Green-, Red-, and Near-Infrared-Emitting Polymer Dot Probes for Simultaneous Multicolor Cell Imaging with a Single Excitation Wavelength

Article abstract of DOI:10.1021/acs.chemmater.0c02172

We report newly synthesized fluorescence resonance energy transfer (FRET)-based green-, red-, and near-infrared (NIR)-emitting polymer dot (Pdot) probes. Fluorescent Pdots (～60 nm) were prepared with a green-emissive conjugated polymer (PPDT-P, donor) alo

Full text of DOI:10.1021/acs.chemmater.0c02172

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Article
Green-, Red-, and NIR-Emitting Polymer Dot Probes for Simultaneous
Multicolor Cell Imaging with a Single Excitation Wavelength
Ji-Eun Jeong, Mohammad Afsar Uddin, Hwa Sook Ryu, Hee-Chang Kim, Minsu Kang,
Joonyoung Francis Joung, Sungnam Park, Sang-Hee Shim, and Han Young Woo
Chem. Mater., Just Accepted Manuscript • DOI: 10.1021/acs.chemmater.0c02172 • Publication Date (Web): 05 Jul 2020
Downloaded from pubs.acs.org on July 7, 2020
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Green-, Red-, and NIR-Emitting Polymer Dot
Probes for Simultaneous Multicolor Cell Imaging
with a Single Excitation Wavelength
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†
‡
†
†,§
†,§
Ji-Eun Jeong, Mohammad Afsar Uddin, Hwa Sook Ryu, Hee-Chang Kim, Minsu Kang,
†
†
†,§,
†,
Joonyoung Francis Joung, Sungnam Park, Sang-Hee Shim, * and Han Young Woo *
†Department of Chemistry, Korea University, Seoul 136-713, Republic of Korea.
‡
Instituto de Ciencia de Materiales de Madrid, Madrid, 20849, Spain.
§
Center for Molecular Spectroscopy and Dynamics, Institute for Basic Science (IBS), Korea
University, Seoul, 02841, Republic of Korea.
*E-mail: hywoo@korea.ac.kr (H. Y. Woo), sangheeshim@korea.ac.kr (S.-H. Shim)
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Abstract
We report newly synthesized fluorescence resonance energy transfer (FRET)-based green-, red-,
and near-infrared (NIR)-emitting polymer dot (Pdot) probes. Fluorescent Pdots (~60 nm) were
prepared with a green-emissive conjugated polymer (PPDT-P, donor) alone or mixing the donor
with a red or NIR-emitting fluorophore (T-DCS or ITIC, acceptor), where an optically inert
matrix polymer (PSMA) was mixed together to minimize the aggregation-caused quenching by
diluting the fluorophores and surface functionalization for further bioconjugation with
antibodies for active targeting. Highly fluorescent green-emissive PPDT-P Pdots were prepared
with a photoluminescence (PL) quantum efficiency of ~30% and the FRET-mediated red and
NIR PL was intensified by 5.3 to 8.5 times (with high FRET ratios of ~9) via the efficient
energy transfer (FRET efficiency of 80-98%) and antenna effect, compared with the signals
obtained via direct excitation of the fluorophores in Pdots. All three types of Pdot/antibody
conjugates were simultaneously immunostained to COS-7 cells (showing minimal cross-
reactivity), reducing the tedious sequential immunostaining process to a single step. Finally, we
obtained high-contrast three-color cell images with little spectral leakthrough by exciting all
the probes simultaneously at a single wavelength at 405 nm without the need for a complicated
or expensive multiple-excitation setup.
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1
. INTRODUCTION
π-Conjugated semiconducting polymers (CPs) have useful optical and electrical properties
and great potential for utilization in optoelectronic devices, chemo-/biosensors, and biological
imaging.1-4 In particular, the intrinsic light harvesting and molecular wire effect of CPs, as well
as the large absorption cross sections, render them applicable as optical platforms in fluorescent
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chemo- and bioassays with high sensitivity. To utilize hydrophobic CPs with poor water-
solubility as optical probes in aqueous biological specimens, CPs are often coprecipitated with
an amphiphilic matrix to produce polymer dots (Pdots).6-9 Pdots have many advantages, such
as easy preparation, high brightness, large absorption, surface functionalization, excellent
photostability, good water stability, and biocompatibility; therefore, they have been studied as
fluorescent probes with molecular specificity to investigate biomolecular interactions and to
visualize subcellular structures and processes.10-14
Applications based on multicolor fluorescence imaging have exhibited a remarkable growth
in basic science and clinical diagnostics.15, 16 To compose a multicolor image, different
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biomolecular species are labeled with different fluorophores ; this renders it necessary to
employ different excitation sources for each fluorophore, which in turn increases the instrument
cost and measurement complexity. Rong et. al. synthesized a series of BODIPY-containing
copolymers by coupling the donor (fluorene, benzothiadiazole, or 4,7-bisthienyl-2,1,3-
benzothiadiazole) and acceptor (BODIPY derivatives) moieties to modulate the intramolecular
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energy transfer for multicolor fluorescence imaging. In this report, green-emissive 520 Pdots
were excited at 405 nm, while red-emissive 600 Pdots and deep red-emissive 690 Pdots were
excited at 488 nm for multicolor imaging. Because 520 Pdots and 600/690 Pdots have the
complementary absorptions in a range of 300-500 nm, these Pdots cannot be excited at a same
wavelength. Spectrally similar fluorophores can be excited with a single excitation source and
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distinguished using spectral detection and linear unmixing. However, the detection system is
expensive, and the resultant image can suffer from a spectral leakthrough. Therefore, it is
desired to have a set of fluorophores with similar absorption spectra and distinguishable
emission spectra to obtain crosstalk-free and high-contrast multicolor imaging with a single
excitation source and standard filter sets. Few researchers have attempted multicolor imaging
with single wavelength excitation. Xing and Wu reported water-soluble carbon dots, the size of
which was controlled by modulating cutting temperatures, showing excitation-independent
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emission. They realized multicolor emission in the range of blue to yellow, upon excitation at
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60 nm. Liu et al. tuned the emission color of aggregation-induced emission (AIE) molecules
based on tetraphenylethylene (TPE) by controlling the degrees of intramolecular charge transfer
through the introduction of different electron-donating and electron-accepting moieties to
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TPE. Two types of AIE-based structures exhibited a large difference in the emission spectra
but a small difference in the absorption spectra. Despite their successful emission at different
wavelengths upon a single irradiation, challenges such as the unavoidable spectral overlap, lack
of surface modification, and limited number of targeting organelles prohibit the structures from
becoming practical multicolor probes.
Fluorescence resonance energy transfer (FRET) can be utilized for multicolor imaging
because a single donor excitation can result in fluorescence emission of various acceptor dyes.
FRET involves non-radiative energy transfer between donor and acceptor molecules via dipole–
dipole coupling and has been widely used for fluorescent and ratiometric sensing.20-22 Because
the FRET efficiency is inversely proportional to the donor–acceptor intermolecular distance
raised to the sixth power, Pdots that encage the fluorophores within the nano-sized particles can
be an appropriate platform to locate donor and acceptor dyes in close proximity. Furthermore,
the photoluminescence (PL) of red- and near-infrared (NIR)-emitting probes can be enhanced
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via the antenna effect of CPs, which can be advantageous for biological imaging owing to the
deep tissue penetration and negligible autofluorescence.
In this study, green, red, NIR-emissive three types of FRET-based Pdot probes were prepared
with different target specificity for multicolor biological imaging (Scheme 1). We successfully
demonstrated a multicolor fluorescence imaging with semiconducting Pdots by single
wavelength excitation, which is the first report to the best of our knowledge. By precipitating
the green-emitting conjugated polymer, poly[(2,5-bis(2-hexyldecanoate)phenylene)-alt-(5,5'-
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(
1,4-phenylene)-bis(thiophen-2-yl))] (PPDT-P) alone or by co-precipitating PPDT-P (as a
FRET donor) with the red-emitting fluorophore, (2E,2'E)-3,3'-(2,5-bis((2-hexyldecyl)oxy)-1,4-
phenylene)-bis(2-([2,2'-bithiophen]-5-yl)acrylonitrile) (T-DCS) or the NIR-emitting
fluorophore, 3,9-bis(2-methylene-(3-(1,1-dicyanomethylene)-indanone))-5,5,11,11-tetrakis(4-
hexylphenyl)-dithieno[2,3-d:2’,3’-d’]-s-indaceno[1,2-b:5,6-b’]dithiophene (ITIC) (as a FRET
acceptor), we prepared green-, red-, and NIR-emitting Pdots. A FRET donor (PPDT-P) showed
a suppressed aggregation-caused quenching (ACQ) in Pdots, showing a PL quantum yield
(
PLQY) of ~30% even in a condensed solid state. Because all the Pdots had PPDT-P as a main
component, the three Pdots had maximum absorption at the same wavelength (determined by
PPDT-P) and could be excited using a single excitation wavelength. The FRET-mediated red
and NIR emissions were significantly enhanced (by factors of 5.3 and 8.5, respectively)
compared with the signals obtained via direct excitation of red and NIR fluorophore in Pdots.
A remarkably high FRET efficiency (80-98%) in red- and NIR-emitting Pdots and the FRET-
mediated emissions with high color purity were obtained. The surface of each Pdot was
functionalized with three types of secondary antibodies for specific targeting into different
subcellular regions. By co-staining COS-7 cells with all three Pdot probes simultaneously,
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multi-color images of tubulin, vimentin, and mitochondria were successfully obtained by
exciting all the probes at a single wavelength.
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Scheme 1. Schematic for multicolor subcellular imaging with single wavelength excitation.
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2
. RESULTS AND DISCUSSION
.1. Material Synthesis and Characterization
Br
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Scheme 2. Synthesis routes for PPDT-P and T-DCS. i) 2-(tributylstannyl)thiophene, Pd(PPh
THF; ii) n-BuLi, Me SnCl; iii) SOCl , pyridine, C16 33OH, DCM; iv) Pd (dba) , tris(o-
tolyl)phosphine, toluene; v) THF, n-BuLi, DMF, –78 ℃; vi) 2-(5-bromothiophen-2-
3
)
4
,
3
2
H
2
3
yl)acetonitrile, t-BuOK, t-BuOH, 50 ℃.; vii) 2-(tributylstannyl)thiophene, Pd
2
(dba) , tris(o-
3
tolyl)phosphine, toluene.
A green-emissive conjugated polymer PPDT-P and a red-emissive fluorophore T-DCS were
newly synthesized, as shown in Scheme 2. A NIR-emissive conjugated molecule ITIC was
purchased from Solarmer (China). The synthesis procedures are described in the Supporting
23
Information. In accordance with a previously reported procedure, 1,4-dibromobenzene (1)
was reacted with 2-(tributylstannyl)thiophene to produce 1,4-bis(thiophen-2-yl)benzene (2)
with a 79% yield, and subsequent stannylation yielded 1,4-bis(5-(trimethylstannyl)thiophen-2-
yl)benzene (M1) with a 75% yield. 1,4-Dibromo-2,5-bis(2-hexyldecanoate)benzene (M2) was
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prepared via the esterification reaction of 2,5-dibromoterephthalic acid (3) and 2-hexyl-1-
decanol together with a catalytic amount of pyridine in dichloromethane (DCM). The green-
emissive polymer PPDT-P was synthesized via Stille coupling of M1 and M2 in toluene with
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tris(dibenzylideneacetone)dipalladium (0) (Pd
2
(dba)
3
) and tri(o-tolyl)phosphine as a catalytic
= 22,600 Da). After formylation
system (78% yield, number-average molecular weight of M
n
of 1,4-dibromo-2,5-bis(2-decyltetradecyloxy)benzene (4), compound 5 was reacted with 2-(5-
bromothiophen-2-yl)acetonitrile in the presence of t-BuOK in t-BuOH to obtain (2E,2'E)-3,3'-
2,5-bis((2-hexyldecyl)oxy)-1,4-phenylene)-bis(2-(5-bromothiophen-2-yl)acrylonitrile) (6).24
(
The red-emissive T-DCS was prepared with a 72% yield via Stille coupling between compound
6 and 2-(tributylstannyl)thiophene in toluene.
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.2. Preparation and optimization of FRET-based multicolor Pdots
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T-DCS (red)
ITIC (near Infrared)
Donkey anti-Rat
OEG-NH2
COMU/NMM
H2O, RT
Donkey anti-Mouse
OEG-NH2
COMU/NMM
H2O, RT
Donkey anti-Rabbit
OEG-NH2
COMU/NMM
H2O, RT
Figure 1. (a) Chemical structures of FRET donor (PPDT-P), acceptor1 (T-DCS), and acceptor2
ITIC). (b) Absorption (solid) and emission (dashed) spectra of PPDT-P, T-DCS, and ITIC in
(
THF. (c) Schematic of preparation of green-, red-, and NIR-emitting Pdots and surface
modification with different antibodies.
To prepare the FRET-based multicolor-emissive Pdots, the FRET donor (PPDT-P) and
acceptors (T-DCS or ITIC) were designed and selected by considering their bandgap and
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spectral overlap between the donor emission and acceptor absorption (Figure 1). In
tetrahydrofuran (THF), the maximum absorption and PL wavelengths of PPDT-P were
observed at λabs = ~410 nm and λPL = ~510 nm, respectively, with a PLQY of ~43%, relative to
a freshly prepared fluorescein solution in water at pH=11. As shown in Figure 1b, the absorption
spectra of the red-emitting T-DCS and NIR-emitting ITIC had peaks at λabs = 495 nm and 655
nm, respectively, exhibiting significant spectral overlap with the emission of PPDT-P. The
maximum PL emission in THF was measured at λPL = 575 nm (PLQY = ~17%) for T-DCS and
at λPL = 732 nm (PLQY = ~3%) for ITIC, allowing the facile tuning of the PL emission ranges
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To examine the optical properties of the three fluorophores in a condensed aggregated phase,
Pdots were first prepared with a single fluorophore. PPDT-P, T-DCS, or ITIC was dissolved in
THF together with poly(styrene-co-maleic anhydride) (PSMA, M ~1600 Da, ~1.3:1 mole ratio
n
of styrene:maleic anhydride) as a matrix, followed by a quick transfer to an aqueous solution
under vigorous sonication, precipitating nano-sized Pdots.25, 26 The PSMA matrix played
several important roles to control the local environment around the fluorophores and particle
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surface. i) By encaging both the FRET donor and acceptor inside the PSMA matrix, they were
kept in close proximity to induce efficient energy transfer. ii) PSMA prevented excessive self-
aggregation of emitting species by diluting the photoactive fluorophore concentration in the
photo-inactive matrix, reducing the ACQ. iii) In the formation of Pdots, the maleic anhydride
units in the PSMA were hydrolyzed to carboxylic acid groups, leading to further surface
functionalization for molecular-specific targeting. To identify the optimum concentration of
PSMA within the Pdots, the precursor solutions of PPDT-P/PSMA, T-DCS/PSMA, and
ITIC/PSMA in THF with various mixing ratios ([PSMA] = 0.02–0.09 mg/mL, [precursor] =
[PSMA] + [PPDT-P, T-DCS, or ITIC] = 0.1 mg/mL) were precipitated into water, providing a
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series of Pdots with different [PSMA]:[fluorophore] ratios. With the increasing [PSMA], the
PLQY of the resulting Pdots increased, indicating the need for excess PSMA to minimize the
self-quenching effect. Finally, the optimum [PSMA] for maximum PL emission was
determined to be 0.08 mg/mL.
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Figure 2. (a, c, e) UV–vis and (b, d, f) PL spectra of PPDT-P/PSMA (a, b), PPDT-P/T-
DCS/PSMA (c, d), and PPDT-P/ITIC/PSMA (e, f) Pdots in an aqueous solution with different
D:A molar ratios. The PL spectra were obtained by exciting the donor polymer (PPDT-P) at
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Compared with the PPDT-P solution in THF, slightly red-shifted absorption and PL maxima
for the PPDT-P Pdots were observed (at 420 and 515 nm, respectively), indicating
agglomerated PPDT-P molecules (Figure 2a and b). In the aggregated phase, T-DCS and ITIC
also exhibited red-shifted PL spectra, with λPL = 695 and 778 nm, respectively (Figure S1).
Intermolecular aggregation of conjugated polymer chains within a Pdot matrix often results in
a significant PL quenching, which should be carefully considered in the design of Pdot-based
fluorescent probes. Interestingly, the PLQY of ~43% was measured for PPDT-P in solution
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THF) and the PPDT-P aggregates in Pdots still maintained a high PLQY of ~30% in a
condensed solid state, indicating that the ACQ is not significant. As shown in Table S1, the
PLQY of PPDT-P/PSMA Pdots was significantly higher or comparable relative to those of
other reported Pdots by careful polymer design to suppress the ACQ. To prevent a serious
aggregation of polymers, we introduced a tilted polymer backbone consisting of repetitive
phenylene and thiophene units as well as branched side-chains.28-30 Because torsional angles
along the main chain have a great effect on the intermolecular packing and PL quenching, the
polymer chain conformation was modulated to optimize the π-conjugation and exciton
migration with minimized ACQ. The incorporation of the phenylene moieties in a polymer
backbone breaks the chain planarity of polythiophenes and avoids a serious intermolecular
packing.31, 32 The energy-minimum conformational analysis of PPDT-P was performed by
density functional theory (DFT) calculation (basis set: B3LYP/6-31g(d,p)) (Figure S2). By
introducing the phenylene units in a polymer backbone, torsional angles between thiophene and
o
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backbone. In addition, the steric hindrance by incorporating 2-hexyldecanoate side-chains onto
a phenylene ring further increases a torsional angle (Φ , Φ , and Φ = ~51°). Thus, PPDT-P
which forms a randomly aggregated structure (amorphous) with suppressed ACQ is a promising
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candidate as an efficient FRET donor to improve the energy transfer even in the highly
aggregated state.
Then, a multicolor set of green-, red-, and NIR-emitting Pdots was prepared by precipitating
the green-emissive PPDT-P (FRET donor) alone or by co-precipitating the mixture of PPDT-P
and red or NIR-emitting fluorophores (FRET acceptor) together with PSMA as a matrix. Ideal
FRET-based multicolor fluorescent probes require bright FRET-induced PL for a high signal-
to-noise ratio as well as a high FRET ratio (Iacceptor/Idonor, where Iacceptor represents the PL
intensity at the λPL of the acceptor and Idonor represents the PL intensity at the λPL of the donor)
for color purity. To satisfy the requirements, the FRET system in the red- and NIR-emitting
Pdots was optimized by controlling the mixing ratio of the FRET donor and acceptor. Donor
and acceptor stock solutions (1 mg/mL) in THF were mixed at various molar ratios of [donor]/
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[acceptor]: 0–4.6 and 0–6.7 for T-DCS- and ITIC-based FRET Pdots, respectively. As a Pdot
matrix, PSMA (0.08 mg/mL) was also introduced. Finally, the precursor solutions with
different mixing ratios (total concentration of 0.1 mg/mL) were quickly transferred to an
aqueous solution under vigorous sonication. As the donor-to-acceptor molar ratio within the
Pdots increased, the absorption of the FRET donor (PPDT-P) at ~420 nm increased, and the
acceptor absorption at ~520 nm for T-DCS or ~620 nm for ITIC decreased gradually (Figure
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Figure 3. Normalized (a) UV–vis and (b) PL spectra of PPDT-P/PSMA, PPDT-P/T-
DCS/PSMA and PPDT-P/ITIC/PSMA Pdots in aqueous solution. PL spectra were obtained by
exciting at 420 nm. Inset in (a) shows the photograph of green (left), red (middle) and NIR
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right)-emissive Pdots under illumination with a handheld UV lamp at 365 nm.
In the presence of PPDT-P, the energy transfer within the PPDT-P/T-DCS/PSMA and PPDT-
P/ITIC/PSMA Pdots was evident from the loss of donor emission and the significant increase
in the acceptor emission upon the excitation of PPDT-P at 420 nm (Figure 2d and f). With
increasing of [PPDT-P], the FRET-induced acceptor PL enhanced via the efficient energy
transfer with gradually blue-shifted emissions, indicating the reduced self-aggregation of the
acceptors. Additionally, the FRET ratio was improved by modulating the donor-to-acceptor
ratio. Finally, the optimum D:A molar ratio that yielded the highest FRET-induced PL intensity
as well as the largest FRET ratio was determined to be 4.6:1 for PPDT-P/T-DCS and 6.7:1 for
PPDT-P/ITIC (Figure 3). In Figure S3, the strong absorption (absorbance ~0.27) at ~420 nm
originates from PPDT-P and the small peaks at ~520 and ~620 nm (absorbance ~0.03 and ~0.02)
are assigned to absorptions from T-DCS and ITIC, respectively. Due to the presence of excess
PPDT-P in red- and NIR-Pdots, the strong light absorption by PPDT-P and following FRET to
the acceptors (T-DCS and ITIC) can occur to yield the intensified FRET-mediated PL by
exciting donor polymer (PPDT-P) at 405 nm, which is known as signal amplification via the
antenna effect.5, 33, 34 The FRET-mediated red and NIR emissions were significantly enhanced
(
by factors of 5.3 and 8.5, respectively) compared with the signals obtained via direct excitation
of red and NIR fluorophores in Pdots at 520 and 620 nm, respectively. With the optimum
blending ratio, high FRET ratios of ~9 were measured for both the PPDT-P/T-DCS/PSMA and
PPDT-P/ITIC/PSMA Pdots. By further increasing the D:A mixing ratio, the FRET-induced PL
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emission was saturated or reduced with a concomitant increase in the PPDT-P emission,
decreasing the FRET ratio (Figure S4).
To determine the quenching efficiency of PPDT-P by each acceptor (T-DCS or ITIC), the
Stern−Volmer plots (Figure S5) were prepared by measuring PL quenching with varying the
quencher concentration.35-37 The obtained results show that each single T-DCS and ITIC
molecule can quench ~56 and ~79 polymer repeating units (RU), respectively, indicating highly
efficient energy transfer from PPDT-P to acceptors. It is worthy to mention that efficient exciton
migration along the conjugated polymer backbone is an important factor to yield the large Ksv
values. Similar results have been reported by Zhou et. al. to show ~60 times greater Ksv for
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conjugated polymers compared to that of small molecular fluorophores. Additionally, the
FRET efficiencies in the Pdots were estimated by measuring the PL decays of the donor (PPDT-
P) in the presence and absence of the acceptors (T-DCS and ITIC) via the time-correlated single
photon counting (TCSPC) method as shown in Figure S6. The amplitude-weighted average PL
lifetime of PPDT-P without acceptors was determined to be 0.547 ns. In the presence of
acceptors, the PL decays of PPDT-P were fitted using the modified FRET model (for the FRET
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system with randomly dispersed donors and acceptors) reported by Fung et. al. The FRET
efficiencies of PPDT-P/T-DCS/PSMA (D:A = 4.6:1) and PPDT-P/ITIC/PSMA (D:A = 6.7:1)
Pdots in aqueous solution were determined to be 80% and 98%, respectively. The high FRET
efficiencies show a good agreement with the measured Ksv, indicating efficient energy transfer
from PPDT-P to the acceptors.
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Figure 4. AFM images of (a) PPDT-P/PSMA, (b) PPDT-P/T-DCS/PSMA, and (c) PPDT-
P/ITIC/PSMA Pdots.
The size and shape of nanoparticles may play an important role in cellular uptake. To
determine the hydrodynamic diameter, dynamic light scattering (DLS) measurements of Pdots
in an aqueous solution were performed using a 633 nm laser source (Figure S7). The average
diameter of the green-, red-, and NIR-emissive Pdots was ~60 nm according to the constrained
39
regularization method for inverting data (CONTIN) analysis. As shown in Figure S7d, all
Pdots show negative zeta potentials, ranging from -37 to -52 mV due to the presence of
negatively charged carboxylate groups on the Pdot surfaces.40, 41 No aggregation or significant
changes in the optical properties were observed for the three types of Pdots even after several
months, indicating the pronounced stability of the dispersed Pdots in aqueous media. Similar
results have been reported to show that colloids with a zeta potential over (+/−) 30 mV have a
42
long-term colloidal stability. The morphology of the Pdots was also characterized using
atomic force microscopy (AFM) (Figure 4). The AFM samples were prepared by dropping ~20
μL of a diluted Pdot solution (stock solution of Pdots (~2.5 μg/mL) was diluted by a factor of
2
0) on a mica substrate. All the Pdots exhibited a spherical shape with an average diameter of
60 nm, which is consistent with the DLS measurements. There were no significant differences
in the particle size or shape for the Pdots with different compositions. Additionally, no
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aggregation or significant changes in the optical properties for the three types of Pdots were
observed, even after several months, indicating the stability of the dispersed Pdots in the
aqueous media. In a previous study, nanoparticles with a size of 20–100 nm were reported to
easily penetrate the plasma membrane of cells to localize in specific organelles with the aid of
a permeabilizing reagent.43-46 The size of the prepared Pdots is expected to be suitable for
further immunostaining.
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The photostability of PPDT-P/PSMA Pdot probes (50 μg/mL) in water was measured under
continuous illumination of an excitation laser light (405 nm, 100 mW) (Figure S8). For
comparison, a fluorescein solution (50 μM) was also prepared in water (pH 11) as a control.
The fluorescence intensity of PPDT-P/PSMA Pdots remained ~90% of their initial intensity
under continuous irradiation for 300 s, while fluorescein showed a severe intensity decrease
within 60 s, suggesting high photostability of the Pdots. The excellent photostability of Pdots
compared to single molecule fluorophores can be attributed to the larger number density of
emitting units inside Pdots. In addition, encapsulation of luminescent fluorophores with a Pdot
matrix also prevents the contact of oxygen (or air), reducing the photooxidation (and/or
photobleaching) of fluorophores.47, 48 We also studied the optical properties of PPDT-P/PSMA
Pdots in phosphate buffered saline (PBS, pH 7.4) solutions with changing bovine serum
albumin (BSA) concentrations (0-3 wt%). As shown in Figure S9, similar absorption and PL
spectra of Pdots were measured in BSA solutions compared to those in water, suggesting their
stability in physiological environments. It is expected that the encapsulation with hydrophilic
PSMA minimizes the direct contact of hydrophobic fluorophores with aqueous buffering media,
prohibiting fluorophores from serious aggregation. Additionally, the emission of PPDT-
P/PSMA Pdots in BSA solution was somewhat increased (by ~10%) with compared to that in
water. Because the isoelectric point of BSA is 4.8 and it must be negatively charged in PBS.
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The surface of Pdots possesses large amounts of carboxylate groups, and the electrostatic
repulsion between BSA and Pdots further stabilizes the Pdot dispersion. The optical properties
of Pdots remained unchanged in BSA buffered solutions even after 1 week. To evaluate the
cytotoxicity of the Pdots, COS-7 cells were incubated with different concentrations of Pdots
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was monitored by optical microscopy. As shown in Figure S10, no obvious morphological
change of COS-7 cells was observed in the presence of 10 μg/mL of PPDT-P/PSMA Pdots after
incubating for 48 h. Although all the experiments in this study were proceeded with fixed cells,
sufficient biocompatibility of the Pdots for live cell imaging was also confirmed.41, 49, 50 Similar
results were also observed in PPDT-P/T-DCS/PSMA and PPDT-P/ITIC/PSMA Pdots.
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.3. Antibody labeling of Pdot surface via COMU coupling
PPDT-P/PSMA Pdot
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b Antibody
PPDT-P/PSMA/antibody conjugates (reaction mixture)
c
d
Elution time
Figure 5. Photographs of separated drops of (a) PPDT-P/PSMA Pdots, (b) secondary antibodies
Donkey anti-Rat), and (c) PPDT-P/PSMA/antibody conjugates depending on the eluting time.
(
Non-fluorescent antibodies were visualized by reacting with Alexa Fluor 568-NHS ester.
Irradiation was applied using a handheld UV lamp (365 nm). (d) PL Intensity ratio of PPDT-
P/PSMA/antibody conjugates depending on the number of post-washing by centrifugal
purification (I/I
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12 nm for PPDT-P/PSMA/antibody conjugates and 602 nm for excess free antibodies,
respectively). The data were obtained from three independent measurements, and the error bars
indicate the standard deviation.
During the formation of Pdots, the hydrophobic polystyrene units of the PSMA point toward
the inside of the particles, while the hydrophilic maleic anhydride units are exposed on the
surfaces. By heating the aqueous solution, the maleic anhydride moieties are hydrolyzed to
carboxyl acids, allowing bio-conjugation with antibodies through the formation of amide bonds.
To demonstrate specific cellular targeting of three Pdots, three types of amine-functionalized
secondary antibodies—Donkey anti-Rat, Donkey anti-Mouse, and Donkey anti-Rabbit—were
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coupled with carboxylic acid functional groups on the surface of PPDT-P/PSMA, PPDT-P/T-
DCS/PSMA, and PPDT- P/ITIC/PSMA Pdots, respectively (Figure 1c). As a coupling reagent,
oxyma-derived uronium salt (1-cyano-2-ethoxy-2- oxoethylidenaminooxy)dimethylamino-
morpholino-carbenium hexafluorophosphate, COMU), which improves a coupling efficiency
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in aqueous media, was introduced to catalyze the formation of amide bonds. First, PPDT-
P/PSMA,PPDT-P/T-DCS/PSMA, and PPDT-P/ITIC/PSMA Pdots were concentrated via
centrifugal filtration (100 μL, 0.35 mg/mL). Then, each concentrated Pdot solution was mixed
6
with polyethylene glycol (5% PEG in deionized water, 4000 Da), freshly prepared COMU
(
100 μL, 5.4 mM), N-methylmorpholine (NMM, 6 μL, 0.2 M) and the amine-containing
secondary antibody to conjugate (30 μL, 1.3 mg/mL) in deionized water. The reaction mixture
was stirred overnight at room temperature. After antibody conjugation, the remaining
carboxylic acid groups were subsequently blocked with amine-containing oligoethylene glycol
(
OEG) chains to prevent the non-specific adsorption of Pdots during immunostaining (Figure
S11). The purification of Pdot/antibody conjugates was optimized via a two-step procedure
involving size-exclusion chromatography (SEC, Nap-5 columns packed with Sephadex G-25
DNA grade resin) and centrifugal filtration.52-54 As shown in Figure 5a and c, the PPDT-
P/PSMA/antibody conjugate exhibited slightly faster elution than the Pdot itself, owing to the
increased size with antibody conjugation. Because the antibodies were colorless and non-
fluorescent, they were reacted with commercially available Alexa Fluor 568 containing a N-
hydroxysuccinimide (NHS) ester group, allowing their elution to be monitored. Because most
of immunoglobulin G (IgG) antibodies show similar molecular weight (~150 kDa), Donkey
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anti-Rat secondary antibodies was chosen and tested for SEC purification. According to the
elution tests of unreacted secondary antibodies (Figure 5b), free antibodies appeared from 7th
th
th
tube, which overlaps with the elution range of the Pdot/antibody conjugates (4 –9 tubes),
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requiring a further purification to remove the excess free antibodies after coupling reaction.
After SEC, the collected mixtures of the Pdot/antibody conjugates and free excess antibodies
were separated via centrifugal filtration (molecular weight cutoff, 300K). The residue was re-
dispersed into phosphate-buffered saline (PBS), and centrifugal filtration was repeated three
times to ensure that no free antibodies remained (Figure 5d). After antibody conjugation, a
broad absorption at ~280 nm appeared in the UV–vis absorption spectra, indicating the
successful coupling of antibodies on the surface of the Pdots (Figure S12).
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.4. Single staining of Pdot/antibody conjugates
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PPDT-P/PSMA
PPDT-P/T-DCS/PSMA
PPDT-P/ITIC/PSMA
Figure 6. Confocal fluorescence images of mitochondria in COS-7 cells with three different
emission colors. Fluorescence signals from (a) PPDT-P/PSMA, (b) PPDT-P/T-DCS/PSMA,
and (c) PPDT-P/ITIC/PSMA Pdots with 525/50 nm, 600/50 nm, and 700/75 nm emission filters,
respectively. All images were obtained by excitation with a 405 nm laser. The scale bars
represent 20 µm.
Prior to the simultaneous immunostaining of all three Pdot/antibody conjugates, an
independent staining test of each Pdot/antibody conjugate was performed to verify the specific
binding to target organelles and spectral leakthroughs. By combining the green-, red-, and NIR-
emitting Pdots with Donkey anti-Rabbit, we prepared three Pdot/antibody conjugates specific
to mitochondria. Mitochondria labeling in COS-7 cells was performed with sequential
incubation of the Rabbit anti-Tom20 primary antibody and three types of Pdot/anti-Rabbit
conjugates. As shown in Figure 6, typical mitochondria images in three different emission
colors were obtained, indicating the specific targeting of Pdot/antibody conjugates via the
immunostaining process. Target specificity of Pdot/antibody conjugates was further
investigated via colocalization analysis of the Pdot conjugates with a commercial mitochondria-
targeting dye (Figure S13). The resultant two-color images of MitoTracker Deep Red and
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PPDT-P/PSMA Pdots/Donkey anti-Rabbit IgG show colocalization of the two different probes
on mitochondria. In contrast, unconjugated Pdots without antibodies yielded nonspecific
staining of COS-7 cells, suggesting that the Pdots did not perturb the antibody specificity
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(
Figure S14). All the FRET-based Pdots were suitable for excitation with the same wavelength
because of their identical absorption at λabs = ~420 nm, which well matched a commercially
available 405 nm laser source. Importantly, the FRET-based Pdots as a multicolor imaging
platform with a single excitation source can be extended to cover more target structures by
modifying the target-specific primary antibodies and paired secondary antibodies conjugated
to the Pdot surface.
Before simultaneous immunostaining of COS-7 cells, the green-emissive PPDT-
P/PSMA/anti-Rat, red-emissive PPDT-P/T-DCS/PSMA/anti-Mouse, and NIR-emissive PPDT-
P/ITIC/PSMA/anti-Rabbit conjugates were first tested for specific targeting of tubulin,
vimentin, and mitochondria, separately. While the excitation laser was fixed at 405 nm, the
emissions of the three Pdots were distinguished using three emission bandpass filters. The green
fluorescence image of tubulin was detected with a 525/50 nm emission filter, and no
recognizable images were obtained with the 600/50 and 700/75 nm filters (Figure S15a–c). The
vimentin and mitochondria images via the FRET-mediated red and NIR emissions were
acquired with a 600/50 and 700/75 nm filter set, respectively and exhibited little spectral
leakthrough. The residual fluorescence of the donor fluorophore was observed in the 525/50
nm emission channel, but it was 7-8% of the signal intensity in the acceptor channels for T-
DCS and ITIC (Figure S15d and g). The color purities of the green-, red-, and NIR-emitting
Pdots allowed fluorescence signals to be obtained only with the corresponding emission filter,
enabling efficient separation of images of different targets with standard filter sets for
constructing multicolor images.
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In addition, the FRET-induced and directly excited confocal fluorescence images were
compared by changing the excitation wavelengths (Figure S16). For cell images acquired by
direct excitation of acceptors, the 561 nm and 637 nm lasers were utilized to excite T-DCS and
ITIC moieties, respectively. In COS-7 cells stained with PPDT-P/T-DCS/PSMA/Donkey anti-
Mouse IgG (vimentin) or PPDT-P/ITIC/PSMA/Donkey anti-Rabbit IgG (mitochondria), red-
emissive vimentin or NIR-emissive mitochondria confocal microscopy images were obtained
by exciting PPDT-P (at 405 nm) or acceptors (T-DCS and ITIC at 561 and 637 nm,
respectively). As shown in Figure S16, the FRET-induced confocal images are much brighter
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advantages of FRET-based multicolor imaging.
2
.5. Simultaneous multicolor imaging with a single wavelength excitation
a
b
c
d
Figure 7. Three-color confocal microscopy images of COS-7 cells co-labeled with all three
Pdot/antibody conjugates by switching the emission filters: (a) tubulin, (b) vimentin, and (c)
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mitochondria. (d) Overlaid images of panels (a)–(c). All images were collected by excitation
with a 405 nm laser. The scale bars are 20 μm.
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Encouraged by the confocal images by separate staining of each type of Pdot, three
Pdot/antibody probes specific for three types of targets were immunostained simultaneously in
COS-7 cells (Figure 7). First, the mixture of primary antibodies of Rat anti-tubulin, Mouse
anti-vimentin, and Rabbit anti-Tom20 (mitochondria) were incubated with COS-7 cells,
allowing secondary antibody-coupled Pdots to target the specific subcellular organelles. All
three types of Pdot/antibody conjugates were simultaneously incubated together because all the
secondary antibodies labeled on the Pdot surfaces were chosen to have minimal cross-reactivity,
except for their target antigen, and came from the same host species (Donkey). It was possible
to achieve highly selective binding of the Pdot/antibody conjugates to the corresponding target
even when different Pdot/antibody conjugates were mixed together without the need for tedious
sequential immunostaining. As shown in Figure 7, multicolor images of the three subcellular
structures from the Pdot/antibody conjugates were successfully obtained by excitation with a
single laser (405 nm) and simply switching the emission filters for green-emissive tubulin, red
fluorescing vimentin and NIR-emitting mitochondria.
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. CONCLUSION
We prepared FRET-based green-, red-, and NIR-emitting Pdots and employed them for
multicolor cell imaging with a single excitation wavelength. The green-emissive conjugated
polymer (PPDT-P) was precipitated alone or co-precipitated with a FRET acceptor (T-DCS or
ITIC), facilitating FRET-mediated red and NIR emission. The FRET donor-acceptor pairs were
carefully optimized to show the remarkably high FRET efficiency (80-98%) and FRET-
mediated emissions with high color purity. The FRET-mediated red and NIR signals were
amplified (via the antenna effect) by a factor of 5.3–8.5 relative to those obtained via the direct
excitation of acceptors, emphasizing the advantages of FRET-based imaging. The covalent
immobilization of target-specific antibodies onto the surface of Pdots yielded active targeting
Pdot/antibody probes. Three Pdot/antibody probes specific for subcellular organelles (tubulin,
vimentin, and mitochondria) were simultaneously co-stained in COS-7 cells, reducing the
tedious sequential immunostaining process to a single step. Highly selective binding of the
Pdot/antibody conjugates to the corresponding targets was observed even when different
Pdot/antibody probes were mixed together. Thus, high-contrast three-colored cell images were
obtained with a single excitation wavelength, by simply changing the emission filter. Our new
strategy for simultaneous multicolor cellular imaging based on FRET-induced emission with a
single excitation is a cost-effective and time-saving multicolor imaging technique for cell
biological investigations. By simply changing the labeling antibodies, it is also possible to
further extend the target molecules for multicolor imaging of other cellular structures.
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4
. EXPERIMENTAL SECTION
.1. Chemicals
4
FRET acceptor, ITIC was purchased from Solarmer (Beijing, China). The amphiphilic
functional polymer PSMA (average M ~1600 Da, ~1.3:1 mole ratio of styrene:maleic
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n
anhydride), COMU, and NMM were purchased from Sigma–Aldrich (St. Louis, MO, USA).
PEG (MW = 4000 Da) was purchased from Tokyo Chemical Industry (TCI) Co., Ltd. (Tokyo,
Japan). The primary antibodies, Rat anti-Tubulin (ab6160) and Rabbit anti-beta Tubulin
(
ab6046) were purchased from Abcam (Cambridge, UK). Rabbit anti-Tom20 (sc-11415) and
Mouse anti-Vimentin (M0725) were purchased from Santa Cruz Biotechnology (Texas, USA),
and Dako (Glostrup, Denmark), respectively. For surface modification for targeting, Donkey
anti-Rat (712-005-153), Donkey anti-Mouse (715-005-151), and Donkey anti-Rabbit (711-005-
1
52) secondary antibodies were purchased from Jackson Immuno Research Laboratories, Inc.
(
(
Pennsylvania, USA). MitoTracker Deep Red was purchased from Thermo Fisher Scientific
Massachusetts, USA). All chemicals and biomolecules were used as received, unless otherwise
specified. NAP-5 gel columns were purchased from GE Healthcare (Chicago, USA).
4.2. Synthesis of PPDT-P and T-DCS
Synthesis of Intermediates
M1 and compound 6 were prepared via previously reported procedures.23, 24 To prepare M2,
a mixture of 2,5-dibromoterephthalic acid (2.5 g, 7.7 mmol) and SOCl
2
(30 mL) was heated
was evaporated, to
under reflux overnight. The solution was cooled, and the excess SOCl
2
which a solution comprising of 2-hexyl-1-decanol (6.4 g, 26.5 mmol) in pyridine (20 mL) was
added dropwise. The mixture was heated at 80 ℃ for 18 h and then cooled to room temperature.
The reaction mixture was poured into a cold 1 M HCl, and the product was extracted with ethyl
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acetate (EA). The organic layer was then washed with saturated NaHCO and brine, and the
3
solvent was evaporated. The product was purified via silica gel chromatography using
1
EA/hexane (1:4, by volume) as the eluent, yielding colorless oil. Yield: 70%. H NMR (500
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MHz, CDCl
3
): δ (ppm) 7.99 (s, 2H), 4.25 (m, 4H), 1.75 (m, 2H), 1.28 (m, 48H), 0.86 (m, 12H).
13C NMR (125 MHz, CDCl
): δ (ppm) 164.62, 136.55, 136.01, 120.03, 69.22, 37.40, 31.97,
3
3
1.88, 31.37, 30.0, 29.67, 29.63, 29.38, 26.78, 26.76, 22.76, 22.73, 14.20, 14.18.
Synthesis of PPDT-P
M1 (121 mg, 0.21 mmol) and M2 (165 mg, 0.21 mmol) were added to a 2 mL microwave
vial. Pd (dba) (2 mol%) as a catalyst and tri(o-tolyl)phosphine (8 mol%) as a ligand were added
2
3
to the above reaction mixture in a glovebox. After the microwave vial was capped, anhydrous
toluene (1.5 mL) was added using a syringe. Polymerization was conducted in a microwave
reactor at 140 ℃ for 60 min. The resulting polymer was precipitated into methanol, and the
precipitate was subjected to Soxhlet extraction with methanol, hexane, and chloroform. The
extracted polymer in chloroform was precipitated into methanol and dried under vacuum,
yielding a final polymer of PPDT-P. Yield: 78%. M = 22,600 Da and polydispersity index
n
1
(
PDI) = 1.8. H NMR (500 MHz, CDCl
3
): δ (ppm) 7.86 (Br, 2H), 7.63 (Br, 4H), 7.32 (Br, 2H),
1
3
7.10 (Br, 2H), 4.13 (Br, 4H), 1.55 (m, 2H), 1.18 (m, 48H), 0.83 (m, 12H). C NMR (125 MHz,
CDCl ): δ (ppm) 167.82, 144.64, 139.61, 133.49, 132.89, 132.52, 131.25, 127.56, 125.63,
23.19, 68.37, 36.79, 31.47, 31.43, 30.63, 29.51, 29.16, 28.90, 26.34, 26.31, 22.26, 22.22, 13.70,
13.68.
3
1
Synthesis of T-DCS
To prepare T-DCS, a 25 mL two-neck round-bottom flask was filled with compound 6 (0.2
g, 0.203 mmol), 2-(tributylstannyl)thiophene (0.19 g, 0.509 mmol), Pd
2
(dba) (4 mol%), and
3
tri(o-tolyl)phosphine (8 mol%). Under nitrogen, anhydrous toluene (10 mL) was added,
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followed by refluxing at 120 ℃ overnight for a Stille coupling reaction. After the reaction was
completed, the reaction mixture was extracted using chloroform and water. The organic layer
was dried over anhydrous MgSO , and the solvent was removed via rotary evaporation. The
4
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reaction mixture was purified via column chromatography (1:1 chloroform/hexane by volume
1
as an eluent) and recrystallization using methanol. Yield: 72%. H NMR (500 MHz, CDCl
3
): δ
(
ppm) 7.88 (s, 2H), 7.86 (s, 2H), 7.33 (d, 2H), 7.31 (d, 2H), 7.25 (d, 2H), 7.31 (d, 2H), 7.08 (dd,
1
3
2H), 4.04 (d, 4H), 1.94-1.87 (m, 2H), 1.56-1.20 (m, 48H), 0.87 (t, 12H). C NMR (125 MHz,
CDCl ): δ (ppm) 151.73, 138.63, 138.52, 136.55, 132.44, 128.09, 128.04, 125.57, 125.47,
24.56, 124.37, 116.94, 110.63, 105.75, 72.20, 38.17, 31.92, 31.89, 31.74, 31.73, 30.16, 29.82,
29.65, 29.40, 27.04, 27.01, 22.71, 14.12, 14.11. MALDI-TOF (C60 ): 988.4862.
3
1
H
80
2 2 4
N O S
4
.3. Preparation of Pdots
Pdots were prepared via a nanoprecipitation method, by modification of previous reports.7, 8,
The stock solutions (1 mg/mL) of FRET donor polymer (PPDT-P), two types of FRET
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acceptors (T-DCS and ITIC) and PSMA were prepared in THF. For green-emissive Pdots, the
PPDT-P (100 μL) and PSMA (400 μL) stock solutions were added into 4.5 mL THF to yield a
solution of [PPDT-P] = 20 μg/mL. A series of red- and NIR-emissive Pdots was prepared by
varying the mixing ratio of donor and acceptor stock solutions, e.g., 80:20 μL, 60:40 μL, 40:60
μL, and 20:80 μL, to form fluorophore solutions with a total volume of 100 μL. Then,
fluorophore solutions for red- and NIR-emissive Pdots ([PPDT-P] = 4–18 μg/mL and [T-DCS
or ITIC] = 2–16 μg/mL) were added to a mixture of PSMA stock solution (400 μL) and THF
(
4.5 mL). After vigorous sonication, 5 mL of the precursor solution was quickly transferred to
0 mL of deionized water in a bath sonicator. The solution was concentrated to 2 mL via
continuous evaporation under a stream of nitrogen gas in a 90 ℃ bath, producing a Pdot solution
1
2
9
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