Monochromophore-Based Phosphorescence and Fluorescence from Pure Organic Assemblies for Ratiometric Hypoxia Detection

Article abstract of DOI:10.1002/ange.202007039

Hypoxia is a parameter related to many diseases. Ratiometric hypoxia probes often rely on a combination of an O₂-insensitive fluorophore and an O₂-sensitive phosphor in a polymer matrix, which require high cost and multi-step synthesis of transition metal complexes. The two-chromophore hypoxia probes encounter unfavorable energy transfer processes and different stabilities of the chromophores. Reported herein is a pure organic ratiometric hypoxia nanoprobe, assembled by a monochromophore, naphthalimide ureidopyrimidinone (BrNpA-UPy), bridged by a bis-UPy-functionalized benzyl skeleton. The joint factors of quadruple hydrogen bonding, the rigid backbone of UPy, and bromine substitution of the naphthalimide derivative facilitate bright phosphorescence (Φ_P=7.7 %, τ_P=3.2 ms) and fluorescence of the resultant nanoparticles (SNPs) at room temperature, which enable accurate, ratiometric, sensitive oxygen detection (K_sv=189.6 kPa^?1) in aqueous solution as well as in living HeLa cells.

Full text of DOI:10.1002/ange.202007039

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Titel: Monochromophore-Based Phosphorescence and Fluorescence
from Pure Organic Assemblies for Ratiometric Hypoxia Detection
Autoren: Li-Zhu Wu, Xiao-Qin Liu, Ke Zhang, Jian-Feng Gao, Yu-Zhe
Chen, and Chen-Ho Tung
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Zitierweise: Angew. Chem. Int. Ed. 10.1002/anie.202007039
Link zur VoR: https://doi.org/10.1002/anie.202007039

10.1002/ange.202007039
Angewandte Chemie
COMMUNICATION
Monochromophore-Based Phosphorescence and Fluorescence
from Pure Organic Assemblies for Ratiometric Hypoxia Detection
Xiao-Qin Liu+, Ke Zhang+, Jian-Feng Gao, Yu-Zhe Chen,* Chen-Ho Tung, and Li-Zhu Wu*
Abstract: Hypoxia is
a
parameter related to many diseases.
ratiometric oxygen detection, would be promising to overcome
the above disadvantages. Particularly, the long-lived character
of organic phosphorescence will ensure the efficient
phosphorescence quenching by molecular oxygen, thus leading
to the sensitive O2 detection. However, most pure organic
compounds could only emit fluorescence at room temperature
Ratiometric hypoxia probes often rely on a combination of an O2-
insensitive fluorophore and an O2-sensitive phosphor in a polymer
matrix, which are comprised by high cost and multi-step synthesis of
transition metal complexes. The two-chromophore hypoxia probes
encounter unfavorable energy transfer processes and different
stabilities of the chromophores. Reported herein is a pure organic
ratiometric hypoxia nanoprobe, assembled by a monochromophore,
naphthalimide ureidopyrimidinone (BrNpA-UPy) bridged by a bis-
UPy functionalized benzyl skeleton. The joint factors of quadruple
hydrogen bonding, the rigid backbone of UPy, and bromine
owing to the spin prohibition of the underlying triplet-singlet
[9,10]
emission.
Recently, delicate strategies such as crystal
formation,[11] host-guest interaction,[12] polymerization [13], as well
as halogen and hydrogen bonding[14] have been employed to
increase the occurrence of triplet excited states and decrease
their nonradiative decay to promote room temperature
phosphorescence (RTP) at solid state. Unfortunately, these
strategies are excluded to be a hypoxia sensor owing to their
poor water solubility/dispersibility to penetrate into biological
systems.
substitution
of
naphthalimide
derivative
facilitate
bright
phosphorescence (ΦP = 7.7%, P = 3.2 ms) and fluorescence of the
resultant nanoparticles (SNPs) at room temperature, which enable
the accurate ratiometric sensitive oxygen detection (Ksv= 189.6 kPa-
1) in aqueous solution as well as in living HeLa cells.
Quantification of molecular oxygen (O2) is of great significance in
complex environments and biological fields, e.g. the lack of
oxygen named as hypoxia is connected with many diseases.
Intracellular O2 levels or the degree of hypoxia provides an
important parameter in the early-stage diagnosis of cancers,
cardiovascular diseases, and stroke.[1] Among currently
available methods (i.e., positron emission tomography (PET)[2],
magnetic resonance imaging (MRI)[3], optical detection),
ratiometric readout of optical signals at two wavelengths is
highly preferable for imaging in vivo O2 levels due to its simple
operation, minimal invasiveness and accuracy [4-8]. To date, most
reported ratiometric oxygen sensors rely on a combination of the
O2-insensitive fluorophore and O2-sensitive phosphorescent
transition metal complex (e.g. Pt(II) porphyrin) in a polymer
matrix. However, undesirable energy transfer and different
stabilities of the fluorophore and metal complex phosphor in one
probe might also compete with readout of oxygen levels.
Meanwhile, transition metal complexes would face multi-step
synthesis, high cost and unclear toxicity.
Scheme 1. Phosphorescence and fluorescence enabled by an organic
monochromophore in supramolecular polymer assembled nanoparticles
(SNPs) for ratiometric oxygen detection. Molecular structures of organic
chromophore (BrNpA-UPy), skeleton molecule (Ph-bisUPy), and UPy.
At present, the accurate ratiometric readout of oxygen
levels using pure organic monochromphore platform remains
largely unexplored. Even for RTP from the self-assembled
organic molecules in aqueous solution, they failed to act as
hypoxia sensor due to the poor oxygen permeability in the
tightly-packed structures.[15] Impressively, Fraser and coworkers
discovered that nanoparticles made from difluoroboron
dibenzoylmethane poly(lactic acid) could emit both fluorescence
and phosphorescence for ratiometric hypoxia imaging.[13b] Kim
and coworkers presented an oxygen detection platform in
aqueous solution and gas phase by a core-shell nanoparticles
with an organic chromophore cross-linked polystyrene as
core.[13c] In these carefully chosen polymer matrixes, the long-
lived organic phosphorescence in water as well as oxygen-
permeabilities have been successfully achieved. Inspired by
these works, we wondered whether one could use
supramolecular polymers of small organic molecules to execute
A
single
organic
chromophore
to
generate
phosphorescence and fluorescence simultaneously for
X.-Q. Liu[+], K. Zhang[+], Prof. Y.-Z. Chen, Prof. C.-H. Tung, Prof. L.-Z. Wu
Key Laboratory of Photochemical Conversion and Optoelectronic
Materials, Technical Institute of Physics and Chemistry, Beijing 100190,
China.
E-mail: chenyuzhe@mail.ipc.ac.cn, lzwu@mail.ipc.ac.cn
X.-Q. Liu, Prof. J.-F. Gao
Department of Chemistry, North University of China, Taiyuan 030051,
China
K. Zhang, Prof. Y.-Z. Chen, Prof. C.-H. Tung, Prof. L.-Z. Wu
University of Chinese Academy of Sciences Beijing 100049, China.
[+] These authors contributed equally to this work.
Supporting information for this article is given via a link at the end of the
document.((Please delete this text if not appropriate))
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10.1002/ange.202007039
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hypoxia detection. Representative 2-ureido-4-[1H] pyrimidinone
(UPy) moiety as quadruple hydrogen-bonded unit has been
established forming supramolecular polymers (a binding
constant of up to 6×107 M-1 in chloroform).[16] The facile
structural and functional tunability, stimuli responsiveness and
good oxygen permeability of these supramolecular polymers[8a-b]
promoted us to explore a small organic molecule based
quadruple hydrogen-bonded assembly for ratiometric O2
detection.
bands at 396 nm and 570 nm under nitrogen, with bright orange-
red emission color observed. The redshift of 174 nm between
these two emission peaks, the nanosecond and microsecond
lifetimes of the two emission bands suggest the assignment of
the higher energy band at 396 nm as fluorescence and stronger
lower energy band at 570 nm as phosphorescence. Furthermore,
the emission band at 570 nm totally disappeared under air,
further indicating its phosphorescence character and oxygen-
sensitivity. Together with the total transparency of Ph-bisUPy at
the excitation wavelength (Figure S3), we believe that a pure
organic assembled nanoparticle with intense phosphorescence
and fluorescence from BrNpA-UPy in aqueous solution at room
temperature have been successfully prepared.
Next, SNPs with different ratios of BrNpA-UPy and Ph-
bisUPy were investigated to identify and optimize the optimal
ratios of BrNpA-UPy on the phosphorescence properties. As
shown in Figures 2a and S5a, the phosphorescence quantum
yield reached the maximum as 7.7% at the ratio of Ph-bisUPy:
BrNpA-UPy = 5:1 under nitrogen, which is the most efficient pure
organic RTP in aqueous solution reported so far. The
phosphorescence lifetime also reached the maximum as 3.2 ms
at ratio of 5:1 under nitrogen (Figure S5b), suggesting the
Herein
a
UPy
functionalized
bromine-substituted
naphthalimide derivative (BrNpA-UPy) as the organic
chromophore, and a bis-UPy functionalized benzyl (Ph-bisUPy)
as the skeleton molecule are designed to fabricate organic
supramolecular polymer assembled nanoparticles (SNPs)
(Scheme 1). Intriguingly, BrNpA-UPy emit only fluorescence in
organic solvent at room temperature, while the water-dispersible
SNPs made of BrNpA-UPy can show phosphorescence and
fluorescence simultaneously at room temperature. The
synergistic interaction of the quadruple hydrogen bonds to form
backbone and the heavy atom effect of bromine are believed
responsible for the observed RTP and fluorescence from BrNpA-
UPy (see below). Most importantly, these SNPs could
ratiometrically and quantitatively detect hypoxia (Ksv=189.6 kPa-1) minimal self-quenching and the rigid molecular environment of
in water and in living cells. To our best knowledge, this is the
first example to realize ratiometric hypoxia sensing based on
supramolecular assemblies of small organic monochromophore.
SNPs at this ratio. As noted above, the efficient long-lived
phosphorescence from SNPs in aqueous solution is in contrast
to negligible RTP observed for BrNpA-UPy in CHCl3 (Figure
S3b).
To further understand the phosphorescence enhancement
of SNPs in aqueous solution, we studied the contribution of the
UPy backbone, heavy atom effect and quadruple hydrogen
bonding. Firstly, control nanoparticles prepared from BrNpA-UPy
(SNP-1) in the absence of Ph-bisUPy by the same miniemulsion
method exhibited a cubic mass (Figure S6a). Under the same
excitation conditions, similar phosphorescence peaks at 570 nm
but with lower intensity and shorter lifetime (τp = 0.2 ms) than
those of SNPs were observed in SNP-1 (Figures 2c, S6 and
Table S1). Obviously, the skeleton molecule Ph-bisUPy plays an
important role in the formation of well-dispersed SNPs for
efficient phosphorescence. Secondly, control nanoparticles
SNP-2 from Ph-bisUPy and a bromine-free compound (NpA-
UPy) were prepared. Upon the introduction of halogen, spin-orbit
Figure 1. (a) SEM and TEM (inset) of SNPs (Ph-bisUPy: BrNpA-UPy = 5:1).
(b) Steady-state emission spectra of SNPs-dispersed aqueous solution under
air and nitrogen (blue: Air; red: N2; λex= 348 nm); Inset: Luminescence
photographs of SNPs-dispersed aqueous solution under 365 nm UV irradiation
under air and nitrogen.
coupling between singlet excited state and triplet excited state
[13b, 20]
can facilitate the intersystem crossing.
Thus, SNP-2
exhibited much lower phosphorescence quantum yield (Φp
=1.2%) than those of SNPs (Figures 2 and S7). At last, control
nanoparticles from Ph-bisUPy doping with phosphor molecule
(BrNpA) in the absence of UPy units (SNP-3, Ph-bisUPy:
BrNpA=5:1) showed decreased phosphorescence quantum yield
and lifetime to 3.9% and 2.5 ms as compared with SNPs under
identical conditions, Figures 2 and S8. The differences of these
nanoparticles are also evident by their emission colors pictures
and the Commission Internationale de L'Eclairage (CIE) 1931
Ph-bisUPy and BrNpA-UPy were facilely synthesized from
[17-18]
cheap starting materials.
SNPs from certain ratios of Ph-
bisUPy and BrNpA-UPy in aqueous solution were prepared by
miniemulsion method (see Supporting Information for details).
Driven by the quadruple hydrogen bonding in chloroform, the
first step is the dimerization of UPy moieties, then the emulsified
organic droplets in water enabled aggregation to form SNPs
[17a, 19]
shown in Scheme 1 and Figure S1.
Morphological
characterization by scanning electron microscopy (SEM) and
transmission electron microscope (TEM) indicated the formation
of stable water-dispersible SNPs with a particle size of ca. 240
nm (Figures 1a and S2). The absorption spectra of the SNPs
exhibited the strong characteristic absorption peak at 275 nm,
and 348 nm, 365 nm for Ph-bisUPy and BrNpA-UPy,
respectively, indicating the successful co-assembly of
chromophore with the skeleton molecules (Figure S3a and 4a).
As shown in Figure 1b, upon excitation at 348 nm, SNPs-
dispersed aqueous solution exhibited two distinct emission
[
]
21
chromaticity diagram (Figure S9).
All of the observations
suggest that synergistic interaction of heavy atom effect and
quadruple hydrogen bonding promote the production of triplet
excited state and suppress the vibrational and rotational
nonradiative decay of organic molecules, thus remarkably
enhancing the phosphorescence and fluorescence from SNPs in
aqueous solution (Figure S10).
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10.1002/ange.202007039
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phosphorescence decreased at higher temperature (Figure S11).
[23]
Similar trends as SNPs at ratio of 5:1, the KSV was obtained
as 90.9 kPa-1 and 81.2 kPa-1 for SNPs at ratios of 10:1 and 30:1,
respectively (Figure S13-14). Furthermore, the electron spin
resonance (ESR) signal corresponding to singlet oxygen adduct
is significantly enhanced upon illuminating mixture of SNPs and
2, 2, 6, 6-tetramethylpiperidine (TEMP) (Figure S15), confirming
the production of singlet oxygen through the triplet-triplet energy
transfer process.
Figure 2. (a) Phosphorescence quantum yields (red) and lifetimes (blue) at
different molar ratios of SNPs. (b) The control nanoparticles SNP-1 from
BrNpA-UPy, SNP-2 from NpA-UPy and Ph-bisUPy (5:1), SNP-3 from BrNpA
and Ph-bisUPy (5:1). (c) Steady-state phosphorescence spectra of SNPs (red
line), SNP-1 (green line), SNP-2 (blue line), and SNP-3 (pink line) in aqueous
solution under N2 (λex=348 nm, delay: 0.1 ms). (d) Phosphorescence quantum
yields and lifetimes of SNPs, SNP-2 and SNP-3 in aqueous solutions under
nitrogen.
Subsequently, the dually emissive SNPs at the ratio of Ph-
bisUPy: BrNpA-UPy = 5:1 in aqueous solutions was employed
for the ratiometric O2 sensing experiment for its optimized
fluorescence/phosphorescence intensity ratio and long lifetime.
As shown in Figure 3a, upon excitation, the phosphorescence of
SNPs at 570 nm is gradually quenched by introduction of
different levels of oxygen, and the fluorescence at 396 nm is
hardly affected by changes in oxygen concentration, which
functions as a perfect internal reference signal for ratiometric
analysis. To quantify the oxygen sensitivity of SNPs, R is
defined as the ratio of phosphorescence intensity (IP) at 570 nm
to that of the fluorescence intensity (IF) at 396 nm (R = IP/IF), the
dependence of the intensity ratio RN2/R as a function of oxygen
partial pressure are analyzed according to Stern-Volmer
equation.
Figure 3. (a) Ratiometric spectral responses of the SNPs-dispersed aqueous
solution at varied oxygen levels (0%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%,
0.8%, 1.0%, 1.5%, 2.0%, 3.0%, 4.0%, 8.0%, 14.0%, 21.0%, 100%). (b) Linear
Stern–Volmer plot of RN2/R as a function of oxygen level. Inset: at large
oxygen levels shown for comparison: more significant curvature approaching
ambient level (21%) (c) Steady-state emission spectra of SNPs in aqueous
solution under nitrogen before and after storage for 3 months at room
temperature in the dark. (d) Reversible oxygen detection of SNPs under
oxygen-free/oxygen-saturated cycles. (λex= 348 nm, c=2.1×10-5 M)
Stabilities are very important factor of an excellent
nanoprobe. As indicated by the measured steady-state
luminescence spectra of SNPs, no obvious spectral changes
were observed after over 3 months’ storage at room temperature,
indicating its excellent storage stabilities (Figure 3c). Moreover,
all signal changes are fully reversible under oxygen-free and
oxygen-saturated cycle (Figure 3d). The photostabilities of the
SNPs were also examined under irradiation by a 365 nm LED
lamp (19 mW·cm-2). Even after 3 hours of exposure, UV-vis
absorption spectra hardly showed significant changes (Figure
S16). The above results indicate this organic monochromophore
based optical ratiometric oxygen nanoprobe possesses very
high oxygen sensitivity, excellent storage stability, optical
stability and cycle stability. When SNPs were applied in the
ꢀ
ꢁꢂ
ꢀ
ꢃ 1 ꢄ ꢅꢆꢇꢈOꢉꢊ
(1)
To our delight, the optimal linear range of the oxygen
nanoprobe was found at the oxygen levels of 0-3% (Correlation
coefficient R2
= 0.994, Figure 3b), which are in good
correspondence with biological hypoxia contexts.[22] Sensitive
ratiometric oxygen detection is closely related to the bright
phosphorescence, long decay lifetime and the oxygen
[4a]
permeability of the material.
As a result, the oxygen
quenching efficiency (Q) is calculated to be 95.5% (See
Supporting Information for details). The Stern-Volmer constant
(KSV), which is another important index to indicate the sensing
ability of SNPs for oxygen, was determined to be 189.6 kPa-1.
This is a significant improvement over the Pt(II) porphyrin based
bichromophoric ratiometric oxygen nanosensor (KSV = 23.6 kPa-
1).[8a-b] What’s more, the R value, independent on the probe
concentration, remained practically unaltered at a determined
oxygen level (Figure S11a). On the other hand, the
phosphorescence lifetimes of SNPs at 570 nm decreased
dramatically with increasing oxygen levels (3.2 ms, 1.2 ms and
94 μs for N2, 1% O2 and 21% O2, Figure S12). These are
microenvironments
with
N-ethylmaleimide
(NEM),
N-
acytelcysteine (NAC), and glutathione (GSH) for biomedical
detection, the phosphorescence intensities were independent of
these small biomolecules (Figure S17), implying our hypoxia
nanoprobe was capable of measuring O2 levels in the
intracellular environment without interference.
Given the advantages of the organic nanoprobe, we applied
it for hypoxia detection and imaging in living cells. Here HeLa
cell was chosen as a biometric model. The cytotoxicity of the
nanoprobe was excellent as demonstrated by a standard CCK-8
cell active reagent assay (Figure S18), which facilitate the
biological application of SNPs. Then, the performance of SNPs
consistent with the collisional quenching of phosphorescence by
[8]
oxygen.
As expected, the intensities of the fluorescence and
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10.1002/ange.202007039
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COMMUNICATION
as ratiometric sensor in hypoxia imaging in HeLa cells was
investigated using confocal laser scanning microscopy (CLSM,
Figure 4). When excited by 405 nm laser, blue and red
luminescence observed in the cytoplasm of the cells indicated
that the SNPs can easily penetrate the cell membrane and enter
into the cytoplasm. The blue (425-475 nm, Figure 4b, 4f) and the
red emission channels (570-620 nm, Figure 4c, 4g) are assigned
to the fluorescence and phosphorescence of the organic
chromophore BrNpA-UPy in SNPs, respectively. At 0%and 21%
of O2 atmosphere, the fluorescence intensity was nearly identical,
while the phosphorescence intensity was significantly enhanced
at 0% O2 than 21% O2. The significant change in the Ired / Iblue
ratio in living cells indicates that the SNPs sensor can be
intracellularly hypoxic inductive (Figure 4d, 4h).
Academy of Sciences (Grant XDB17000000), the Key Research
Program of Frontier Sciences of the Chinese Academy of
Sciences (Grant QYZDY-SSW-JSC029), and K. C. Wong
Education Foundation is gratefully acknowledged. The authors
thank Dr. Ye Tian (TIPC) for her help in cytotoxicity experiment.
Keywords: Supramolecular chemistry • Self-assembly • Pure
organic phosphorescence and fluorescence • Oxygen probe •
Ratiometric detection
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Figure 4. Confocal luminescence images of HeLa cells incubated with SNPs
(1.1× 10−6 M in water) at 21% O2 (a-d) and 0% O2 (e-h) partial pressure. (a, e)
Images of bright-field; (b, f) Images of blue channel (425-475 nm); (c, g)
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monochromophore based ratiometric hypoxia nanoprobe from
quadruple-hydrogen bonded assembled nanoparticles. These
nanoparticles can emit efficient long-lived phosphorescence (ΦP
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BrNpA-UPy, which could function as oxygen indicator and
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substitution facilitate bright phosphorescence in aqueous
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possess excellent oxygen permeability to accurately detect
hypoxia ratiometrically in aqueous solution and in living HeLa
cells as well. We believe this unique pure organic hypoxia
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Financial support from the National Natural Science Foundation
of China (Grants 21871280 and 21861132004), the Ministry of
Science and Technology of China (Grant 2017YFA0206903),
the Strategic Priority Research Program of the Chinese
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10.1002/ange.202007039
Angewandte Chemie
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COMMUNICATION
An organic hypoxia nanoprobe
Xiao-Qin Liu+, Ke Zhang+, Jian-
Feng Gao, Yu-Zhe Chen*, Chen-
Ho Tung, and Li-Zhu Wu*
assembled
hydrogen
efficient
by
bonds,
quadruple-
shows
long-lived
(ΦP=7.7%,
Page No. – Page No.
phosphorescence
τP=3.2 ms) and fluorescence
from a monochromophore at
room temperature for oxygen
detection in water and living
cells. This is the first example
to realize ratiometric hypoxia
sensing by supramolecular
assemblies of an organic
monochromophore.
Monochromophore-based
phosphorescence
fluorescence from pure organic
assemblies for ratiometric
hypoxia detection
and
This article is protected by copyright. All rights reserved.