Near-Infrared-Emitting Boron-Difluoride-Curcuminoid-Based Polymers Exhibiting Thermally Activated Delayed Fluorescence as Biological Imaging Probes

Article abstract of DOI:10.1002/anie.202103965

Near-infrared-emitting polymers were prepared using four boron-difluoride-curcuminoid-based monomers using ring-opening metathesis polymerization (ROMP). Well-defined polymers with molecular weights of ≈20 kDa and dispersities <1.07 were produced and exhi

Full text of DOI:10.1002/anie.202103965

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Accepted Article
Title: Near-Infrared Emitting Boron Difluoride Curcuminoid-Based
Polymers Exhibiting Thermally Activated Delayed Fluorescence
as Biological Imaging Probes
Authors: Nathan R. Paisley, Sarah V. Halldorson, Michael V. Tran,
Rupsa M Gupta, Saied Kamal, W. Russ Algar, and Zachary
M. Hudson
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Angewandte Chemie International Edition
10.1002/anie.202103965
RESEARCH ARTICLE
Near-Infrared Emitting Boron Difluoride Curcuminoid-Based
Polymers Exhibiting Thermally Activated Delayed Fluorescence
as Biological Imaging Probes
Nathan R. Paisley†[a], Sarah V. Halldorson†[a], Michael V. Tran , Rupsa Gupta , Saeid Kamal , W.
[a]
[a]
[a]
[
a]
[a]
Russ Algar* , and Zachary M. Hudson*
[
a]
Mr. N.R. Paisley, Ms. S.V. Halldorson, Mr. M.V. Tran, Ms. R. Gupta, Dr. S. Kamal, Prof. W.R. Algar, prof. Z.M. Hudson.
Department of Chemistry
The University of British Columbia
2036 Main Mall, Vancouver, British Columbia, Canada, V6T 1Z1
E-mail: algar@chem.ubc.ca, zhudson@chem.ubc.ca
†
These authors contributed equally to this work.
Supporting information for this article is given via a link at the end of the document.
Abstract: Near-infrared emitting polymers were prepared using four
boron-difluoride curcuminoid-based monomers using ring-opening
metathesis polymerization (ROMP). Well-defined polymers with
molecular weights of ~20 kDa and dispersities < 1.07 were produced
and exhibited near-infrared (NIR) emission in solution and in the solid
state with photoluminescence quantum yields (ΦPL) as high as 0.72
and 0.18, respectively. Time-resolved emission spectroscopy
revealed thermally-activated delayed fluorescence (TADF) in
polymers containing highly planar dopants, whereas room-
temperature phosphorescence dominated with twisted species.
Density functional theory demonstrated that rotation about the donor-
acceptor linker can give rise to TADF, even where none would be
expected based on calculations using ground-state geometries.
Incorporation of TADF-active materials into water-soluble polymer
dots (Pdots) gave NIR-emissive nanoparticles, and conjugation of
these Pdots with antibodies enabled immunofluorescent labeling of
SK-BR3 human breast cancer cells.
infrared (NIR) TADF emitters based on 2,1,3-benzothiadiazole
and 1,8-naphthalimide have previously been applied as
bioimaging agents, however they all require excitation with blue
or green light and have modest photoluminescence quantum
yields (ΦPL).[
3,12,13]
In 2018, Adachi and coworkers described a
boron difluoride curcuminoid (BFC) that exhibited TADF with
emission longer than 700 nm and ΦPL above 0.50.[7] Using this
framework, NIR OLEDs were achieved with external quantum
efficiencies of nearly 10%, as well as amplified spontaneous
emission with a peak wavelength of 750 nm. BFCs have
previously been used as NIR-emitting biological probes,
suggesting that TADF-active BFCs would be promising for
bioimaging applications.[
14]
Given the hydrophobicity of these dyes, however, additional
steps would be required to provide solubility in aqueous media. It
is possible to form aggregated suspended particles of BFC dyes
by adding them directly to water from concentrated organic
solvent.[15] Unfortunately, the resulting particles suffer from
aggregation-induced quenching and can disaggregate causing
dye leakage into the hydrophobic regions of cells. Though
functionalization of the BFC framework with hydrophilic groups
may improve aqueous solubility, TADF materials typically exhibit
strong solvatochromism, such that high-polarity solvents such as
water can red-shift and broaden emission spectra while also
Introduction
Materials exhibiting thermally activated delayed fluorescence
(
TADF) are rapidly emerging as useful probes for biological
imaging.[ TADF materials exhibit both short (ns) and long (µs-
1]
reducing
Φ
PL
.
Alternatively, hydrophobic dyes can be
ms) emissive lifetimes as a result of rapid interconversion
between the lowest singlet (S ) and triplet (T ) excited states,
1 1
incorporated into water-soluble nanoparticles, removing the need
for elaborate chemical functionalization.[16] For example, BFC
permitting the use of time-gated microscopy to remove
background autofluorescence from biological samples.[2,3] This is
achieved by minimizing the singlet-triplet energy gap of the
material (ΔEST), such that ambient thermal energy can facilitate
materials have been loaded into porous silica nanoparticles or
[17,18]
bovine serum albumin as water-soluble matrices.
This
method provides nanoparticles with low cytotoxicity, though
leakage from the nanoparticle into the surrounding media can
reduce dye localization, alter photophysical properties, and even
damage cells through the production of singlet oxygen. Other
approaches have incorporated small-molecule TADF dyes into
polymer nanoparticles, relying on the hydrophobicity of the
nanoparticle core to keep the dyes localized.[19]
Alternatively, hydrophobic polymers exhibiting TADF can be
coprecipitated with amphiphilic polymers in water, giving
luminescent nanoparticles termed polymer dots (Pdots). Pdots
are water-soluble nanoparticles containing ≥50% semiconducting
[4]
1 1
reverse intersystem crossing (RISC) from T to S . This property
also allows for the efficient harvesting of both singlet and triplet
excitons in devices such as organic light-emitting diodes
(
OLEDs)[5,6] and organic lasers,[7–9] and has led to some of the
most efficient examples of such devices reported thus far.
In bioimaging applications, the use of dyes with excitation
and emission wavelengths within the biological transparency
window (650–1350 nm) is also highly advantageous.[10,11]
Unfortunately, few TADF materials are known that emit at these
wavelengths, and even fewer can be excited in this range. Near-
1
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Angewandte Chemie International Edition
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RESEARCH ARTICLE
[
20]
polymer by weight or volume, and have emerged as powerful
acetone adduct 1 affords 3-hydroxyethyl 3-oxobutanoate (2),
which is then coupled with exo-5-norbornenecarboxylic acid using
N,N′-dicyclohexylcarbodiimide (DCC) to give 3. Acylation with
[
20–26]
biosensing,[27–30] and photodynamic
Characterized by improved photostability and high
probes for bioimaging,
therapy.[
31–33]
brightness, Pdots can also be functionalized for cell targeting and
therapy, providing a versatile platform for theranostics.[34] As a
result, Pdots have recently found application in single-particle
2
acetyl chloride in the presence of MgCl furnishes the target β-
diketone 4 in 70% yield. A three-step reaction is then used to form
the desired BFC in one pot. First, the dioxaborine ring is formed
by coordination of borontrifluoride diethyletherate, after which the
donor-functionalized benzaldehyde is added in a condensation
reaction promoted by n-butylamine. In this case, the donors 4-(di-
p-tolylamino)benzaldehyde (Dta-CHO), 4-(9,9-dimethylacridin-
10(9H)-yl)benzaldehyde (Acr-CHO), 4-(10H-phenoxazin-10-
[35]
[36,37]
tracking in cells,
lateral flow or blot-style assays,
and
immunolabeling of cancer cells.[38] Despite this growing interest,
Pdots with NIR emission remain rare.[39–41]
Here, we describe a series of BFC-based monomers
exhibiting either TADF or room-temperature phosphorescence
(
RTP) in the deep red/NIR region. Copolymerization by ring-
yl)benzaldehyde)
(Poz-CHO),
or
the
aldehyde
of
opening metathesis polymerization (ROMP) with a carbazole-
based host gives polymers with molecular weights of
approximately 20 kDa and dispersities ≤ 1.07. Using arylamine
donors with varying degrees of rotational restriction, we elucidate
the structure-property relationships leading to either TADF or RTP
in donor-acceptor BFCs. These results are supported by steady-
state and time-resolved spectroscopy, density functional theory,
and X-ray crystallographic analysis. Finally, these polymers are
used to prepare NIR-emitting Pdots with high quantum yield,
which are then demonstrated in immunofluorescent labeling
experiments with SK-BR3 human breast cancer cells.
hexamethylazatriangulene (Hmat-CHO) were employed, giving
the final BFC monomers in 45–50% yield. A carbazole-based host
monomer CzBN is synthesized by DCC coupling of exo-5-
norbornenecarboxylic
acid
with
(4-(9h-carbazol-9-
yl)phenyl)methanol (see Figure 2).
Solid-state molecular structures for each monomer were
obtained from single crystal X-ray diffraction (Figure 1). The BFC
backbone is nearly co-planar, with the dihedral angle about the
C=C bonds and dioxaborine ring ranging from 172–178˚. Similarly,
the dihedral angle between the C=C bond and adjacent phenyl
rings were ~175˚ for all structures. Within the donor moiety,
however, significant differences in planarity were observed.
Without any rotational restriction, the terminal N-tolyl groups in
DtaB twist out of the backbone plane with a dihedral angle of 15˚.
PozB and AcrB contain oxygen and methylene bridges,
respectively, which join the terminal aryl groups into a single
heterocyclic coplanar unit. The planarity of these terminal
heterocycles results in large increases in dihedral angle with the
BFC acceptor, with values of 84˚ and 76˚ for AcrB and PozB,
respectively. In HmatB, additional methylene bridging groups
Results and Discussion
BFCs functionalized at the meso-carbon of the dioxaborine ring
can be readily obtained through the synthesis of modified β-
diketones prior to BF coordination. Here, a meso-functionalized
2
β-diketone containing an exo-norbornene moiety for ROMP is
prepared in three steps (Scheme 1). Ring opening of the dikenene
Scheme 1. Synthetic route to BFC-based norbornene monomers.
2
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RESEARCH ARTICLE
result in the flattening of the entire BFC structure. It should be
noted that, in AcrB, both equatorial (Figure 1) and axial
conformations of the donor group are present. Molecular packing
of AcrB and PozB show ~3.7 and ~3.5 Å π-π stacking
interactions between the bridging phenyl groups, however, no
such interactions are observed in DtaB or HmatB (Figures S9–
S12).
ROMP of CzBN with BFC dopant at –20 °C gave
monomodal molecular weight distributions by gel permeation
chromatography at >95% conversion after 24 h (Figure 2A).
Polymer purification was performed by preparatory size exclusion
chromatography (SEC) to remove any residual monomer and Ru
catalyst. No product retention was observed on the polystyrene-
divinylbenzene resin used for this procedure, such that a single
column may be reused many times. To optimize red/NIR emission,
Figure 2. (A) General synthetic route to BFC-doped polymers. (B) Emission of
DtaB-doped polymers. Measured in the solid state, spin-cast on glass slides;
λ
ex = 313 nm. (C) Representative refractive index trace (3 wt. % doped CzBN-
co-DtaB). (D) Toluene solutions of BFC-doped polymers under room light and
65 nm illumination (CzBN-co-AcrB and CzBN-co-PozB show no emission
3
under 365 nm illumination and are not shown).
a series of copolymers of CzBN and DtaB were first prepared
(
monomer:initiator = 50:1), with DtaB concentrations of 1, 2, 3, 4,
or 6 wt. % (CzBN-co-DtaB). Dispersities ranged from 1.04 to 1.06
with number-average molecular weights (M ) ranging from 17.2 to
0.0 kDa (Table S1). In solution, a low-energy absorption band is
observed at 613 nm (CH Cl ) and emission is observed at 694 nm
in toluene) for all polymers. Photoluminescence quantum yields
PL) show oxygen dependence with values under nitrogen
n
2
2
2
(
(
Φ
ranging from 0.16 (1 wt. %) to 0.58 (4 wt. %), ~1.9-fold enhanced
from values obtained under air. Spin-cast films display
concentration-dependent emission with λmax shifting from 719 to
739 nm as the concentration of dopant is increased (Figure 2B).
Films display reduced ΦPL compared with solution data, and
minimal intensity enhancement under inert atmosphere with ΦPL
ranging from 0.14 to 0.18 (6 and 3 wt. %, respectively). This solid-
state data is comparable with other boron-based NIR emitters,
including boron azobenzene-based polymers (solid state ΦPL
0
=
.06).[42] As the photophysical properties in films most closely
approximate the aggregated chromophores found in Pdots, 3
wt. % was chosen as the optimal doping concentration. Using
these conditions, CzBN-co-AcrB, CzBN-co-PozB, and CzBN-
co-HmatB were synthesized from AcrB, PozB, and HmatB,
respectively, with dispersities ranging from 1.06 to 1.07 with M
n
values of 18.2 to 18.4 kDa (Table 1).
2 2
Absorbance features in CH Cl solution below 350 nm are
dominated by the CzBN host and no significant differences
between CzBN-co-DtaB, CzBN-co-AcrB, CzBN-co-PozB, and
CzBN-co-HmatB are observed in this range (Figure S13). CzBN-
co-AcrB and CzBN-co-PozB both have features at 440 nm and
lower intensity bands at 548 and 583 nm, respectively (Figure 3,
Figure 1. Crystal structures of BFC-based monomers with thermal ellipsoids at
the 50% probability level. Hydrogen atoms have been omitted for clarity. Boron
=
pink, carbon = grey, nitrogen = blue, oxygen = red, fluorine = yellow. Selected
average dihedral angles are shown.
3
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RESEARCH ARTICLE
Table 2). CzBN-co-DtaB and CzBN-co-HmatB have a single
strong absorption band at 614 and 646 nm, respectively.
in all cases. Spin-cast films show minimal host emission, with
maxima at 730, 687, 714, and 752 nm for CzBN-co-DtaB, CzBN-
co-AcrB, CzBN-co-PozB, and CzBN-co-HmatB, consistent with
trends in donor strength (Hmat > Dta > Poz > Acr) (Figure 3;
Table 2). Under vacuum, ΦPL values of 0.18 (CzBN-co-DtaB),
0.06 (CzBN-co-AcrB), 0.05 (CzBN-co-PozB), and 0.12 (CzBN-
co-HmatB) were determined, with little difference observed under
an air atmosphere.
The delayed lifetimes of these polymers were then
measured using spin-cast films (Figure 4 top; Table 2). Data for
all polymers were best fit with triexponential curves, consistent
with previous research showing that excited-state dimer formation
is common in BFC-based materials.[7] Under vacuum, CzBN-co-
Table 1. Properties of 3 wt. % doped polymers.
M
(
n
Polymer
Đ[a]
Da)[a]
CzBN-co-DtaB
CzBN-co-AcrB
CzBN-co-PozB
CzBN-co-HmatB
19900
18300
18200
18400
1.05
1.06
1.06
1.07
[a] Measured by triple detection SEC in tetrahydrofuran (THF).
Density functional theory (DFT) calculations at the ωB97X-
D*/6-31+G(d) level along with natural transition orbital (NTO)
analysis was used to gain insight into the nature of these
transitions. Time-dependent DFT calculations typically show
multiple orbital contributions for each transition; NTO analysis
combines these multiple transitions into ideally one particle-hole
DtaB and CzBN-co-HmatB have delayed lifetimes with values of
4.7 and 5.2 μs, respectively, accounting for >75% of the decay.
CzBN-co-AcrB and CzBN-co-PozB, however, have longer
decays with significant contribution from lifetimes on the order of
milliseconds. Under air, CzBN-co-DtaB and CzBN-co-HmatB
show decays with reduced intensity indicating the quenching of
(
excited state-ground state) pair. NTO analysis shows that the
transitions in CzBN-co-AcrB and CzBN-co-PozB are primarily
due to charge transfer from the terminal heterocycle to the
coplanar BFC backbone (Figure S14). Absorption in CzBN-co-
DtaB and CzBN-co-HmatB is predominantly π-π* in character
with electron density spread across the BFC with larger
coefficients on the donor in the ground state (Figure S14). In the
S state, the electron distribution becomes more centralized with
1
reduced contribution from the donor and larger coefficients on the
dioxaborine ring.
Emission from CzBN-co-DtaB and CzBN-co-HmatB in
toluene is observed at λmax = 694 and 717 nm, respectively, while
no dopant emission was detected for CzBN-co-AcrB and CzBN-
co-PozB (Figure S13). In degassed toluene, ΦPL values of 0.33
(
CzBN-co-DtaB) and 0.30 (CzBN-co-HmatB) were determined,
which are enhanced 2.2- and 1.5-fold from aerated conditions,
respectively. Host emission is observed from all polymers in
toluene solution due to inefficient energy transfer to the BFC-
based dopants. It should be noted that improved values of ΦPL are
obtained when the dopant is excited directly (0.72 for CzBN-co-
DtaB and 0.58 for CzBN-co-HmatB with 605 nm excitation). Low
temperature time-gated emission studies were conducted in an
Figure 3. Normalized absorbance (dashed) in CH
PL, solid) of (A) CzBN-co-DtaB, (B) CzBN-co-AcrB, (C) CzBN-co-PozB, (D)
CzBN-co-HmatB in the solid state, spin-cast on glass slides; λex = 340 nm.
2 2
Cl and photoluminescence
(
attempt to directly measure the energy of the T
1
state in these
materials, however no phosphorescence data could be detected
Figure 4. Top: Time-resolved fluorescence emission for (A) CzBN-co-DtaB, (B) CzBN-co-AcrB, (C) CzBN-co-PozB, and (D) CzBN-co-HmatB under vacuum
inert) and air. Bottom: Temperature dependence of the time-resolved fluorescence emission for the corresponding polymers under N . Measured in the solid state,
(
2
spin-cast on glass slides; λex = 313 nm. Arrows indicate the change in lifetime on cooling.
4
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RESEARCH ARTICLE
Table 2. Photophysical data for CzBN-co-DtaB, CzBN-co-AcrB, CzBN-co-PozB, and CzBN-co-HmatB.
abs
opt
DFT
em
air / Φvac[f]
Polymer
λmax
(
Egap / Egap
HOMO / LUMO
(eV)
λmax
Φ
τ
air / τvac
nm)[a]
(eV)
[b,c]
[d,e]
(nm)
[f]
(μs)
[g]
CzBN-co-DtaB
CzBN-co-AcrB
CzBN-co-PozB
CzBN-co-HmatB
340, 614
1.92 / 2.47
2.67 / 2.56
2.69 / 2.54
1.81 / 2.30
-5.3 / -3.4
-5.4 / -2.7
-5.2 / -2.5
-5.3 / -3.5
730
687
714
752
0.16 / 0.18
0.05 / 0.06
0.05 / 0.05
0.10 / 0.12
4.7, 102, 508
323, 2270, 6790
465, 2180, 5290
5.2, 60, 343
340, 440, 548
340, 440, 583
340, 646
[
a] Measured in CH
2
Cl
2
at 0.01 mg mL–1. [b] Calculated from a Tauc plot of the UV-Vis spectrum in CH
2
Cl
2 1
. [c] Energy corresponding to the S excited state from
opt
TDA-DFT at the ωB97XD*/6-31+G(d) level. [d] HOMO = -(E1/2, ox + 4.8 eV). [e] LUMO = (HOMO + E ). [f] Measured in the solid state, spin-cast on glass slides;
gap
λ
ex = 340 nm. [g] Measured in the solid state, spin-cast on glass slides; λex = 313 nm.
Figure 5. (A) S
0
optimized geometry of HmatB (left; Φ
= 90°, Φ = 90°; ΔEST = 0.015 eV). (B) Vertical excited state transition energies of the T
from 180–90°. (B,C) Calculated by a series of TDA-DFT calculations at the ωB97X-D*/6-31+G(d) level of HmatB structures with rotations about Φ
1 2
= 179°, Φ =177°; ΔEST = 0.416 eV) where rotations of the donor groups gives a reduced singlet triplet gap
(
right; Φ
1
2
1
, T
2
, S
1
, and S
2
excited states and (C) ΔEST of HmatB over Φ
1
and Φ
2
1
and Φ
2
from 180–90° in 15° increments. Calculated points were connected by a surface to aid understanding.
triplet states by molecular oxygen. No reduction in decay intensity
is observed for CzBN-co-AcrB and CzBN-co-PozB, indicating a
reduced sensitivity of these triplet states to oxygen.
HmatB should occur following a similar mechanism as low-
energy rotations about the donor olefin dihedral angle occur at ~3
–1
1 1
cm . To study this effect, a scan of the S and T energy levels
Temperature-dependent microsecond emission lifetimes
were then obtained at 300 K and 77 K (Figure 4 bottom). Both
CzBN-co-DtaB and CzBN-co-HmatB showed reduced decay
intensity at 77 K compared to 300 K. This is consistent with TADF
as at 77 K there is minimal thermal energy to promote RISC. At
lifetimes longer than ~600 μs, higher-intensity emission was
detected at 77 K due to phosphorescence becoming viable from
reduced molecular motion.
In a system similar to DtaB, Adachi and coworkers
reasoned that nonadiabatic effects produce the minimal ΔEST
required for efficient TADF.[7] A ΔEST value higher than that
considered to give effective TADF (~0.2 eV) was calculated;
however, a scan of the excited-state potential energy surface
showed that rotations within the triarylamine donor resulted in a
was performed by changing the donor olefin dihedral angles from
180 to 90˚ in 15˚ increments and computing the vertical excitation
energies at each angle (Figure 5). It was found that a large
reduction in ΔEST occurs as both dihedral angles approach 90˚,
1 2
reaching a minimum energy of 0.015 eV when Φ = Φ = 90˚.
Moreover, with dihedrals of ≲120˚ ΔEST is reduced to 0.2 eV. This
suggests that nonadiabatic pathways are essential to RISC in
HmatB. This also demonstrates that a material having a large
value for ΔEST in its lowest-energy conformation does not, in itself,
rule out TADF behaviour.
Surprisingly, CzBN-co-AcrB and CzBN-co-PozB show the
opposite trend at low temperature with higher emission intensity
at 77 K (Figure 4 bottom). This trend is characteristic of
phosphorescence as reduced thermal energy limits molecular
vibrations and non-emissive decay pathways become inefficient.
Room-temperature phosphorescence (RTP) is common in
materials containing heavy atoms but it is uncommon in organic
compounds. Boron difluoride β-diketonates, however, have been
1 2 1 2
conical intersection of the S , S , T , and T states. TADF from
DtaB assuredly occurs by a similar mechanism and DFT
calculations show the presence of low-energy rotations (~32 cm–
1)
within the triarylamine donor. We reasoned that TADF from
5
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RESEARCH ARTICLE
shown to exhibit RTP in solid films, though phosphorescence is
not typically observed in solution.[43–45] The long-lived emission
from films of CzBN-co-AcrB and CzBN-co-PozB but the absence
of emission from toluene solutions is consistent with this RTP
behaviour. In contrast to DtaB and HmatB, AcrB and PozB have
large donor-acceptor dihedral angles in the ground state and
extremely low S → S oscillator strengths (<0.001) by DFT.
0 1
Moreover, DFT frequency analysis shows the absence of
rotations that would lessen the heterocycle-phenyl dihedral angle
0 1
and increase the strength of the S → S transition. This effect
has been observed in TADF emitters in which functional groups
have been incorporated to increase steric hindrance and
introduce a heightened barrier to rotation.[46–49] Even in materials
with a small ΔEST, this rotational restriction decreases coupling
1 1
between T and S and lowers the efficiency of RISC, allowing
[
48]
phosphorescence to become a viable decay pathway. CzBN-
co-AcrB and CzBN-co-PozB demonstrate such an increase in
phosphorescence on cooling.
BFC materials have previously been explored for their two-
photon fluorescence properties that enable both NIR excitation
and emission.[50] We examined the two-photon emission of CzBN-
co-HmatB due its high ΦPL and the high degree of planarity in the
HmatB dopant, which is known to enhance two-photon cross-
section. Emission spectra were recorded as a function of the
excitation wavelength from 1100–1500 nm (Figure S19).
Maximum emission response was observed at 1150 nm,
indicating that HmatB dyes have potential for further exploration
as two-photon fluorescent dyes with excitation in the biological
transparency window.
Figure 7. (A) Illustration of CzBN-co-DtaB Pdot-bioconjugates binding to HER2
antigens on a SK-BR3 cell. (B) Brightfield (top row), epifluorescence (middle
row) and merged (bottom row) images of fixed SK-BR3 cells immunolabeled
with Pdot-bioconjugates. There was negligible non-specific binding in the
absence of either anti-HER2 or anti-IgG1 (see Figure S25).
Figure 6. (A) Encapsulation of CzBN-co-DtaB in the amphiphilic polymer
PSMA to produce water-soluble polymer dots. (B) AFM height images of
CzBN-co-DtaB Pdots on HOPG substrates (scale bar = 1 μm). (C) Size
distribution of CzBN-co-DtaB Pdots in aqueous solution (blue) and curve fit
We next incorporated CzBN-co-DtaB and CzBN-co-
HmatB into water-soluble Pdots to explore their applications in
immunofluorescent labeling. CzBN-co-DtaB and CzBN-co-
HmatB were chosen as they gave the highest ΦPL of the
(
(
dashed line). (D) Normalized absorbance (dashed) and photoluminescence
PL, solid) of CzBN-co-DtaB Pdots in water at 0.01 mg mL– . (E) Time-
1
resolved emission of CzBN-co-DtaB Pdots under N
2
(inert) and air.
6
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RESEARCH ARTICLE
compounds studied. Pdots were prepared by dissolving either
CzBN-co-DtaB or CzBN-co-HmatB with the amphiphilic polymer
poly(styrene-co-maleic anhydride) (PSMA; PS11-co-MA ) in THF,
6
followed by rapid injection into water under sonication, and
removal of all volatile organics in vacuo (Figure 6A). CzBN-co-
DtaB particles were found to have a spherical morphology by
atomic force microscopy (AFM) with an average diameter of 42 ±
Pdot-bioconjugates, was 5.1 ± 4.5. Similarly, the CzBN-co-
HmatB Pdot-bioconjugates provided specific labeling albeit at a
slightly lower SBR of 1.1 ± 0.9 (SNR: 11  9), and a CR of 3.6 ±
3.1 (Figure S26). It is unknown if the large-sized fraction of Pdots
(>150 nm) and small-sized fraction of Pdots (< 100 nm) in the
blocking buffer immunolabeled the cells with different efficiencies.
This polydispersity in blocking buffer may be a point for further
optimization in future studies.
13 nm (Figure 6B). The mean hydrodynamic diameters of these
Pdots, as determined by nanoparticle tracking analysis (NTA),
was 67 ± 18 nm (mode = 65 nm; CzBN-co-DtaB) and 61 ± 22 nm
Lastly, we sought to demonstrate the utility of the Pdots for
time-gated photoluminescence measurements using time-gated
emission spectra. (Figure 8) First, CzBN-co-DtaB Pdots were
mixed with Cy5, a widely used red fluorescent dye with a
nanosecond-scale emission lifetime and no reported TADF.
Prompt spectra showed a combination of Pdot and Cy5 emission
with approximately equal intensity, whereas time-gated spectra
(50 µs delay) showed Pdot emission with no detectable Cy5
emission. Next, Pdots were diluted in bovine serum, which has
significant autofluorescence under UV illumination. Both serum
autofluorescence and Pdot emission were observed in prompt
measurements. In contrast, the serum autofluorescence was
negligible in time-gated spectra, whereas Pdot emission was still
readily observed. Similar results were observed in analogous
experiments with CzBN-co-HmatB Pdots (Figure S27). Overall,
this data illustrates the potential advantages and synergy of a
combination of NIR emission and TADF-enabled time-gating for
low-background biological imaging.
(
mode = 58 nm; CzBN-co-HmatB) (Figure 6C, Figure S20).
These hydrodynamic sizes are consistent with the AFM results,
for which the particles were measured after drying. The low-
energy absorption maxima of the Pdots is red-shifted in both
cases from those of the unencapsulated dyes, with λmax = 625 nm
(
CzBN-co-DtaB) and 665 nm (CzBN-co-HmatB) (Figure 6D,
Figure S20). The emission of CzBN-co-DtaB and CzBN-co-
HmatB experience only small red shifts in emission from λmax
30 and 752 nm in films to 731 and 764 nm in Pdots, respectively.
Lifetime data indicates that TADF behaviour is retained (Figure
E, Figure S20), and the photophysical properties of the dyes
=
7
6
remain relatively unchanged between degassed and aerated
conditions (ΦN2 = 0.26 τN2 = 0.86 μs vs ΦAir = 0.26 τAir = 0.94 μs for
CzBN-co-DtaB Pdots; ΦN2 = 0.21 τN2 = 0.95 μs vs ΦAir = 0.21 τAir
=
1.8 μs for CzBN-co-HmatB Pdots), ideal for imaging
applications. Additional long-lived components to the Pdot
lifetimes (>30 ms) account for <5% of the observed emission
2
under air and N . (Table S8)
Next, we investigated the use of the CzBN-co-DtaB and
CzBN-co-HmatB Pdots for specific extracellular immunolabeling
of HER2 on SK-BR3 breast cancer cells as a model system.
To this end, the surface carboxyl groups of the Pdots were
conjugated to secondary goat anti-mouse IgG1 antibodies via
EDC-mediated coupling. We chose an indirect immunolabeling
strategy with a secondary antibody (see Figure 7A) instead of
direct immunolabeling because we anticipated less steric
hindrance from the Pdot toward binding to HER2 on the cell
membrane. Other studies with Pdots have also utilized this
strategy or a similar one.[51-53] After conjugation with secondary
antibodies, the mean hydrodynamic diameters of the CzBN-co-
DtaB and CzBN-co-HmatB Pdots increased by ~26–28 nm
(
Figure S24). This size increase was also confirmed by agarose
gel electrophoresis. It should be noted that the Pdot size
distribution widened with an increase in average hydrodynamic
diameter to >150 nm when the antibody conjugates were
redispersed in the protein-based blocking buffer used for cellular
imaging. There was no observed loss of colloidal stability in the
blocking buffer, even after ~2 months of storage at 4 ºC. We
hypothesize that the blocking buffer may form a protein corona
around some of the Pdots.
The antibody-conjugated Pdots (from Figure S23) were
tested with HER2-positive SK-BR3 cells. The cells were first
incubated with primary mouse anti-HER2 antibodies followed by
incubation with the Pdot immunoconjugates. Figure 7B shows the
successful labeling of SK-BR3 cells with the CzBN-co-DtaB Pdot-
bioconjugates. A signal-to-background ratio (SBR) of 1.5 ± 1.3
and signal-to-noise ratio (SNR) of 15  13 in the presence of
primary anti-HER2 were determined. In the absence of anti-HER2,
there was negligible nonspecific binding of the Pdot-
immunoconjugates, with a SBR of 0.3 ± 0.04 (SNR: 3  0.4). The
contrast ratio (CR), which demonstrates the specificity of the
Figure 8. (A) Illustration of the delay time and integration time for time-gated
measurements, superimposed on the TADF component of the time-resolved
CzBN-co-DtaB emission (from Figure 6E). (B) Prompt (0 µs delay; 100 µs
integration time) and time-gated (50 µs delay; 500 µs integration time)
photoluminescence emission spectra for Cy5 dye and a mixture of Cy5 and
CzBN-co-DtaB Pdots. (C) Prompt (0 µs delay; 100 µs integration time) and
time-gated (50 µs delay; 250 µs integration time) photoluminescence emission
spectra for CzBN-co-DtaB Pdots diluted in bovine serum. The buffer blank
highlights the serum autofluorescence in the prompt spectra and its absence
from the time-gated spectra.
7
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RESEARCH ARTICLE
Conclusion
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Entry for the Table of Contents
Polymers exhibiting near-infrared thermally activated delayed fluorescence (TADF) or room temperature phosphorescence are
prepared from four boron difluoride curcuminoid-based monomers. Incorporation of TADF-active materials into water-soluble polymer
dots (Pdots) and subsequent antibody conjugation enabled near-infrared immunofluorescent labeling of SK-BR3 human breast cancer
cells.
1
0
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