100 kDa), and concentrated to 0.5 mL by using
the centrifuge, and then were separated from free biomolecules
by gel ltration using Sephacryl HR-300 gel media.
Cell culture
Synthesis of polymer P4. To a 100 mL ask was added The breast cancer cell line MCF-7 was ordered from American
monomer B (0.1 mmol, 55.8 mg), monomer F (0.02 mmol, 19 Type Culture Collection (ATCC, Manassas, VA, USA). Cells were
mg), monomer G (0.08 mmol, 51.4 mg), toluene (4 mL), Na2CO3 cultured at 37 ꢀC, 5% CO2 in Eagles minimum essential
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medium supplemented with 10% Fetal Bovine Serum (FBS),
50 U mLꢁ1 penicillin, and 50 mg mLꢁ1 streptomycin. The cells
were pre-cultured prior to experiments until conuence was
reached. The cells were harvested from the culture ask by
briey rinsing with culture media followed by incubation with
5 mL of trypꢀsin–EDTA solution (0.25 w/v% trypsin, 0.53 mM
EDTA) at 37 C for 5–15 min. Aer complete detachment, the
cells were rinsed, centrifuged and resuspended in 1ꢂ PBS
buffer. The cell concentration was determined by microscopy
using a hemocytometer.
Acknowledgements
We gratefully acknowledge support from the Swedish Founda-
tion for Strategic Research (SSF) within the Nano-X program
(Grant no. SSF [A3 05:204] and Swedish government strategic
faculty grant in material science (SFO, MATLIU) in Advanced
Functional Materials (AFM); and the National Institutes of
Health (CA147831 and GM085485) of USA and the University of
Washington.
Notes and references
Flow cytometry
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