A platinum(II)-acetylide-based conjugated polyelectrolyte for hypoxia imaging via ratiometric and time-resolved luminescence microscopy

Article abstract of DOI:10.1016/j.jorganchem.2018.10.014

A platinum(II)-acetylide-based conjugated polyelectrolyte has been designed and synthesized by using polyfluorenes as an O₂-insensitive fluorophore and Pt(II) complex as an O₂-sensitive phosphor, which can generate conjugated polyelectrolyte nanoparticle (CPE-nanoparticle) in the aqueous solution owing to their amphiphilic structures. The CPE-nanoparticle displays good sensitivity to O₂ concentration and can detect oxygen levels reversibly. The intracellular ratiometric O₂ sensing performance of the CPE-nanoparticle has been demonstrated by the remarkable variation in the I_green/I_blue ratio values (0.18–0.85) in HeLa cells under different O₂ levels. Furthermore, O₂ level detection was carried out through time-resolved luminescence imaging (TRLI) to demonstrate the accuracy of the probe based on the CPE-nanoparticle. The CPE-nanoparticle shows a high phosphorescence quantum yield (19.98%) and oxygen quenching efficiency (0.975), which are superior to the existing O₂ probe. The CPE-nanoparticle has been successfully applied in photoluminescence lifetime imaging and time-gated luminescence imaging for monitoring intracellular O₂ levels.

Full text of DOI:10.1016/j.jorganchem.2018.10.014

Accepted Manuscript
A platinum(II)-acetylide-based conjugated polyelectrolyte for hypoxia imaging via
ratiometric and time-resolved luminescence microscopy
Guo Li, Tianci Huang, Mingjuan Xie, Xiangxiang Zhang, Qi Yu, Shujuan Liu, Tianshe
Yang, Qiang Zhao
PII:
S0022-328X(18)30764-2
DOI:
https://doi.org/10.1016/j.jorganchem.2018.10.014
JOM 20599
Reference:
To appear in: Journal of Organometallic Chemistry
Received Date: 31 August 2018
Revised Date: 15 October 2018
Accepted Date: 18 October 2018
Please cite this article as: G. Li, T. Huang, M. Xie, X. Zhang, Q. Yu, S. Liu, T. Yang, Q. Zhao, A
platinum(II)-acetylide-based conjugated polyelectrolyte for hypoxia imaging via ratiometric and
time-resolved luminescence microscopy, Journal of Organometallic Chemistry (2018), doi: https://
doi.org/10.1016/j.jorganchem.2018.10.014.
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ACCEPTED MANUSCRIPT
2
Graphical Abstract
A platinum(II)-acetylide-based conjugated
polyelectrolyte for hypoxia imaging via
ratiometric and time-resolved
luminescence microscopy
Guo Li, Tianci Huang, Mingjuan Xie, Xiangxiang Zhang,
Qi Yu, Shujuan Liu, Tianshe Yang, Qiang Zhao*
Key Laboratory for Organic Electronics and Information Displays & Jiangsu Key Laboratory for Biosensors, Institute
of Advanced Materials (IAM), Nanjing University of Posts and Telecommunications (NUPT), 9 Wenyuan Road,
Nanjing 210023, China.
We designed and synthesized a platinum(II)-acetylide-based conjugated polyelectrolyte, which can form conjugated
polyelectrolyte dots (CPE-Pdots) in water owing to their amphiphilic structures. The CPE-Pdots display good
sensitivity to O2 concentration and can detect oxygen levels reversibly. The intracellular ratiometric O2 sensing
performance of the CPE-Pdots has been demonstrated by the remarkable variation in the Igreen/Iblue ratio values in HeLa
cells under different O2 levels.
The CPE-Pdots have been successfully applied in photoluminescence lifetime imaging and time-gated luminescence imaging for
monitoring intracellular O2 levels, which will remarkably improve the accuracy and enhance the signal-to-noise ratio.

1
ACCEPTED MANUSCRIPT
Journal of Organometallic Chemistry
journal homepage: www.elsevier.com
A platinum(II)-acetylide-based conjugated polyelectrolyte for hypoxia imaging via
ratiometric and time-resolved luminescence microscopy
Guo Li, Tianci Huang, Mingjuan Xie, Xiangxiang Zhang, Qi Yu, Shujuan Liu,* Tianshe Yang, Qiang Zhao*
Key Laboratory for Organic Electronics and Information Displays & Jiangsu Key Laboratory for Biosensors, Institute of Advanced Materials (IAM), Nanjing
University of Posts and Telecommunications (NUPT), 9 Wenyuan Road, Nanjing 210023, China.
Corresponding E-mail: iamqzhao@njupt.edu.cn (Q. Zhao), iamsjliu@njupt.edu.cn (S. Liu)
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
A platinum(II)-acetylide-based conjugated polyelectrolyte has been designed and synthesized by
using polyfluorenes as an O2-insensitive fluorophore and Pt(II) complex as an O2-sensitive
phosphor, which can generate conjugated polyelectrolyte dots (CPE-Pdots) in the aqueous
solution owing to their amphiphilic structures. The CPE-Pdots display good sensitivity to O2
concentration and can detect oxygen levels reversibly. The intracellular ratiometric O2 sensing
performance of the CPE-Pdots has been demonstrated by the remarkable variation in the
Igreen/Iblue ratio values in HeLa cells under different O2 levels. Furthermore, O2 level detection
was carried out through time-resolved luminescence imaging (TRLI) to demonstrate the
accuracy of the probe based on the CPE-Pdots. The CPE-Pdots have been successfully applied
in photoluminescence lifetime imaging and time-gated luminescence imaging for monitoring
intracellular O2 levels, which will remarkably improve the accuracy and enhance the
signal-to-noise ratio.
Available online
Keywords:
Hypoxia
Conjugated polyelectrolyte
Pt(II) complex
Ratiometric imaging
Time-resolved photoluminescence imaging
2018 Elsevier Ltd. All rights reserved.
1. Introduction
advantages that are suitable to be used as bio-probes, including
the tunable emission, good cell membrane permeability and high
Cellular oxygen (O2), which plays a crucial role in many
physiological process, is one of the most vital components in
biological systems.[1] Hypoxia, defined as oxygen-deprived
condition, is a distinct feature of multifarious disease including
tumors,[2] stroke,[3] and retinal disease.[4] In some solid tumors,
the median oxygen concentration has been reported to be around
4%, and locally, it can be as low as 0%.[5] Therefore, accurate
detection of tumor hypoxia is not only great importance for
disease diagnosis , but also can be used to evaluate therapeutic
effects. Therefore, attention has been dedicated to the
development of hypoxia probe.
quantum yields. The triplet excited state of PTMCs is usually
responsive to the triplet ground state of molecular oxygen
through energy transfer.[27,28] PTMCs have attracted considerable
interest in imaging and sensing the oxygen level in vitro and in
vivo.[29-34] However, most of these oxygen probes are usually
water insoluble and based on the single variation of
phosphorescent emissive intensity, which is easily influenced by
the complex intracellular microenvironment and external
experimental conditions, such as excitation power and probe
concentration. Therefore, ratiometric probes, whose ratio
represents the variation value of the emission intensities at two
paths, can improve the accuracy for hypoxia sensing,[32,35] as well
as eliminate photobleaching and excitation power fluctuation.[36]
Conjugated polyelectrolytes (CPEs) is a good choice, owing to
their distinguished merits such as strong absorbance, easy
modification, high fluorescent efficiency, good photostability and
biocompatibility.[37-41] In addition, the charged side chains of
CPEs allow them good solubility in aqueous solution, which is
beneficial for bioimaging and biosensing in vitro and in vivo.[42,43]
Considering the merits of both PTMCs and CPEs, herein, a
dual-emissive conjugated polyelectrolytes containing platinum
acetylide has been designed and synthesized for hypoxia sensing.
A long-lived phosphorescent platinum(II) complex has been
introduced into conjugated polyelectrolyte as the O2-sensitive
phosphorescent moieties, and O2-insensitive fluorescence from
To date, immunostaining,[6-8] magnetic resonance imaging,[9,10]
positron emission tomography imaging,[11-13] near infrared
tomographic imaging[14,15] and optical imaging[16,17] are available
approaches to detect hypoxic regions in vitro and in vivo. As a
non-invasive optical technology, optical imaging has been an
outstanding method to map oxygen in biological samples owing
to the high sensitivity and spatial resolution.[18,19] However,
extremely low intracellular oxygen level and long incubation
time may be required for most of these probes.[20-22] Moreover,
the binding of these probes to cancer cells is also obviously
impaired by the low pH of the tumor microenvironment.[23,24] All
the limitations compromise the application of these probes in
accurately detection hypoxia in cancer cells and tumor tissues.
Phosphorescent transition metal complexes (PTMCs), which
possess long lifetime, can be used as an effective probe in
imaging to eliminate short-lived autofluorescence and increase
the signal-to-noise ratio by time-resolved photoluminescence
technique (TRPT).[25,26] Additionally, PTMCs own distinctive
polyfluorene moieties as
a
reference. Therefore, the
dual-emissive CPEs dots have been successfully synthsized as O2
probes. The ratiometric measurements of hypoxia were carried
out in living cells. In addition, O2 levels in living cells have been

2
investigated through time-resolved luminescence imaging
techniques, including photoluminescence lifetime imaging
microscopy (PLIM) and time-gated luminescence imaging
(TGLI). These results indicated that CPE-dots are ideal
luminescent probes in monitoring O2 concentrations in living
biological samples.
results of zeta-potential was 31.00 mV, facilitating the
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interaction with cell membrane potential through electrostatic
interactions.
2. Experimental section
All materials and regents were used as received from
commercial sources unless stated otherwise. The solvents (NEt3
and CH2Cl2) were purified under reflux over CaH2 for several
hours, distilled at these conditions. The structure of P1 was
1
characterized by H NMR and gel permeation chromatography
(GPC). Transmission electron microscopy (TEM) was conducted
on a JEOL transmission electron microscope (JEM-2100).
Average particle size was measured by dynamic light scattering
(DLS) on Zetasizer Nanoseries (Nano ZS90). Zeta-potential was
measured by Zeta-plus zeta potential analyzer. The UV-visible
absorption spectra were recorded using a Shimadzu UV-3600
UV-VIS-NIR spectrophotometer. Oxygen concentration was
monitored
by
Mass
Flow
Controller
or
oxygen
Scheme 1. Synthetic route of the target conjugated polyelectrolyte P2 and the
intermediate P1.
concentration-changeable multigas incubator during the spectra,
lifetime and cell imaging measurements.
The conjugated polyelectrolyte P1 was obtained in the
presence of catalytic amount of CuI via reaction between
trans-PtCl2(PBu3)2 and the diethynyl ligand (Scheme 1). M1
(302 mg, 0.50 mmol), Pt1 (335 mg, 0.50 mmol) and CuI (9.5 mg,
0.05 mmol) in distilled 40 mL Et3N/CH2Cl2 (1:1, v/v) were
stirred for 0.5 h at room temperature under nitrogen. After the
reaction was finished, the solvent was evaporated under reduced
pressure. After concentrating the CH2Cl2 solution, yellow solid
was collected by precipitation into a large excess of methanol.
Condensed trimethylamine (3 mL) was added dropwise to a
solution of the polymer P1 (150 mg) in THF (20 mL) at -78 °C
using dry ice-acetone bath. The mixture was stirred for 24 h at
room temperature. After removing most of the solvent, acetone
was added to precipitate P2. The resulting polymers were dried
under vacuum with a yield of 65%.
3.2 Photophysical properties
The photophysical properties of P2 have been investigated by
UV-vis absorption and luminescence spectra. As shown in the
absorption spectra of Fig. 2b, the absorption peak of P2 was
centered at 396 nm in aqueous solution, which was ascribed to
the π-π* transition of the fluorene unit and Soret bands of Pt(II)
complex (Fig. 2b). In the emission spectra of P2, two major
emission bands at 415 and 550 nm were observed in degassed
aqueous solution, which were assigned to polyfluorene main
chain and Pt(II) complex, respectively. The high-energy band of
the emission spectra is attributed to the fluorescence emission
1
from π-π* state of fluorene with short lifetime (τF = 0.33 ns),
while the emission band at 550 nm is from the long-lived
phosphorescence from Pt complex (τP = 5.96 µs). Moreover, P2
show no Förster resonance energy transfer (FRET) between the
polyfluorenes and Pt(II) complex according to the excitation
spectra (Figure S3).
3. Results and discussion
3.1 Synthesis and characterization
To develop a new and excellent dual-emissive long-lived
hypoxia probes for biosensing and bioimaging, Pt coordination
centers is directly introduced onto the fluorene π-conjugated
backbones. The synthetic routes and the chemical structures of
the CPEs were illustrated in Scheme 1 and Scheme S1 in the
Supporting Information (SI). The platinum acetylide-based
polymer P1 containing fluorene unit was obtained in the presence
of CuI as catalyst via reaction between trans-PtCl2(PBu3)2 and
the diethynyl ligand. The structure of P1 was characterized by 1H
NMR. The molecular weight of conjugated polyeletrolyte was
characterized by GPC (Mn ≈ 54 724 and Mw ≈ 96 596 with
3.3 Luminescence response to O2 content
To investigate the oxygen-responsive ability of P2, the
emission spectra of the CPE-Pdots under different oxygen
concentrations in aqueous solution were measured, and the
results were shown in Fig. 2c. Under 21% O2, the emissions at
415 nm and 430 nm of CPE-Pdots, which was attributed to
polyfluorene units, were stronger than those at 550 nm and 590
nm from Pt(II) complex. When the O2 level decreased, the
intensity of fluorescence at 415 nm showed negligible change,
while the green emission intensity at 550 nm enhanced
remarkably, revealing the phosphorescence sensitivity of Pt(II)
complex to O2. Therefore, a ratiometric oxygen probe was
achieved in aqueous solution based on the phosphorescent
CPE-Pdots.
polydisperisity index PDI
= 1.76). The target platinum
acetylide-based polyelectrolyte P2 was prepared via the
quarternization of the precursor P1.
To investigate the size and morphology of P2, TEM and DLS
measurements were carried out and the results were shown in Fig.
2a and Figure S2. The size of CPE-Pdots was about 3 nm, which
was formed through self-assembly, owing to the amphiphilic
structures of P2 with hydrophobic backbones and hydrophilic
side chains. As show in Figure S2b, DLS measurement shows
that CPE-Pdots possessed an average hydrodynamic diameter of
about 12 nm, which is larger than that measured by TEM because
of the shrinkage at dry state on copper grid. In addition, the
The quantitative oxygen sensing performance of the
CPE-Pdots was investigated through the emission as shown in
Fig. 2c. The oxygen level can be calculated according to the
Stern-Volmer equation:
ꢀꢁꢂ
= 1 + ꢃꢄꢅꢆꢇꢈ
(1)
ꢀꢁ

3
where Ksv is the constant of quenching rate, pO is the oxygen
CPE-Pdots under different concentration (Figure S6). The result
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2
level, RI = (I5500/I4150) and RI = (I550/I415) are the ratios of the
phosphorescence (550 nm) intensity of Pt(II) complex to the
fluorescence (415 nm) intensity of the polyfluorene units in the
absence and presence of O2, respectively.
of cell viabilities (%) versus incubation concentrations (0-100
µM) of the CPE-Pdots in PBS buffer at 37 °C for 72 h is
elucidated in Figure S6. The cell viability was more than 78%
when CPE-Pdots (100 µM) was added in the incubation medium
0
f
o
r
7
2
h
,
Fig. 3. Confocal luminescence imaging and ratiometric luminescence
imaging (λex = 405 nm) of Hela cells incubated with the polymer (20 µg mL-1)
at 21% or 2.5% O2 concentrations. In luminescence imaging, the emission
channels at wavelengths of 410–460 nm and 520–580 nm were collected,
respectively. In ratiometric imaging, the ratio of emission intensity at 520–
580 nm to that at 410–460 nm was chosen as the detected signal.
demonstrating that the CPE-Pdots have low cytotoxicity and
excellent biocompatibility for potential biological applications.
Fig. 2. (a) A TEM image of CPE-Pdots in aqueous solution. (b) Absorption
spectrum of CPE-Pots (15 µg mL-1) in aqueous solution. (c) Emission spectra
of CPE-Pdots (15 µg mL-1) in aqueous solution under different O2
concentrations excited at 365 nm. (d) Plot of RI0/RI as a function of O2
concentration. (e) Phosphorescence decays of CPE-Pdots in aqueous solution
saturated with N2 or air, monitored at 550 nm from the Pt(II) complex excited
3.6 Ratiometric measurement of hypoxia in vitro
To investigate the capability of hypoxia sensing quantitatively
in living cells, ratiometric measurement was carried out by using
CPE-Pdots. HeLa cells (human cervical carcinoma cells) were
cultured at 37 °C with the CPE-Pdots in PBS at pH 7.4 under
21%, 10%, 5% and 2.5% oxygen levels for 4 h. Under excitation
at 405 nm, the fluorescence and phosphorescence signals were
collected at blue (410-460 nm) and green (520-580) channel,
respectively. As shown in Fig. 3 and Figure S7, the emission at
the blue channel almost remained a certain value under different
oxygen concentrations, while the phosphorescence emission from
Pt(II) complex enhanced with decreasing oxygen level. The ratio
at 405 nm. (f) Plot of τ0/τ as a function of O2 concentration (λex = 405 nm).
0
As shown in Fig. 2d and Figure S4, RI /RI and pO2 revealed a
good linear relationship and the Ksv value is calculated to be 1.17
× 10-2 mmHg-1. Moreover, small fluctuation was found at 415 nm
under different O2 concentrations (Fig. 2c), demonstrating that
the ratio of the emissive intensity at 550 nm and at 415 nm of the
CPE-Pdots relied on O2 level. Thus, the O2 concentration can be
calculated based on the value of Ksv.
of phosphorescence signal (Igreen) to fluorescence signal (Iblue
)
a
l
s
o
3.4 Lifetime response to O2
In addition, the O2 sensing was also investigated by lifetime
measurements. The oxygen level can be calculated according to
the Stern-Volmer equation:
τ
ꢉ
= 1 + ꢃꢄꢅOꢈ
(2)
τ
where Ksv is the quenching constant, pO2 is the oxygen level, τ0
and τ are the phosphorescence lifetimes in the absence and
presence of O2, respectively.
As shown in Fig. 2e, the luminescence lifetime at 550 nm of
CPE-Pdots in degassed conditions was 30.52 µs. The lifetime
decreased to 5.96 µs when the O2 level enhanced to 21%. In
addition, τ0/τ and pO2 had a good linear relationship, as shown in
Fig. 2f. According to the slope of the trend line, the Ksv value is
calculated to be 2.71 × 10-2 mmHg-1. However, the fluorescence
lifetime at 415 nm of the CPE-Pdots remains unchanged at
different O2 levels (Figure S5). As a result, the CPE-Pdots are
able to used as a hypoxia probe through luminescence lifetime
measurements.
3.5 Cell cytotoxicity
To evaluate the cytotoxicity of the CPE-Pdots in living cells,
MTT (methyl thiazolyl tetrazolium) assay was carried out for

4
Fig. 4. (a) Photoluminescence lifetime images (λex = 405 nm) of Hela cells
Academic Program Development of Jiangsu Higher Education
Institutions (YX03001).
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incubated with the polymer (20 µg mL-1) at 37 °C for 2 h at 21% and 5% O2
concentrations, respectively. The magnification of the objective lens is 60×.
The luminescence signals were collected in the range of 520–600 nm. (b)
Time-gated luminescence intensity images (λex = 405 nm) of Hela cells
incubated with the polymer 20 µg mL-1 at 37 °C for 2 h at 21% or 5% O2
concentrations with different time delays. The magnification of the objective
lens is 60×. The luminescence signals were collected in the range of 410–580
nm
Supplementary data
Supplementary data (details of the CPEs synthesis, NMR, cell
culture) associated with this article can be found in Supporting
Information.
References
increased from 0.18 to 0.85 along with the decreasing of oxygen
level from 21% to 2.5%, which allows us to monitor the
concentration of oxygen.
[1] J. Aragonés, P. Fraisl, M. Base, P. Carmeliet, Cell Metab. 9 (2009)
11-22.
[2] W. R. Wilson, M. P. Hay, Nat. Rev. Cancer. 11 (2011) 393-410.
[3] K. Prass, A. Scharff, K. Ruscher, D. Lowl, C. Muselmann, I. Victorov,
K. Kapinya, U. Dirnagl, A. Meisel, Stroke. 34 (2003) 1981-1986.
[4] R. N. Frank, N. Engl. J. Med. 350 (2004) 48-58.
[5] P. Vaupel, K. Schlenger, C. Knoop, M. Hockel, Cancer Res. 51 (1991)
3316-3322.
[6] S. Kizaka-Kondoh, M. Inoue, H. Harada, M. Hiraoka, Cancer Sci. 94
(2003) 1021-1028.
[7] A. L. Harris, Nat. Rev. Cancer. 2 (2003) 38-47.
[8] E. K. Rofstad, H. Rasmussen, K. Galappathi, B. Mathiesen, K. Nilsen,
B. A. Graff, Cancer Res. 62 (2002) 1847-1853.
[9] T. Inoue, E. Kozawa, H. Okada, K. Inukai, S. Watanabe, T. Kikuta, Y.
Watanabe, T. Takenaka, S. Katayama, J. Tanaka, H. Suzuki, J. Am. Soc.
Nephrol. 22 (2011) 1429-1434.
3.7 TRLI images of intracellular O2 concentration
Considering the phosphorescence lifetime of CPE-Pdots
showed a good linear relationship with oxygen level, lifetime
imaging was also conducted to monitor the intracellular oxygen
level by using CPE-Pdots as a lifetime-based probe. The mean
emission lifetime τ was approximately 130 ns (21% O2) and 360
ns (2.5% O2), demonstrating the sensitive response of the lifetime
of CPE-Pdots towards O2 levels (Fig. 4a). The PLIM results
indicated that the emission signals from CPE-Pdots could be
distinguished
from
the
interference
of
short-lived
autofluorescence. Therefore, it is feasible to use the CPE-Pdots
as a potential lifetime probe for discriminating normoxia and
hypoxia in living cells.
[10] S. Iwaki, K. Hanaoka, W. Piao, T. Komatsu, T. Ueno, T. Terai, T.
Nagano, Bioorg. Med. Chem. Lett. 22 (2012) 2798-2802.
[11] S. Apisarnthanarax, K. S. C. Chao, Rad. Res. 163 (2005) 1-25.
[12] J. L. J. Dearlin, J. S. Lewis, G. E. D. Mullen, M. J. Welch, P. J. J.
Blower, Biol. Inorg. Chem. 7 (2002) 249-259.
[13] D. J. Yang, S. Wallace, A. Cherif, C. Li, M. B. Gretzer, E. E. Kim, D.
A. Podoloff, Radiology 194 (1995) 795-800.
[14] B. M. Fenton, S. F. Paoni, J. Lee, C. J. Koch, E. M. Lord, Brit. J.
Cancer 79 (1999) 464-471.
[15] H. L. Liu, Y. L. Song, K. L. Worden, X. Jiang, A. Constantinescu, R.
P. Mason, Appl. Opt. 39 (2000)5231-5243.
[16] K. Kiyose, K. Hanaoka, D. Oushiki, T. Nakamura, M. Kajimura, M.
Suematsu, H. Nishimatsu, T. Yamane, T. Terai, Y. Hirata, T. Nagano, J. Am.
Chem. Soc. 132 (2010) 15846-15848.
[17] G. M. Palmer, A. N. Fontanella, S. Q. Shan, G. Hanna, G. Q. Zhang,
C. L. Fraser, M. W. Dewhirst, Nat. Protoc. 6 (2011) 1355-1366.
[18] G. Zhang, G. M. Palmer, M.W. Dewhirst, C. L. Fraser, Nat. Mater. 8
(2009) 747-751.
[19] S. Zhang, M. Hosaka, T. Yoshihara, K. Negishi, Y. Iida, S. Tobita, T.
Takeuchi, Cancer Res. 70 (2010) 4490-448.
Furthermore, to eliminate the short-lived autofluorescence in
complicated conditions in living cells, TGLI imaging was used to
monitor the intracellular oxygen level (Fig. 4b). Without delay
time, the signals of images showed a similar intensity at 21% and
5% O2 concentrations, owing to the reference fluorescence from
short lifetime moieties. However, by exerting a 250 ns decay
time, the signal of the images at 21% O2 concentration
disappeared whereas the signal of the images at 5% O2
concentration was still observed, which ascribed to the long-lived
phosphorescence from Pt(II) complex of the CPE-Pdots. These
results indicated that hypoxia sensing could be more accurate by
exerting a long delay time. Therefore, TGLI imaging can avoid
anti-interference effectively and improve the signal to noise ratio
significantly on the basis of long-lived phosphorescence signal.
[20] G. E. Arteel, R.G. Thurman, J. M. Yates, J. A. Raleigh, Br. J. Cancer.
72 (1995) 889-895.
4. Conclusion
[21]
M. W. Gross, U. Karbach, K. Groebe, A. J. Franko, W.
In summary, a dual emissive and biocompatible hypoxia probe
CPE-Pdots has been developed for the high sensitive and reliable
imaging of hypoxia. The phosphorescence emissive intensity and
lifetime from CPE-Pdots showed an excellent sensitivity to
oxgen level. The probe can be readily used for ratiometric
measurement, allowing for the quantitative evaluation of the
hypoxia concentration in living cells with low cytotoxicity.
Furthermore, the CPE-Pdots can employ TRLI techniques to
monitor O2, which can remarkably improve the sensitivity and
accuracy. We believe that the CPE-Pdots can provide a specific
tool for hypoxia imaging, and will enrich the design of
ratiometric and lifetime-based probes for monitoring hypoxia in
living cells. Further work will focus on developing efficiently
NIR-excitable hypoxia probes in vivo.
Mueller-Klieser, Int. J. Cancer. 61 (1995) 567-573.
[22] M. R. Horsman, L. S. Mortensen, J. B. Petersen, M. Busk, J.
Overgaard, Nat. Rev. Clin. Oncol. 9 (2012) 674-687.
[23] M. M. Kleiter, D. E. Thrall, D.E. Malarkey, D. Y. W. Lee, S. C. Chou,
J. A. Raleigh, Int. J. Radiat. Oncol. Biol. Phys. 64 (2006) 592-602.
[24] Y. G. Wang, K. J. Zhou, G. Huang, C. Hensley, X. N. Huang, X. P.
Ma, T. Zhao, B. D. Sumer, R. J. DeBerardinis, J. M. Gao, Nat. Mater. 13
(2014) 204-212.
[25] Q. Zhao, F. Y. Li, C. H. Huang, Chem. Soc. Rev. 39 (2010) 3007-3030.
[26] K. Y. Zhang, Q. Yu, H. Wei, S. J. Liu, Q. Zhao, W. Huang, Chem.
Rev. 118 (2018) 1170-1839.
[27] D. B. Papkovsky, R. I. Dmitriev, Chem. Soc. Rev. 42 (2013)
8700-8732.
[28] C. F. Wu, B. Bull, K. Christensen, J. McNeill, Angew. Chem. 121
(2009) 2779-2783； Angew. Chem. Int. Ed. 48 (2009) 2741-2745.
[29] T. Yoshihara, Y. Yamaguchi, M. Hosaka, T. Takeuchi, S. Tobita,
Angew. Chem. Int. Ed. 51 (2012) 4148-4151.
[30] S. J. Zhang, M. Hosaka, T. Yoshihara, K. Negishi, Y. Iida, S. Tobita,
T. Takeuchi, Cancer. Res. 70 (2010) 4490-4498.
Acknowledgments
[31] T. C. Huang, X. Tong, Q. Yu, H. R.Yang, S. Guo, S. J. Liu, Q. Zhao,
K. Y. Zhang, W. Huang, J. Mater. Chem. C. 4 (2016) 10638-10645.
[32] Q. Zhao, X. B. Zhou, T. Y. Cao, K. Y. Zhang, L. J. Yang, S. J. Liu, H.
Liang, H. R. Yang, F. Y. Li, W. Huang, Chem. Sci. 6 (2015) 1825-1831.
[33] H. F. Shi, X. Ma, Q. Zhao, B. Liu, Q. Y. Qu, Z. F. An, Y. L. Zhao, W.
Huang, Adv. Funct. Mater. 24 (2014) 4823-4830.
This work was supported by the National Natural Science
Foundation of China (51473078, 21501098 and 21671108),
National Program for Support of Top-Notch Young Professionals,
Scientific and Technological Innovation Teams of Colleges and
Universities in Jiangsu Province (TJ215006), and Priority
[34] W. Lv, T. S. Yang, Q. Yu, Q. Zhao, K. Y. Zhang, H. Liang, S. J. Liu,

5
F. Y. Li, W. Huang, Adv. Sci. 2015, 2, 1500107.
[35] X. C. Zheng, X. Wang, H. Mao, W. Wu, B. R. Liu, X. Q. Jiang, Nat.
Commun. 6 (2015) 5834-6834.
ACCEPTED MANUSCRIPT
[36] I. Sanchez-Barragan, J. M. Costa-Fernandez, M. Valledor, J. C.
Campo, A. Sanz-Medel, Trends Anal. Chem. 25 (2006) 958-967.
[37] X. L. Feng, L. B. Liu, S. Wang, D. B. Zhu, Chem. Soc. Rev. 39 (2010)
2411-2419.
[38] F. D. Feng, F. He, L. L. An, S. Wang, Adv. Mater. 20 (2008)
2959-2964.
[39] C. L. Zhu, L. B. Liu, Q. Yang, F. T. Lv, S. Wang, Chem. Rev. 112
(2012) 4687-4735.
[40] H. Jiang, P. Taranekar, J. R. Reynolds, K. S. Schanze, Angew. Chem.
Int. Ed. 48 (2009) 4300-4316.
[41] A. Herland, O. Inganäs, Macromol. Rapid Commun. 28 (2007)
1703-1713.
[42] K. Lee, J. Lee, J. E. Jeong, A. Kronk, K. S. J. Elenitoba-Johnson, M. S.
Lim, J. Kim, Adv. Mater. 24 (2012) 2479-2484.
[43] X. L. Feng, G. M. Yang, L. B. Liu, F. T. Lv, Q. Yang, S. Wang, D. B.
Zhu, Adv. Mater. 24 (2012) 637-641.

ACCEPTED MANUSCRIPT
Highlights
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A platinum(II)-acetylide based conjugated polyelectrolyte is prepared, which can
form conjugated polyelectrolyte dots (CPE-Pdots) in water owing to their
amphiphilic structures.
The intracellular ratiometric O2 sensing performance of the CPE-Pdots has been
demonstrated by the remarkable variation in the Igreen/Iblue ratio values in HeLa
cells under different O2 levels.
The CPE-Pdots have been successfully applied in photoluminescence lifetime
imaging and time-gated luminescence imaging for monitoring intracellular O2
levels, which will remarkably improve the accuracy and enhance the
signal-to-noise ratio.