Polymer

Chemistry
View Article Online
PAPER View Journal
Published on 24 September 2020. Downloaded by University of New England on 10/10/2020 2:01:37 PM.

A fluorescent sensor for intracellular Zn2+

Cite this: DOI: 10.1039/d0py01054e
based on cylindrical molecular brushes of
poly(2-oxazoline) through ion-induced emission†
Shanshan Chen,a,b Tingting Sun, c
Zhigang Xie, c
Dewen Dong *a,b and
Ning Zhang *a,d

Received 23rd July 2020,
Accepted 24th September 2020
DOI: 10.1039/d0py01054e
rsc.li/polymers
Mobile divalent zinc, Zn(II), is an essential cofactor involved in mammalian biological processes. To under-
stand the intrinsic action mechanism of mobile zinc, the development of a Zn2+-selective fluorescent
probe that can detect mobile zinc via biological imaging is of great significance. We present here a novel
Zn2+-selective fluorescent sensor based on poly(2-oxazoline) (POx) cylindrical molecular brushes with
good water solubility and biocompatibility. The POx molecular brushes were end-capped by a Zn2+-
responsive functionality, which can give efficient blue emission upon coordination with zinc ions.
Fluorescence investigation indicates that the POx molecular brushes exhibit superior selectivity toward
Zn2+ in comparison with most competitive heavy and transition metal ions. Due to the ease of cellular
uptake that results from their unique molecular contour, POx molecular brushes are capable of detecting
intracellular Zn2+ ions as demonstrated by living cell fluorescence studies.

Introduction poorly understood. A Zn2+-selective fluorescent sensor offers a

Zinc is the second most abundant transition metal ion in the
human body after iron, and plays an important role in many
biological activities such as brain function, gene transcription,
cellular metabolism and immune function.1,2 Zinc is also
involved in pathological processes such as Alzheimer’s disease
and diabetes.3 It is therefore of importance to clarify the exact
concentration and even the subtle variation of zinc. These bio-
logical zinc ions are either tightly bound to proteins having a
catalytic or structural function, or loosely bound to proteins
reversibly with dissociation and association rates commensu-
rate with the requirements in regulation, transport, transfer,
sensing, signaling, and storage.4–6 This mobile zinc, which is
the main target of fluorescent sensors, exists in some tissues
such as the central nervous system, pancreas, retina, and intes-
tines,7 and is crucial in mammalian physiology. To date, the
action of mobile zinc within and between live cells has been
a
CAS Key Laboratory of High-Performance Synthetic Rubber and Its Composite
Materials, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences,
Changchun 130022, China. E-mail: zhangn380@nenu.edu.cn
b
University of Science and Technology of China, Hefei 230026, China
c
State Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of
Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, China
d
Department of Chemistry, Northeast Normal University, 5268 Renmin Street,
Changchun 130024, China
† Electronic supplementary information (ESI) available: The characterization
spectra of the synthesized polymers. See DOI: 10.1039/d0py01054e
widely applicable and practical tool in detecting such mobile
zinc. In the past decades, various Zn2+-responsive fluorescent
sensors have been developed to image mobile zinc in the live
cell environment.8 Arenesulfonamides of 8-aminoquinoline,
such as TSQ9 and Zinquin,10 are the first reported sensor
molecules for cellular Zn2+, and since then various fluorescent
sensors have been established, e.g. TSQ derivatives and
peptide or protein-based sensors.11–13 Recently, Xu et al.
demonstrated that naphthalimide-based fluorescent sensors
could efficiently detect Zn2+ by forming an imidic acid tauto-
mer after binding to Zn2+.14 Reaction-based fluorescent
sensors are another alternative which can exhibit different
responses before and after exposure to Zn2+.15
Until now, most of the reported fluorescent sensors
detected zinc ions with poor sensitivity or selectivity due to
their similar affinities for other heavy and transition metal
ions. For instance, some available Zn2+ fluorescent sensors
have difficulty in discriminating between Zn2+ and Cd2+ since
they are in the same group of the periodic table and have the
intrinsic d10 shell which leads to similar properties. It is still a
significant challenge to develop a highly selective Zn2+ fluo-
rescent sensor that can exclude interference of some heavy and
transition metal ions such as Fe2+, Ni2+, Cu2+, Hg2+ and Cd2+,
especially one that can distinguish between Zn2+ and Cd2+
with different emission wavelengths.14
At present, various kinds of Zn2+ fluorescent sensors can be
classified into: (a) organic molecule luminescent probes (such

This journal is © The Royal Society of Chemistry 2020 Polym. Chem.


Paper
Published on 24 September 2020. Downloaded by University of New England on 10/10/2020 2:01:37 PM.
Scheme 1 Schematic illustration for the synthesis of P(IPOx-g-EtOx).
as the derivatives of quinolone, indole, naphthol, furan and
fluorescein);16–18 (b) luminescent nanomaterials (such as
carbon dots, semiconductor quantum dots and upconversion
nanoparticles);19–22 and (c) polymer-based fluorescent
probes,23,24 which use internal charge transfer (ICT), chela-
tion-enhanced fluorescence (CHEF), photo-induced electron
transfer (PET) or fluorescence resonance energy transfer
(FRET) mechanisms.25–28 However, most of them are either
poisonous, biologically incompatible, less membrane per-
meable and selective, or water-insoluble, which may greatly
limit the intracellular detection of Zn2+ ions from human cells.
It is still a challenge to design a sensor with highly selective,
sensitive, biologically compatible and water-soluble Zn2+ fluo-
rescent probes. To cope with the abovementioned dilemma,
water-soluble polymer-based sensors with good biocompatibil-
ity might meet all the demands compared to the common
fluorescent probes.
Kaur et al. developed a polymer-based Schiff base fluo-
rescent sensor which can detect Zn2+ in a real microbial cell.29
It demonstrated that the polymer-based sensor is feasible for
intracellular Zn2+. A proper polymer chosen for the sensor
seems to be essential for diverse biological and medical
applications.
Hydrophilic poly(2-oxazoline)s (POx) came into focus as a
potential alternative to conventional biocompatible polymers
due to their good water solubility and biocompatibility.30
Living cationic ring-opening polymerization used in the prepa-
Polym. Chem.
View Article Online
Polymer Chemistry
ration of POx guarantees the tailoring of molecular architec-
tures. Thus, POx with various topologies such as linear,
branched and brush-like can be obtained with the aid of living
cationic ring-opening polymerization (LCROP) and modern
synthetic tools. In 2009, we reported for the first time the
preparation of molecular brushes of poly(2-oxazoline)s via the
combination of living anionic polymerization and LCROP.
Furthermore, polymers with the molecular brush architecture
possess unique characteristics in thermo-responsiveness, anti-
fouling efficacy and drug delivery in comparison with their
linear counterparts. Moreover, cylindrical molecular brushes31
have remarkable advantages in tissue penetration, cellular
uptake and faster body clearance compared to general polymer
objects.32
A new enaminitrile receptor to Zn2+ was designed to func-
tionalize the poly(2-oxazoline) molecular brush as a Zn2+-
responsive fluorescent sensor (Scheme 1).
Experimental section
Materials and methods
All chemicals were purchased from Aladdin (China) or Merck
and were used as received unless otherwise stated. Methyl tri-
flate (MeOTf ), 2-isopropenyl-2-oxazoline (IPOx), 2-ethyl-2-oxa-
zoline (EtOx), acetonitrile (ACN) and toluene were dried by
refluxing over CaH2 under a dry nitrogen atmosphere and sub-
This journal is © The Royal Society of Chemistry 2020

Polymer Chemistry
sequently distilled under vacuum prior to use. Cp2YbMe was
prepared according to the literature.33 All these purified mono-
mers and catalysts were stored inside a glovebox freezer at
−30 °C.
Characterization
1
H NMR spectra were recorded on a Varian Inova 300 (300 MHz)
instrument at room temperature. Chemical shifts for 1H were
Published on 24 September 2020. Downloaded by University of New England on 10/10/2020 2:01:37 PM.
referenced to internal solvent resonances and are reported as
parts per million relative to SiMe4. Gel permeation chromato-
graphy (GPC) measurements were conducted with a PL-GPC120
system equipped with a Waters Styragel column (HT3) using
DMF as the eluent, and the molecular weights were calibrated
with polystyrene standards. Emission spectra were recorded
using a Fluoromax-4 spectrofluorometer (Horiba Jobin Yvon
Inc., France) with an excitation wavelength of 365 nm. Confocal
laser scanning microscopy (CLSM) images were obtained using
a Zeiss LSM 700 (Zurich, Switzerland) microscope.
Synthetic procedures
PAN-1: (E)-3-Ethoxy-2-( pyridin-2-yl)acrylonitrile was prepared
according to a literature procedure with minor modifi-
cations.32 An oven-dried 50 mL round-bottom flask equipped
with a condenser was purged with nitrogen three times, and
then 2-pyridylacetonitrile (0.95 mL, 8.46 mmol, 1 equiv.),
triethyl orthoformate (2.15 mL, 12.70 mmol), ZnCl2 (catalytic
amount), and acetic anhydride (10 mL) were added. The deep
reddish mixture was stirred in an oil bath at 120 °C under a
nitrogen atmosphere for 12 h, followed by evaporation of all
volatiles at 40 °C under vacuum to yield a dark brown oil,
which could be used in the next step without the need for
further purification. The product was stored in a freezer.
tert-Butyl (E)-(2-((2-cyano-2-( pyridin-2-yl)vinyl)amino)ethyl)
carbamate was synthesized by a method adapted from a litera-
ture procedure.34 (E)-3-Ethoxy-2-( pyridin-2-yl)acrylonitrile
(174 mg, 1 mmol) N-boc-ethylenediamine (240 mg, 1.5 mmol)
and 2 mL EtOH were added in an oven-dried round-bottom
flask, and refluxed for 8 h under Ar. Following silica gel
column chromatography and successive recrystallization, the
product was isolated as a white solid (198 mg, 68.7%). 1H
NMR (300 MHz, CDCl3): δ = 10.41 (brs, 1H, NHCHv), 8.38 (d,
1H, NCH), 7.64 (td, 1H, NCHCHCH), 7.42 (d, 1H,
NCHCHCHCH), 7.16 (d, 1H, NCHCH), 6.98 (t, 1H, NHCH),
4.76 (s, 1H, CH2NH), 3.45 (m, 4H, CH2CH2), 1.44 (s, 9H, Boc).
(E)-3-((2-Aminoethyl)amino)-2-( pyridin-2-yl)acrylonitrile was
synthesized according to a literature procedure with minor
modifications.35 A solution of tert-butyl (E)-(2-((2-cyano-2-
( pyridin-2-yl)vinyl)amino)ethyl)carbamate (1 g, 3.5 mmol) in
30 mL of dichloromethane was added dropwise to 20 mL of tri-
fluoroacetic acid at 0 °C and the mixture was stirred for 2 h at
room temperature. After evaporation of the solvent, the residue
was dissolved in an aqueous solution of sodium hydroxide and
extracted with dichloromethane. The collected organic layer
was dried over potassium carbonate and evaporated to
dryness. Then the residue was purified by column chromato-
graphy (340 mg, 51.7%). 1H NMR (300 MHz, DMSO-d6): δ =
This journal is © The Royal Society of Chemistry 2020
View Article Online
Paper
10.28 (brs, 1H, NHCHv), 8.34 (d, 1H, NCH), 7.78 (td, 1H,
NCHCHCH), 7.57 (d, 1H, NCHCHCHCH), 7.23 (d, 1H,
NCHCH), 7.09 (t, 1H, NHCHv), 3.56 (m, 2H, CH2CH2), 2.50
(m, 2H, CH2CH2), 1.23 (s, 2H, NH2).
P(IPOx-g-EtOx): P(IPOx-g-EtOx) was prepared according to
previously published procedures with modifications.36
Rare earth metal-mediated group-transfer polymerization
Poly(2-isopropenyl-2-oxazoline). Under a dry nitrogen atmo-
sphere, 2-isopropenyl-2-oxazoline (IPOx) (600 mg, 5.4 mmol),
Cp2YbMe and 5 mL toluene were stirred for 4 h at room temp-
erature and the reaction was quenched by the addition of
MeOH. Then the resulting mixture was precipitated into dry
diethyl ether. The product was reprecipitated twice using
chloroform and ether. After filtration and drying under
vacuum, a colorless powder was obtained (480 mg, 80%). GPC:
PDI = 1.22, Mn = 18 kg mol−1.
Macroinitiator salt
Poly(2-isopropenyl-2-oxazolium triflate). Under dry and inert
conditions, PIPOx (100 mg, 1.0 equiv. of oxazoline unit) and
MeOTf (177 mg, 1.2 equiv.) were added to 4 mL of dry aceto-
nitrile at approximately −35 °C. After stirring overnight at 0 °C,
the mixture was poured into cold and dry diethyl ether to pre-
cipitate the oxazolinium salt. The precipitate (270 mg, 91%)
was washed twice with cold ether.
Poly(2-isopropenyl-2-oxazoline-g-2-ethyl-2-oxazoline). The macro-
initiator salt (120 mg, 1 equiv.) was dissolved in 5 mL of dry
acetonitrile, and 860 mg of 2-ethyl-2-oxazoline (EtOx) (20
equiv.) was added. The polymerization solution was heated in
an oil bath to 80 °C and stirred for 6 h. The mixture was equili-
brated to room temperature, and 242 mg (3 equiv.) of (E)-3-((2-
aminoethyl)amino)-2-( pyridin-2-yl)acrylonitrile was added.
After stirring the reaction mixture overnight at room tempera-
ture, an excess of finely ground potassium carbonate (8 equiv.)
was added, and the mixture was allowed to stir overnight. The
solid was filtered and then precipitated three times into dry
diethyl ether. The product was freeze-dried (water) and
obtained by dialysis purification (membrane MWCO 4 kDa)
against water for 72 h to separate the product from minor por-
tions of homopolymer side products. GPC: PDI = 1.47, Mn =
188 kg mol−1.
Cell lines and cell culture
Human cervical carcinoma (HeLa) cells were grown in
Dulbecco’s modified Eagle’s medium (DMEM, GIBCO) sup-
plemented with 10% heat-inactivated fetal bovine serum (FBS,
GIBCO), and the culture medium was replaced once every day.
MTT assays
Cells harvested in the logarithmic growth phase were seeded
in 96-well plates at a density of 2 × 103 cells per well and incu-
bated in DMEM for 24 h. The medium was then replaced by
P(IPOx-g-EtOx) at various concentrations. The incubation was
continued for 48 h. Then, 20 µL of MTT solution in PBS at a
concentration of 5 mg mL−1 was added and the plates were
incubated for another 4 h at 37 °C, followed by the removal of
the culture media containing MTT and addition of 150 µL of
Polym. Chem.
 View Article Online

Paper Polymer Chemistry

dimethyl sulfoxide (DMSO) to each well to dissolve the forma- Table 1 Characterization of PIPOx and molecular brushes
zan crystals formed. Finally, the plates were shaken for 5 min,
and the absorbance of the formazan product was measured at Polymer Mn (kg mol−1) Mw/Mn nb ma
490 nm using a microplate reader. PIPOx 18.5b 1.34b 180 —
P(IPOx-g-EtOx) 188.3b 1.42b 180 18
Confocal laser scanning microscopy (CLSM) a
Calculated from end-group analysis based on 1H NMR data.
b
Cellular uptake by HeLa cells was examined using CLSM. HeLa Calculated from GPC traces. n: Average degree of polymerization of
the backbone. m: Average degree of polymerization of the side chains.
Published on 24 September 2020. Downloaded by University of New England on 10/10/2020 2:01:37 PM.

cells were seeded in six-well culture plates (a clean cover slip

was put in each well) at a density of 2 × 105 cells per well and
allowed to adhere for 24 h. The medium was then replaced by
P(IPOx-g-EtOx) solutions at a concentration of 2 mg mL−1.
After incubation for 4 h at 37 °C, the supernatant was carefully
removed and the cells were washed three times with PBS.
Subsequently, different concentrations of ZnCl2 in the culture
media were added to the cells, and the cells were incubated for
2 h at 37 °C. Following washing three times with PBS, the cells
were treated with 500 nM PHK26 for 30 min at 37 °C. After
washing with PBS, the cells were fixed with 1 mL of 4% paraf-
ormaldehyde in each well for 10 min at room temperature and
washed twice with PBS again. The treated cells were imaged by
confocal laser scanning microscopy.

Results and discussion

The synthesis of the end-capping enaminitrile molecule, i.e.
3-((2-aminoethyl)amino)-2-( pyridin-2-yl)acrylonitrile (PAN-1), is
provided in the Experimental section according to the litera-
ture.34 Poly(2-isopropenyl-2-oxazoline-g-2-ethyl-2-oxazoline), i.e.
the P(IPOx-g-EtOx) molecular brush, was synthesized analo-
gously to our previous report as outlined in Scheme 1.
According to the literature, the poly(2-isopropenyl-2-oxazoline)
backbone with low polydispersity was synthesized via the
rare-earth-metal-mediated group-transfer polymerization
(REM-GTP) of 2-isopropenyl-2-oxazoline (IPOx) catalyzed by bis
(cyclopentadienyl)methylytterbium (Cp2YbMe).36 After conver-
sion to the macroinitiator by the reaction with methyltriflate
(MeOTf ), the grafting-from LCROP of 2-ethyl-2-oxazoline
(EtOx) led to the formation of molecular brushes with poly(2-
ethyl-2-oxazoline) (PEtOx) side chains. End groups were intro-
duced by terminating the growing side chain living species
with PAN-1. The primary amine group of PAN-1 quenched the
polymerization as shown in Fig. S1(B).† The structures of
PAN-1, the PIPOx backbone and molecular brushes are shown
in Scheme 1. The structures of these polymers were verified by
proton nuclear magnetic resonance (1H NMR) and gel per-
meation chromatography (GPC). The characterization data are
presented in Table 1 and Fig. S1.†
An ideal metal ion fluorescent sensor should be capable of
detecting a metal ion in the presence of other relevant metal
ions. Initially, we examined the selective sensing of P(IPOx-g-
EtOx) toward various representative metal ions in single and
multicomponent systems, such as transition metal ions (Cu2+,
Zn2+, Ni2+, Fe3+), alkali metal ions (K+), heavy metal ions (Cd2+,
Ag+, Hg2+), and alkaline earth metal ions (Ca2+). Fig. 1A shows
the fluorescence response of P(IPOx-g-EtOx) to a single metal
Fig. 1 (A) Fluorescence spectra for P(IPOx-g-EtOx) (2 mg mL−1)
aqueous solution in the presence of various metal ions (100 µM) (exci-
tation at 365 nm). Inset: Visual color change observed with the addition
of different metal ions as observed under UV light (λ = 365 nm). (B)
Fluorescence responses of P(IPOx-g-EtOx) aqueous solution (2 mg
mL−1) to various metal ions in the absence/presence of Zn2+. Excitation
at 365 nm. Blue bar: P(IPOx-g-EtOx) with different metal ions. Red bar:
P(IPOx-g-EtOx) and Zn2+ with different metal ions.
ion system in aqueous solution (deionized water) upon exci-
tation at 365 nm. It was interesting to note that selective and
large fluorescence enhancement was observed upon addition
of Zn2+ to the aqueous solution of P(IPOx-g-EtOx), whereas
other relevant competing metal ions had no noteworthy var-
iance in the fluorescence spectra. The photographs of P(IPOx-
g-EtOx) with different metal ions verified their sensitivity by
strong blue fluorescence, as shown in the inset of Fig. 1A. The
preference of P(IPOx-g-EtOx) toward Zn2+ over other ions is
evident. It is worth mentioning that Cd2+, having properties
analogous to Zn2+ owing to its intrinsic d10 shell structure in
the same group, did not exhibit obvious fluorescence in the
presence of P(IPOx-g-EtOx). As a result, P(IPOx-g-EtOx) had a
remarkable inclination to distinguish Zn2+ from Cd2+ in
common solution.

Polym. Chem. This journal is © The Royal Society of Chemistry 2020

 View Article Online

Polymer Chemistry Paper

In order to further examine the selectivity of the fluorescent

response of P(IPOx-g-EtOx) to Zn2+, we also carried out multi-
component metal ion analysis. A single metal ion (Cu2+, K+,
Cd2+, Ag+, Zn2+, Ni2+, Fe3+, Hg2+, Ca2+) was added to nine equal
divisions of the P(IPOx-g-EtOx) aqueous solution, respectively.
Evidently, only the solution upon addition of Zn2+ exhibited
strong blue fluorescence (Fig. 1B, blue bars). However, when
equal amounts of Zn2+ (100 µmol L−1) and other metal ions
Published on 24 September 2020. Downloaded by University of New England on 10/10/2020 2:01:37 PM.

(100 µmol L−1) were combined to form a new dual-metal com-

petitive aqueous solution system, strong blue fluorescence
appeared for all the solutions upon the addition of P(IPOx-g-
EtOx) (2 mg mL−1). The fluorescence intensity as a function of
the dual-metal ion composition is outlined in Fig. 1B (red
Fig. 2 The Job plot of PAN-1 evaluated from the fluorescence with a
bars). The presence of Cu2+, Ag+ or Fe3+ in Zn2+ solution total concentration of 50 µmol L−1.
resulted in a slight decrease of the fluorescence intensity upon
the addition of P(IPOx-g-EtOx), in comparison with other dual-
metal competitive systems. It seems that several ions resulted
in partial fluorescence quenching. In particular, the inherently
quenching metals, such as Cu2+, Ag+ and Fe3+, could bind
tightly to the ligand and result in intracomplex quenching.37,38
These results demonstrate that P(IPOx-g-EtOx) has a good
selection behavior for Zn2+ with no/minor interference from
other metal ions. Moreover, the good water solubility of
P(IPOx-g-EtOx) provided an advantage for its application in
biological imaging as well.
Interestingly, the P(IPOx-g-EtOx) sensor with Zn2+ exhibited
Scheme 2 Plausible mechanism of PAN-1 sensing Zn2+ based on
good photostability39 with the fluorescence intensity maxima complexation.
being reduced by 23%, after one week of exposure to sunlight
(Fig. S2†).
In light of the fluorescence enhancement upon the addition
of Zn2+, we examined the molecular brush of poly(2-oxazoline)
without enaminitrile end-capping toward Zn2+. There was no
noteworthy enhancement as observed in the fluorescence
spectra (Fig. S3†). It indicates that the presence of the coordi-
nating sites (PAN-1) in the side chain end makes POx mole-
cular brushes potential candidates in detecting Zn2+.
To determine the binding stoichiometry of the sensor-Zn2+
complex, we performed a conventional Job’s plot (continuous
variation analysis) experiment employing PAN-1 to coordinate
with Zn2+. We found that PAN-1 was highly selective to Zn2+
compared to other relevant metal ions (Fig. S4†). The Job’s
plot spectrum in Fig. 2 describes the maximum fluorescence
intensity change as a function of the metal mole fraction (XM).
The sum concentration of Zn2+ and PAN-1 was fixed at 50 µM.
The maximum fluorescence intensity change was observed at a Fig. 3 Fluorescence spectra of P(IPOx-g-EtOx) aqueous solution (2 mg
mL−1) in the presence of Zn2+ with different concentrations. The exci-
mole fraction of 0.495 (Fig. 2), indicating that PAN-1 coordi-
tation wavelength was maintained at 365 nm. Inset a: Relative fluor-
nates with the Zn2+ ion in a 1 : 1 complex model. We measured escence intensity as a function of the Zn2+ concentration. Inset b: Visual
the mass spectra of the complexes formed between PAN-1 and photography of P(IPOx-g-EtOx) and P(IPOx-g-EtOx)-Zn2+ under UV
Zn2+ (Fig. S5†), and the ionic peaks at 251 were assigned to light (λ = 365 nm).
[PAN-1 + Zn2+–H+]+, which confirmed the formation of the zinc
complex with PAN-1. Based on the above results and the pre-
vious report, a plausible complex model is proposed in response of P(IPOx-g-EtOx) toward Zn2+ was associated with a
Scheme 2.40,41 sharp color change from colorless to blue which could be
Subsequently, a fluorescence titration experiment was easily observed by the naked eye. Incremental addition of Zn2+
carried out to further interpret the interaction between P(IPOx- ions resulted in a systematic increase in the emission intensity
g-EtOx) and Zn2+. As shown in inset (b) of Fig. 3, the spectral of P(IPOx-g-EtOx) at 450 nm (Fig. 3). It was found that the

This journal is © The Royal Society of Chemistry 2020 Polym. Chem.

 View Article Online

Paper Polymer Chemistry

emission intensity increased linearly with the increase of the
Zn2+ concentration as shown in the inset of Fig. 3.
We attributed the significant fluorescence enhancement
upon the addition of Zn2+ to internal charge transfer
(ICT).41–44 Through the coordination with Zn2+, chelation
inhibits the free rotation of the signal bond and increases the
rigidity of the molecular structure of the grafted enaminitrile-
Published on 24 September 2020. Downloaded by University of New England on 10/10/2020 2:01:37 PM.
for applications under physiological temperature and pH
conditions.
The morphological structure effect of polymer nano-
materials on their biological performance is significant;
however, only a small number of studies have been carried
out.49–54 Since the PIPOx backbone and the side chains were
prepared by living REM-GTP and LCROP, the synthesized

P(IPOx-g-EtOx) through the formation of a five-membered P(IPOx-g-EtOx) adopted a stretched conformation due to the
ring and a six-membered ring. As a result, electron delocaliza- repulsion of polyoxazoline side chains leading to a unique
tion from the N atom of the amino group (donor) to the molecular contour, which was verified by AFM measurements
cyano moiety (acceptor) is much more viable (Scheme 2), as shown in Fig. 4.
leading to an enhancement of the charge separation and To assess the biosafety property of P(IPOx-g-EtOx), we per-
dipole moment which regulate the energy of the ICT state.45 formed in vitro cytotoxicity investigation against HeLa cells by
Consequently, significant fluorescence enhancement takes 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
place upon the addition of Zn2+. At the same time, a small (MTT) assay after 24 h of incubation at different concen-
red shift in the fluorescence emission spectrum is observed trations of P(IPOx-g-EtOx). As shown in Fig. S9,† no apparent
(Fig. 3). cytotoxicity was observed for P(IPOx-g-EtOx), indicating the
The limit of detection (LOD) for the sensor toward an ion is excellent biocompatibility of P(IPOx-g-EtOx).
a significant parameter for practical applications, which is With a new Zn2+-responsive sensor in hand, which demon-
generally calculated by 3σ/k, where σ is the standard deviation strated the ability to detect Zn2+ with excellent selectivity in
of the blank measurements and k is the slope of the cali- in vitro studies, we used HeLa (human cervical carcinoma)
bration curve line. The calculated LOD of P(IPOx-g-EtOx) is cells to investigate exogenous zinc ions in living cells.55–57 In
8.9 µM according to the calibration plots (Fig. S6†). A linear fluorescence cell imaging studies, the cells were incubated
relationship between the fluorescence intensity and Zn2+ con- with P(IPOx-g-EtOx) (4 mg mL−1) for 4 h, followed by enriched
centration can be observed. Zn2+ for 2 h, and evident blue fluorescence was observed
For further identification of the working range of the mole- (Fig. 5c). At the same time, the fluorescence of the bright-field
cular brush P(IPOx-g-EtOx) sensor, we carried out the fluo-
rescent response analysis of P(IPOx-g-EtOx) toward Zn2+ within
the concentration range of 0–50 μM (Fig. S7†). The minimum
concentration at which P(IPOx-g-EtOx) could detect Zn2+ in the
experiment was found to be 10 μM. It indicates that P(IPOx-g-
EtOx) may be suitable for the qualitative detection of Zn2+ in
many environmental and biological systems.
The sensitivity and physiological conditions for the sensing
process are important for practical applications. It is very
essential to ascertain the parameters and optimal analytical
conditions, such as temperature and pH. Successively, we
investigated the effect of temperature and pH on the fluo-
rescence of P(IPOx-g-EtOx) in the presence of Zn2+ ions. The
fluorescence measurements were conducted using a P(IPOx-g-
EtOx) aqueous solution with a concentration of 2 mg mL−1
Fig. 4 AFM scan of the molecular brush P(IPOx-g-EtOx). The polymer
containing 100 µM Zn2+ at different temperature and pH was deposited by dip-coating from a dilute chloroform solution (0.2 mg
values. As shown in Fig. S8a,† with the addition of Zn2+, the mL−1) onto the mica surface.
fluorescence intensity is almost unchanged at the temperature
range of 30–50 °C. As shown in Fig. S8b,† the fluorescence
intensity of P(IPOx-g-EtOx) in the presence of Zn2+ is almost
stable in the pH range of 6.0–8.0 due to the formation of the
enaminitrile-Zn2+ complex, which is appropriate for its appli-
cation under physiological pH conditions. Interestingly, the
fluorescence intensity decreases in the acidic ( pH < 6) region.
This is possibly due to the protonation of the pyridinium
moiety of enaminitrile, which decreases the coordination
Fig. 5 Confocal microscopy images of the HeLa cells. (a) Bright-field
ability of P(IPOx-g-EtOx).46,47 Furthermore, the decreased fluo-
image. (b) Cells treated with 500 nM of PHK26 for 30 min at 37 °C. λex =
rescence intensity and blue-shift in the basic ( pH > 8) region 561 nm. (c) Cells incubated with 4 mg mL−1 P(IPOx-g-EtOx) for 4 h at
may be due to ICT which hindered the complexation.48 These 37 °C, followed by 1 mg mL−1 ZnCl2 for 2 h. λex = 405 nm. (d) Merged
results further demonstrate that P(IPOx-g-EtOx) is appropriate image. Scale bar = 50 µm.

Polym. Chem. This journal is © The Royal Society of Chemistry 2020


Polymer Chemistry
image and the stained cells image had a good coincidence
degree (Fig. 5). These results clearly demonstrated that P(IPOx-
g-EtOx) with cell membrane permeability can detect intracellu-
lar Zn2+.
In order to further study the intracellular fluorescence
response of P(IPOx-g-EtOx) toward Zn2+, we investigated
P(IPOx-g-EtOx) to detect zinc ions in live cells by incubating
HeLa cells with different concentrations of Zn2+ (0, 40 µM,
Published on 24 September 2020. Downloaded by University of New England on 10/10/2020 2:01:37 PM.
200 µM, 500 µM) for 2 h at 37 °C prior to imaging. There was
barely any perceptible fluorescence after incubating with
P(IPOx-g-EtOx) in the absence of Zn2+ (Fig. 6d); however, the
addition of 500 µM exogenous Zn2+ to the cells caused a notice-
able change with evident blue fluorescence (Fig. 6a) being
observed. The intracellular fluorescence decreased with a step-
wise decrease of the Zn2+ concentration from Fig. 6. Clearly, the
fluorescence changed significantly with P(IPOx-g-EtOx) of a
specific concentration in response to different Zn2+ concen-
trations in HeLa cells. These cell experiments indicate that the
long and flexible P(IPOx-g-EtOx) has huge potential in biological
applications as a Zn2+ sensor and in live cell imaging.
Fig. 6 Confocal microscopy images of P(IPOx-g-EtOx) as a function of
the concentration of Zn2+ in HeLa cells. The cells were treated with
P(IPOx-g-EtOx) (2 mg mL−1) for 4 h, (a) 500 µM ZnCl2 for 2 h; (b)
200 µM ZnCl2 for 2 h; (c) 40 µM ZnCl2 for 2 h; and (d) in the absence of
ZnCl2. (1) The cell membranes (red) were stained with PHK26. λex =
561 nm. (2) The cells (blue) were incubated with P(IPOx-g-EtOx) and
zinc ions. λex = 405 nm. (3) Merged images. Scale bar = 50 µm.
This journal is © The Royal Society of Chemistry 2020
View Article Online
Paper
Conclusions
We have designed and synthesized a new fluorescent sensor
P(IPOx-g-EtOx) for Zn2+ sensing which contains a new enamini-
trile receptor. The sensor displays excellent fluorescent selecti-
vity for Zn2+ among competitive metal ions, especially Cd2+.
Furthermore, this polymer-based sensor possesses a wormlike
conformation which endows the sensor with cell membrane
permeability. Thus, the sensor can monitor intracellular zinc
ions. In view of its good water solubility and biocompatibility,
the POx sensor exhibits great potential in the biological field.
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
The financial support of this work by the National Natural
Science Foundation of China (51673194 and 51973026) and
the Department of Science and Technology of Jilin Province
(20180101170JC) is greatly acknowledged.
Notes and references
1 J. M. Berg and Y. G. Shi, Science, 1996, 271, 1081–1085.
2 C. F. Walker and R. E. Black, Annu. Rev. Nutr., 2004, 24,
255–275.
3 M. Shyamal, P. Mazumdar, S. Maity, S. Samanta,
G. P. Sahoo and A. Misra, ACS Sens., 2016, 1, 739–747.
4 L. C. Costello, C. C. Fenselau and R. B. Franklin, J. Inorg.
Biochem., 2011, 105, 589–599.
5 E. D. Gundelfinger, T. M. Boeckers, M. K. Baron and
J. U. Bowie, Trends Biochem. Sci., 2006, 31, 366–373.
6 S. L. Sensi, J. Neurosci., 2011, 31, 16076–16085.
7 C. J. Frederickson, J.-Y. Koh and A. I. Bush, Nat. Rev.
Neurosci., 2005, 6, 449–462.
8 S. Fu, X. Wan, C. Du, H. Wang, J. Zhou and Z. Wang,
Prostate, 2019, 79, 1406–1413.
9 C. J. Frederickson, E. J. Kasarskis, D. Ringo and
R. E. Frederickson, J. Neurosci. Methods, 1987, 20, 91–103.
10 P. D. Zalewski, I. J. Forbes and W. H. Betts, Biochem. J.,
1993, 296, 403–408.
11 K. M. Hendrickson, J. P. Geue, O. Wyness, S. F. Lincoln and
A. D. Ward, J. Am. Chem. Soc., 2003, 125, 3889–3895.
12 M. D. Shults, D. A. Pearce and B. Imperiali, J. Am. Chem.
Soc., 2003, 125, 10591–10597.
13 K. Hanaoka, K. Kikuchi, H. Kojima, Y. Urano and
T. Nagano, J. Am. Chem. Soc., 2004, 126, 12470–12476.
14 Z. Xu, K. H. Baek, H. N. Kim, J. N. Cui, X. H. Qian,
D. R. Spring, I. Shin and J. Yoon, J. Am. Chem. Soc., 2010,
132, 601–610.
15 W. Chyan, D. Y. Zhang, S. J. Lippard and R. J. Radford,
Proc. Natl. Acad. Sci. U. S. A., 2014, 111, 143–148.
Polym. Chem.

Paper
16 Y. Mikata, Y. Nodomi, A. Kizu and H. Konno, Dalton Trans.,
2014, 43, 1684–1690.
17 E. Tomat and S. J. Lippard, Inorg. Chem., 2010, 49, 9113–
9115.
18 H. Woo, Y. You, T. Kim, G. J. Jhon and W. Nam, J. Mater.
Chem., 2012, 22, 17100–17112.
19 J. Peng, W. Xu, C. L. Teoh, S. Han, B. A. Kim, J. C. Er,
Published on 24 September 2020. Downloaded by University of New England on 10/10/2020 2:01:37 PM.
L. Wang, L. Yuan and X. Liu, J. Am. Chem. Soc., 2015, 137,
2336–2342.
20 H. B. Ren, B. Y. Wu, J. T. Chen and X. P. Yan, Anal. Chem.,
2011, 83, 8239–8244.
21 S. Roy, A. Baral, R. Bhattacharjee, B. Jana, A. Datta,
S. Ghosh and A. Banerjee, Nanoscale, 2015, 7, 1912–1920.
22 S. Sahu, B. Behera, T. K. Maiti and S. Mohapatra, Chem.
Commun., 2012, 48, 8835–8837.
23 S. He, S. T. Iacono, S. M. Budy, A. E. Dennis, D. W. Smith
and R. C. Smith, J. Mater. Chem., 2008, 18, 1970–1976.
24 Y. Xu, J. Meng, L. Meng, Y. Dong, Y. Cheng and C. Zhu,
Chem. – Eur. J., 2010, 16, 12898–12903.
25 W. H. Ding, W. Cao, X. J. Zheng, D. C. Fang, W. T. Wong
and L. P. Jin, Inorg. Chem., 2013, 52, 7320–7322.
26 X. Sun, Y. W. Wang and Y. Peng, Org. Lett., 2012, 14, 3420–
3423.
27 D. Maity and T. Govindaraju, Chem. Commun., 2012, 48,
1039–1041.
28 Y. Lu, S. Huang, Y. Liu, S. He, L. Zhao and X. Zeng, Org.
Lett., 2011, 13, 5274–5277.
29 K. Kaur, M. Kaur, A. Kaur, J. Singh, N. Singh, S. K. Mittal
and N. Kaur, Inorg. Chem. Front., 2014, 1, 99–108.
30 N. Zhang, S. Huber, A. Schulz, R. Luxenhofer and
R. Jordan, Macromolecules, 2009, 42, 2215–2221.
31 S. Chen, L. Hou, Q. Wang, D. Dong and N. Zhang, Polym.
Chem., 2018, 9, 5470–5475.
32 Z. Zhang, C. Liu, C. Li, W. Wu and X. Jiang, Research, 2019,
2391486.
33 U. B. Seemann, J. E. Dengler and B. Rieger, Angew. Chem.,
Int. Ed., 2010, 49, 3489–3491.
34 Y. Ren, S. Xie, E. S. Grape, A. K. Inge and O. Ramstrom,
J. Am. Chem. Soc., 2018, 140, 13640–13643.
35 E. Kawabata, K. Kikuchi, Y. Urano, H. Kojima, A. Odani
and T. Nagano, J. Am. Chem. Soc., 2005, 127, 818–819.
36 N. Zhang, S. Salzinger, B. S. Soller and B. Rieger, J. Am.
Chem. Soc., 2013, 135, 8810–8813.
Polym. Chem.
View Article Online
Polymer Chemistry
37 C. Du, S. Fu, X. Ren, X. Wang, Z. Wang, J. Zhou and
H. Wang, New J. Chem., 2018, 42, 3493–3502.
38 E. U. Akkaya, M. E. Huston and A. W. Czarnik, J. Am. Chem.
Soc., 1990, 112, 3590–3593.
39 C. Du, S. Fu, X. Wang, A. C. Sedgwick, W. Zhen, M. Li,
X. Li, J. Zhou, Z. Wang, H. Wang and J. L. Sessler, Chem.
Sci., 2019, 10, 5699–5704.
40 X. Wen, Q. Wang and Z. Fan, J. Lumin., 2018, 194, 366–373.
41 S. R. Bhatta, B. Mondal, G. Vijaykumar and A. Thakur,
Inorg. Chem., 2017, 56, 11577–11590.
42 X. Xia, F. Zeng, P. Zhang, J. Lyu, Y. Huang and S. Wu, Sens.
Actuators, B, 2016, 227, 411–418.
43 L. J. Zhang, Z. Y. Wang, J. T. Liu, J. Y. Miao and B. X. Zhao,
Sens. Actuators, B, 2017, 253, 19–26.
44 X. Zhao, Y. Liu, Y. Wu, X. Ren, D. Zhang and Y. Ye, Sens.
Actuators, B, 2017, 253, 488–494.
45 S. R. Bhatta, B. Mondal, G. Vijaykumar and A. Thakur,
Inorg. Chem., 2017, 56, 11577–11590.
46 K. Tayade, B. Bondhopadhyay, K. Keshav, S. K. Sahoo,
A. Basu, J. Singh and A. Kuwar, Analyst, 2016, 141, 1814–1821.
47 Y. S. Yang, C. M. Ma, Y. P. Zhang, Q. H. Xue, J. X. Ru,
X. Y. Liu and H. C. Guo, Anal. Methods, 2018, 10, 1833–
1841.
48 D. Noy, I. Solomonov, O. Sinkevich, T. Arad, K. Kjaer and
I. Sagi, J. Am. Chem. Soc., 2008, 130, 1376–1383.
49 A. Li, H. P. Luehmann, G. Sun, S. Samarajeewa, J. Zou,
S. Zhang and K. L. Wooley, ACS Nano, 2012, 6, 8970–8982.
50 H. Cabral, Y. Matsumoto, K. Mizuno, Q. Chen,
M. Murakami, M. Kimura and N. Nishiyama, Nat.
Nanotechnol., 2011, 6, 815–823.
51 E. Blanco, H. Shen and M. Ferrari, Nat. Biotechnol., 2015,
33, 941–951.
52 M. Müllner, K. Yang, A. Kaur and E. J. New, Polym. Chem.,
2018, 9, 3461–3465.
53 H. Li, H. Liu, T. Nie, Y. Chen, Z. Wang, H. Huang and
Y. Chen, Biomaterials, 2018, 178, 620–629.
54 M. Müllner, Macromol. Chem. Phys., 2016, 217, 2209–2222.
55 Z. H. Fu, X. Han, Y. Shao, J. Fang, Z. H. Zhang, Y. W. Wang
and Y. Peng, Anal. Chem., 2017, 89, 1937–1944.
56 Y. Dai, K. Yao, J. Fu, K. Xue, L. Yang and K. Xu, Sens.
Actuators, B, 2017, 251, 877–884.
57 X. Feng, Y. Li, X. He, H. Liu, Z. Zhao, R. T. Kwok and
B. Z. Tang, Adv. Funct. Mater., 2018, 28, 1802833–1802841.
This journal is © The Royal Society of Chemistry 2020