Fluorescence turn-on assay for glutathione reductase activity based on a conjugated polyelectrolyte with multiple carboxylate groups

Article abstract of DOI:10.1039/c0jm02400g

In this work, an anionic conjugated polyelectrolyte (CPE) that features four carboxylate groups on each repeat unit, PPE-(COOK)₄, has been designed and synthesized by integrating the two synthetic strategies for conjugated poly(phenylene ethynylene) and dendritic polyamidoamine. Through the installation of multiple functional groups, the proposed CPE has been greatly improved in many aspects, such as the good water solubility, slight aggregation, and relatively high quantum yield. Thanks to the close resemblance in chemical structures between the polymer's pendant groups and EDTA, the resulting PPE-(COOK)₄ has also been found to show stronger complexation with metal ions, and has further been demonstrated to be an excellent fluorescent probe for Cu²⁺ with both high sensitivity and remarkable specificity. Moreover, by taking advantage of the distinct fluorescence recovery rates of PPE-(COOK)₄/Cu²⁺ caused by GSH and by GSSG, a novel fluorescence turn-on assay for glutathione reductase (GR) has successfully been developed. With a minimum detectable enzyme concentration of 0.2 mU/mL, this newly proposed GR activity assay is highly sensitive and robust as compared to most spectrophotometric and fluorescent methods. The Royal Society of Chemistry 2010.

Full text of DOI:10.1039/c0jm02400g

View Article Online / Journal Homepage / Table of Contents for this issue
PAPER
www.rsc.org/materials | Journal of Materials Chemistry
Fluorescence turn-on assay for glutathione reductase activity based on
a conjugated polyelectrolyte with multiple carboxylate groups†
a
Hongliang Fan,ab Tao Zhang, Shaowu Lvc and Qinhan Jinab
*
Received 25th July 2010, Accepted 3rd September 2010
DOI: 10.1039/c0jm02400g
In this work, an anionic conjugated polyelectrolyte (CPE) that features four carboxylate groups on
each repeat unit, PPE-(COOK)4, has been designed and synthesized by integrating the two synthetic
strategies for conjugated poly(phenylene ethynylene) and dendritic polyamidoamine. Through the
installation of multiple functional groups, the proposed CPE has been greatly improved in many
aspects, such as the good water solubility, slight aggregation, and relatively high quantum yield.
Thanks to the close resemblance in chemical structures between the polymer’s pendant groups and
EDTA, the resulting PPE-(COOK)4 has also been found to show stronger complexation with metal
ions, and has further been demonstrated to be an excellent fluorescent probe for Cu2+ with both high
sensitivity and remarkable specificity. Moreover, by taking advantage of the distinct fluorescence
recovery rates of PPE-(COOK)4/Cu2+ caused by GSH and by GSSG, a novel fluorescence turn-on assay
for glutathione reductase (GR) has successfully been developed. With a minimum detectable enzyme
concentration of 0.2 mU/mL, this newly proposed GR activity assay is highly sensitive and robust as
compared to most spectrophotometric and fluorescent methods.
Moreover, from a pharmaceutical perspective, GR inhibitors are
shown to possess the anticancer and antimalarial activity per se,5
Introduction
Glutathione (GSH), a tripeptide of g-L-glutamyl-cysteinyl-
and the inhibition of GR has also been employed as a powerful
glycine, is the most important non-protein thiol for both animals
tool in many other studies.6 Therefore, there exists an increasing
and plants. With the typical concentration in the millimolar
need for the sensitive and selective assays of GR activity.
range,1 this oligopeptide is also of the greatest abundance in
However, the classic spectrophotometric methods based on the
living cells, and serves many essential roles as a major antioxi-
changes of NADPH or DTNB,7 although still widely used, are
dant, including the protection of cells against various oxidative
actually less sensitive and vulnerable, so that many contributions
stresses, xenobiotic metabolism, intra-cellular signal trans-
have turned to the fluorogenic probes.8
duction, and gene regulation.2 In contrast to GSH, the oxidized
In recent years, fluorescent conjugated polymers (CPs) that
glutathione (GSSG) generated from the detoxification of
possess many predominant optoelectronic properties have drawn
peroxides is unable to fulfill these functions. Altered levels of
great attention in highly sensitive bio- and chemo-sensors. As
GSH have been linked to a number of diseases, such as liver
compared with typical fluorescent dyes, the rapid transfer of
damage, Alzheimer’s disease, cancers, and AIDS.3 Thus, a fairly
excitation along the whole backbone of CPs to energy/electron
high concentration of GSH and the overall GSH/GSSG ratio
receptors can result in a remarkable amplification of the optical
ranging from 30 to 3004 must be steadily maintained through
signal.9 Thereupon, the biosensors based on CPs will intrinsically
both the de novo biosynthesis of GSH and the enzyme-mediated
be endowed with high sensitivity, and thus extensive efforts have
recycling of GSSG. Responsible for the latter is glutathione
recently been focused on the functional CPs and their related
reductase (GR), a ubiquitous flavoenzyme that catalyzes the
sensors.10,11 In particular, the water-soluble conjugated poly-
NADPH-dependent reduction of GSSG back to GSH (Scheme
electrolytes (CPEs),12,13 that integrate the unique properties of
1b), so as to keep a healthy ‘‘redox buffer’’ in living cells.
CPs with the electrostatic behavior of polyelectrolytes, have
proven more advantageous in biological assays and provoked
aResearch Center for Analytical Instrumentation, Institute of
special interest in fluorescent sensors for metal ions,14 saccha-
Cyber-Systems and Control, State Key Lab of Industrial Control
rides,15 proteins,9c,16,17 enzymes,18,19 DNA,20–22 and RNA.23
Technology, Zhejiang University, Hangzhou, 310058, P. R. China.
Nevertheless, an important issue concerning this kind of
material is the strong hydrophobicity of the rigid conjugated
backbone, especially in the poly(phenylene ethynylene) deriva-
tives, which tends to result in relatively poor water solubility,
severe aggregation and low quantum yields.12a All these
phenomena have been evidenced in our previous work, in which
a dicarboxylate-PPE was found to form heavily aggregated
clusters, and as a consequence suffered from weak and unstable
emission.24b Herein, inspired by the works of Kim and Xu et al.,13
E-mail: zhtao@zju.edu.cn; Fax: +86 0571 88208382; Tel: +86 0571
88208383
bDepartment of Chemistry, College of Science, Zhejiang University,
Hangzhou, 310021, P. R. China
cKey Laboratory for Molecular Enzymology & Engineering, the Ministry
of Education, Jilin University, Changchun, 130021, P. R. China
† Electronic supplementary information (ESI) available: ESI-MS spectra
of monomers M2 and M3, fluorescence quenching of PPE-(COOK)4
caused
by
MV2+
and
Cu2+
,
fluorescence
recovery
of
PPE-(COOK)4/Cu2+ induced by GSH, and effects of various proteins
on the emission of PPE-(COOK)4. See DOI: 10.1039/c0jm02400g
This journal is ª The Royal Society of Chemistry 2010
J. Mater. Chem., 2010, 20, 10901–10907 | 10901

View Article Online
Scheme 1 (a) Chemical structure of PPE-(COOK)4; (b) GR-catalyzed reduction of GSSG back to GSH; c) schematic illustration of the proposed GR
activity assay.
an anionic CPE that features multiple carboxylate groups
(Scheme 1a) and therefore good water solubility has been
designed and synthesized through a facile synthetic route. Due to
the strong similarity in chemical structures between the poly-
mer’s pendant groups and EDTA, the resulting PPE-(COOK)4
can form stronger complexes with metal ions, and serves as an
excellent fluorescent probe for Cu2+ with both high sensitivity
and remarkable specificity. As reported previously by Liu et al.,
Cu2+-induced fluorescence quenching of CPEs has offered
a unique platform for the sensitive assay of various enzymes.19 In
this work, by taking advantage of the distinct fluorescence
recovery rates of PPE-(COOK)4/Cu2+ caused by GSH and
GSSG, a highly sensitive fluorescence turn-on assay for GR
activity has successfully been developed (Scheme 1c).
all purchased from Bio Basic Inc. and used as received. b-Nico-
tinamide adenine dinucleotide 20-phosphate reduced tetrasodium
salt hydrate (NADPH, 95%) and 1,3-bis(2-chloroethyl)-1-nitro-
sourea (BCNU) were obtained from Sigma-Aldrich Chemical
Co. The stock solutions of GR and NADPH were both freshly
prepared and cooled on ice before use. NMR and FTIR spectra
were obtained by using a Bruker Advance-500 Spectrometer and
a Perkin-Elmer SPECTRUM ONE FTIR (CIS), respectively.
Mass spectra were recorded with an Analytica of Branford
ESI-TOF mass spectrometer. Gel permeation chromatography
(GPC) analysis was carried out on a Waters 2690 HPLC
equipped with 3 Styragelꢀ columns (HT3-HT5, 7.8 ꢀ 300 mm)
using THF as eluent and polystyrene as standard. UV-visible
absorption spectra were measured on a Shimadzu UV-2550
spectrophotometer. All fluorescence experiments were carried
out at ambient temperature by using a Shimadzu RF-5301
spectrometer. Unless otherwise specified, the excitation
wavelength was 400 nm, and the ‘‘fluorescence intensity’’ was
referenced to the maximum emission of PPE-(COOK)4 at 510
nm.
Experimental section
Materials and instruments
1,4-Di(ethyl 4-oxybutyrate)-2,5-diiodobenzene (M1) was
synthesized according to a previously reported procedure.24 1,4-
Diethynylbenzene (96%) and (PPh3)4Pd were purchased from
Sigma-Aldrich Chemical Co. and Hangzhou Kaida Metal
Polymer synthesis
Catalyst
&
Compounds Co. Ltd. (China), respectively.
Monomer M2. 1,4-Di(ethyl 4-oxybutyrate)-2,5-diiodobenzene
(9.44 g, 16 mmol) was dissolved in a mixture of chloroform (60
mL) and methanol (80 mL). Then excess ethylenediamine was
Glutathione reductase (GR) from baker’s yeast, reduced gluta-
thione (GSH, 98%) and oxidized glutathione (GSSG, 95%) were
10902 | J. Mater. Chem., 2010, 20, 10901–10907
This journal is ª The Royal Society of Chemistry 2010

View Article Online
added. The resulting mixture was stirred at 30 ꢁC for 5 days.
After cooling to room temperature, the reaction mixture was
introduced into cold water (300 mL), where a quantitative
precipitate was formed. The final product was collected via
vacuum filtration as a white powder and washed with cold water
several times (76%). dH(500 MHz, DMSO-d6) 1.91(quintet, 4H),
2.27(t, 4H), 2.57(t, 4H), 3.06(q, 4H), 3.96(t, 4H), 7.32(s, 2H),
7.79(t, 2H); vmax/cmꢂ1 3304, 3082, 2935, 1637, 1552, 1488, 1464,
1351, 1265, 1215, 1059, 1039, 941, 846, 821, 754, 586, 490; m/z
(ESI) 619.26 [M + H].
Fluorescence turn-on experiments
A given quantity of GR was firstly added into a solution of
GSSG (0.3 mM) and NADPH (3 mM) in a total 540 mL PBS (10
mM, pH 7.4), and then incubated at ambient temperature. With
a 1 min interval, 30 mL of the resulting mixture was transferred
into a solution of PPE-(COOK)4 (5 mM) and Cu2+ (5 mM) in PBS
(10 mM, pH 7.4), then its fluorescence spectrum was recorded.
The emission intensity of PPE-(COOK)4/Cu2+ at 510 nm was
plotted as a function of the incubation time. Herein, a total of
five GR concentrations (0.2, 0.5, 1.0, 1.5 and 2.0 mU/mL) were
examined.
Monomer M3. Compound M2 (5.2 g, 8.4 mmol) was firstly
dissolved in a mixture of DMSO (20 mL) and methanol (60
mL), then methyl acrylate (10 mL) was added. The resulting
mixture was stirred at 30 ꢁC for 72 h. After cooling to room
temperature, the reaction mixture was transferred into cold
water, where a quantitative precipitate was formed. The final
product was collected via centrifugation, which was then
washed with water several times, and dried in a vacuum oven to
The inhibition experiments were almost the same as the above
procedure except that GR (2.0 mU/mL) was pre-treated with
BCNU in the presence of BSA (20 mg mLꢂ1) for 10 min.
Results and discussion
Synthesis and optical properties
In general, the proposed CPE that bears four carboxylate groups
on each repeat unit, PPE-(COOK)4, was prepared by integrating
the two synthetic strategies for conjugated poly(phenylene
ethynylene) (PPE) and dendritic polyamidoamine (PAMAM).
As shown in Scheme 2, an important aryl halide, 1,4-di(ethyl 4-
oxybutyrate)-2,5-diiodobenzene, was firstly obtained as the
starting material.24b Followed by the exhaustive amidation
reaction with ethylenediamine, and by the subsequent Michael
addition with methyl acrylate, a key monomer that took on four
ester groups (M3) could readily be afforded with 83% yield.
These two typical steps for PAMAM dendrimer have both been
fully validated with 1H NMR and ESI-MS spectra (Fig. S1,
ESI†). Then, the precursor polymer (P1) was prepared via the
Heck–Cassar–Sonogashira–Hagihara coupling reaction,10b,25
with a number-average molecular weight (Mn) of 27285 accord-
ing to the GPC characterization. Ultimately, an alkali induced
saponification of polymer P1 was carried out in methanol to
obtain the target polyelectrolyte, PPE-(COOK)4. Moreover, it is
also believed that there will emerge a series of ‘‘hyper-branched’’
obtain
a
white powder (83%). dH(500 MHz, CDCl3)
2.16(quintet, 4H), 2.42(t, 8H), 2.48(t, 4H), 2.53(br, 4H),
2.73(t, 8H), 3.34(q, 4H), 3.68(s, 12H), 4.00(t, 4H), 6.57(br, 2H),
7.18(s, 2H); vmax/cmꢂ1 3298, 2949, 2831, 2204, 1736, 1647, 1517,
1437, 1377, 1265, 1213, 1041, 837, 721, 544; m/z (ESI) 963.55
[M + H].
Polymer P1. The precursor polymer that possesses four ester
groups on each repeat unit (P1) was prepared via a similar
procedure reported previously.24 Monomer M3 (3.175 g, 3.3
mmol), 1,4-diethynylbenzene (409.5 mg, 3.25 mmol), Pd(PPh3)4
(46.08 mg, 40 mmol) and CuI (7.64 mg, 40 mmol) were placed in
the reaction flask. Meanwhile, a mixture of chloroform (80 mL),
triethylamine (12 mL) and diisopropylamine (12 mL) was added
to a second flask, which was connected to the former one via
a tubule. The whole system was deoxygenated by a gentle flow
of argon for 30 min. The solvent mixture was transferred into
the reaction flask, which was then stirred at 50 ꢁC for 3 days.
After cooling to room temperature, the resulting solution was
slowly added into diethyl ether (500 mL), where a quantitative
precipitate was formed. A high speed centrifuge was used to
collect the polymer precipitate as an orange powder (88%). The
polymer was washed with water and ethanol several times.
dH(500 MHz, CDCl3) 2.24(quintet, 4H), 2.38(t, 8H), 2.51(br,
8H), 2.70(t, 8H), 3.33(q, 4H), 3.63(s, 12H), 4.12 (t, 4H), 6.63 (br,
2H), 7.03(s, 2H), 7.51(d, 4H); vmax/cmꢂ1 3298, 2949, 2831, 2204,
1736, 1647, 1518, 1437, 1377, 1265, 1213, 1041, 837, 721, 588,
544.
PPE-(COOK)4. The ultimate conjugated polyelectrolyte,
PPE-(COOK)4, was obtained through the alkali induced
saponification of P1 according to a procedure described in the
literature.13a,24b In brief, a solution of P1 (166.4 mg, 0.2 mmol)
and KOH (2.0 g) in a mixture of CH3OH (200 mL) and H2O (2
mL) was refluxed for 24 h. Then, diethyl ether (300 mL) was
added, followed by centrifugation to yield PPE-(COOK)4 as an
orange solid (150 mg). dH(500 MHz, CD3OD) 2.16(quintet, 4H),
2.36(t, 8H), 2.51(t, 4H), 2.70(t, 4H), 2.88(t, 8H), 3.37(q, 4H), 4.12
(t, 4H), 7.13(s, 2H), 7.54(d, 4H); vmax/cmꢂ1 3421, 2945, 2725,
1631, 1402, 1215, 1124, 1009, 833, 704, 633, 619.
Scheme 2 Synthetic route towards PPE-(COOK)4.
J. Mater. Chem., 2010, 20, 10901–10907 | 10903
This journal is ª The Royal Society of Chemistry 2010

View Article Online
CPEs with more carboxylate/amino groups by iterating the
foregoing amidation and Michael addition steps at either the pre-
or post-polymerization stage.
excitingly, PPE-(COOK)4 was also found to show high selectivity
towards Cu2+ in PBS. As seen in Fig. 2, most of the metal cations
impose nearly no effects on the emission of PPE-(COOK)4, even
though their concentrations are increased up to 20 mM. In
contrast, Cu2+ at a lower concentration of 3 mM can quench the
fluorescence by >90%, and a high KSV value of 4.8 ꢀ 106 Mꢂ1 has
been obtained through the titration of PPE-(COOK)4 with Cu2+
in 10 mM PBS (Fig. S4, ESI†), providing a limit of detection as
low as 10 nM. These results clearly indicate that PPE-(COOK)4 is
fully competent to serve as an excellent fluorescent probe for
Cu2+, which is characterized by the concurrent high sensitivity
and remarkable specificity.
As expected, the resulting PPE-(COOK)4 with multiple
carboxylate groups is very soluble in aqueous solution, which is
much superior to that of a similar CPE with fewer carboxylate
groups (PPE-OBS,24b also see ESI† for chemical structure). In
PBS (10 mM, pH 7.4), PPE-(COOK)4 exhibits a strong absorp-
tion at 420 nm and a green-blue fluorescence at 510 nm (Fig. 1).
The relatively high quantum yield (ꢃ11%) and blue-shifted
emission peak, as compared to those of typical PPEs,12 are
mainly related to the light aggregation of PPE-(COOK)4 because
of its good water solubility and the enhanced electrostatic
repulsion between these heavily charged macromolecules.
Additionally, there is no evident fluorescence decline as observed
for PPE-OBS in a short period,24 nor is the quenching efficiency
(e.g. by methyl viologen, MV2+) badly lowered in spite of the
steric effects (Fig. S2, ESI†). Thus, it can be seen that the newly
proposed CPE has been greatly improved in various aspects
through the installation of multiple functional groups on the
backbone.
Glutathione induced fluorescence recovery
As an excellent Cu2+ probe, PPE-(COOK)4 can certainly be
extended into a wide range of applications. For instance, a fluo-
rescence turn-on assay for GSH can readily be established since
the free thiols can form the complex with Cu2+ more stably. As
a result, upon titrating a pre-quenched solution of PPE-
(COOK)4/Cu2+ with GSH, its fluorescence can be gradually
restored with a maximum recovery rate over 85% as the [GSH]
exceeds 6 mM (Fig. S5, ESI†). Most important of all, there exists
a notable distinctness in the degrees of fluorescence turn-on
caused by GSH and by GSSG at the same concentrations
(according to GSH molecules). As shown in Fig. 3a, GSH can
restore the fluorescence of PPE-(COOK)4/Cu2+ more efficiently
than its oxidized form (GSSG) does. In addition, these two
recovery rates, RGSH and RGSSG, can both be precisely tuned
through the variation of [Cu2+] and [GSH/GSSG]. Actually, the
Given a close look at the chemical structure, there actually
exists a strong resemblance between the pendant groups of PPE-
(COOK)4 and EDTA. Therefore, a tighter complexation of PPE-
(COOK)4 with metal ions can be expected. This has been partly
evidenced by the sensitive quenching of PPE-(COOK)4 caused by
Cu2+ even in the presence of phosphate (10 mM PBS), whereas
there is nearly no fluorescence decrease in the case of a dicar-
boxylate-PPE under the same conditions (Fig. S3, ESI†). More
Fig. 1 Normalized absorption and emission spectra of PPE-(COOK)4 in
PBS (10 mM, pH 7.4).
Fig. 3 a) GSH/GSSG induced fluorescence turn-on of PPE-(COOK)4 (5
mM)/Cu2+. Their respective recovery rates caused by GSH (RGSH, left)
and GSSG (RGSSG, right) are listed in ‘‘couples’’ corresponding to the
concentrations of Cu2+ and GSH/GSSG. b) The ratio of RGSH to RGSSG
as a function of [GSH/GSSG] at three Cu2+ concentrations. In both
charts, [GSSG] has been normalized according to GSH molecules.
Fig. 2 Fluorescence quenching of PPE-(COOK)4 (5 mM) caused by
various metal ions in PBS (10 mM, pH 7.4). The concentrations for most
cations are 20 mM with the exception of [Cu2+] ¼ 3 mM.
10904 | J. Mater. Chem., 2010, 20, 10901–10907
This journal is ª The Royal Society of Chemistry 2010

View Article Online
largest fluorescence turn-on caused by GSH occurs at a relatively
low [Cu2+], whereas a higher ratio of RGSH to RGSSG can be
obtained by properly increasing the content of Cu2+. As seen in
Fig. 3b, under the optimized conditions ([Cu2+] ¼ 5 mM, [GSH] ¼
6 mM), a maximum ratio of 6.7 between RGSH and RGSSG has
been achieved without the notable decrease of RGSH (Fig. 3a).
Undoubtedly, these results are highly inspiring for designing
a novel fluorescence turn-on assay of GR activity, since this
enzyme can catalyze the reduction of GSSG back to GSH.
of the GR-catalyzed reduction of GSSG. In contrast, other
control experiments have revealed that none of these three
species (GSSG, NADPH and GR) imposed a notable influence
on the emission of PPE-(COOK)4/Cu2+, neither separately nor in
a combination of the two species (Fig. 4b). Therefore, the
observed fluorescence recovery can only be attributed to the
generation of GSH during the GR-catalyzed reduction of GSSG,
which is consistent with the sensing mechanism shown in Scheme
1c. Moreover, the strong fluorescence enhancement of more than
10-fold demonstrates clearly
a high fluorescence turn-on
response, and thus to a large extent affords the capability for the
sensitive assay of GR activity.
Fluorescence turn-on assay for GR activity
As shown in Fig. 5, the emission intensity changes of PPE-
(COOK)4/Cu2+ at 510 nm versus the incubation time have been
recorded and compared at five GR concentrations. It reveals
clearly that the fluorescence recovery of PPE-(COOK)4/Cu2+ is
highly dependent upon the GR concentration, i.e., an increase in
the quantity of GR leads to not only a faster reaction rate at the
initial stage, but also a higher level of fluorescence turn-on. As
a consequence, the GR-catalyzed reduction of GSSG can readily
be monitored, and the enzyme itself can be assayed quantita-
tively. To the best of our knowledge, this newly proposed GR
activity assay, with a detectable enzyme concentration as low as
0.2 mU/mL, is at least comparable or even superior to most other
fluorescence techniques.8 Additionally, it is believed to be more
robust than the classic spectrophotometric methods,7 since the
fluorescence recovery is independent of the vulnerable yet excess
coenzyme NADPH.
The overall strategy of the proposed GR activity assay is pre-
sented in Scheme 1c. In general, PPE-(COOK)4 is used as the
signal reporter, which is specifically quenched by Cu2+ in
advance. At the beginning, there is nearly no fluorescence turn-
on in the presence of GSSG at an optimized concentration. In
contrast, upon the introduction of GR and the coenzyme
NADPH, GSSG undergoes a catalytic reduction to generate
GSH, and the latter can efficiently restore the fluorescence of
PPE-(COOK)4 by pulling Cu2+ away from the polymer. There-
upon, the activity of GR can readily be monitored by recording
the fluorescence recovery.
Herein, in order to verify the above sensing protocol, a series
of control experiments have firstly been carried out. Fig. 4a
displays the fluorescence spectral changes of PPE-(COOK)4/Cu2+
as a function of the incubation time for the GR-catalyzed reac-
tion. In these experiments, fluorescence spectra were recorded
after adding a mixture of GSSG (3 mM), NADPH (30 mM) and
GR (1.5 mU/mL) into the PPE-(COOK)4/Cu2+ solution with a 2
min interval. As expected, the emission of PPE-(COOK)4/Cu2+
underwent a fast increase during the first 10 min, and then leveled
off as the incubation time reached 15 min, which indicated an end
Fig. 5 Emission intensity of PPE-(COOK)4/Cu2+ (5 mM/5 mM) at 510
nm versus the incubation time for GR-catalyzed reduction of GSSG.
[GSSG] ¼ 3 mM, [NADPH] ¼ 30 mM.
Fig. 4 a) Fluorescence spectra of PPE-(COOK)4/Cu2+ as a function of
incubation time, [GR] ¼ 1.5 mU/mL. b) Effects of GSSG, NADPH and
GR (10 mU/mL) on the emission intensity of PPE-(COOK)4/Cu2+ at 510
nm. [PPE-(COOK)4] ¼ [Cu2+] ¼ 5 mM, [GSSG] ¼ 3 mM, [NADPH] ¼ 30
mM.
Fig. 6 Fluorescence turn-on response of PPE-(COOK)4/Cu2+ (5 mM/5
mM) versus the concentration of BCNU. [GR] ¼ 2 mU/mL, [GSSG] ¼ 3
mM, [NADPH] ¼ 30 mM.
This journal is ª The Royal Society of Chemistry 2010
J. Mater. Chem., 2010, 20, 10901–10907 | 10905

View Article Online
Furthermore, by taking advantage of the distinct fluorescence
recovery rates of PPE-(COOK)4/Cu2+ caused by GSH and
GSSG, a new fluorescence turn-on assay for GR activity has
successfully been developed. As the first GR activity assay based
on CPEs, it is highly sensitive and robust as compared to most
fluorescent and spectrophotometric methods. At present, the
assay of GR activity in a continuous and real-time manner on
microfluidic chips is under investigation.
Acknowledgements
Fig. 7 Emission intensity changes of PPE-(COOK)4/Cu2+ (5 mM/5 mM)
caused by various proteins in the presence of GSSG (3 mM) and NADPH
(30 mM). The concentrations of most proteins are 10 mg mLꢂ1 with the
exception of [GR] ¼ 2 mU/mL (ꢃ2.34 mg mLꢂ1).
This work is supported by the National Natural Science Foun-
dation of China (20805042), Natural Science Foundation of
Zhejiang Province (Y4090154), Fundamental Research Funds
for the Central Universities (2009QNA5006), and Innovation
Method Fund of China (2008IM040800).
Given that an effective GR activity assay should be capable of
monitoring the inhibition process, a typical inhibitor 1,3-bis(2-
chloroethyl)-1-nitrosourea (BCNU) has been examined, which is
the most commonly used irreversible GR inhibitor.26 As shown
in Fig. 6, the fluorescence turn-on response was apparently
decreased in the presence of BCNU with varying concentrations,
which demonstrated clearly an efficient restraint of the GR-
catalyzed reduction of GSSG. It should be noted that the
incomplete inhibition of GR in this experiment was actually
because of the fairly poor water solubility of this inhibitor. Thus,
the proposed sensor protocol can certainly be used in screening
potential drugs based on the inhibition of GR, e.g., anticancer or
antimalarial medicines.
References
1 H. F. Gilbert, Methods Enzymol., 1995, 251, 8.
2 A. Meister and M. E. Anderson, Annu. Rev. Biochem., 1983, 52, 711.
3 D. M. Townsend, K. D. Tew and H. Tapiero, Biomed. Pharmacother.,
2003, 57, 145.
4 C. Hwang, A. J. Sinskey and H. F. Lodish, Science, 1992, 257, 1496.
5 C. Biot, H. Bauer, R. H. Schirmer and E. Davioud-Charvet, J. Med.
Chem., 2004, 47, 5972.
6 M. Gunaratnam and M. H. Grant, Chem.-Biol. Interact., 2001, 134,
191.
7 (a) F. Tietze, Anal. Biochem., 1969, 27, 502; (b) I. Rahman, A. Kode
and S. K. Biswas, Nat. Protoc., 2007, 1, 3159.
8 (a) H. Maeda, H. Matsuno, M. Ushida, K. Katayama, K. Saeki and
N. Itoh, Angew. Chem., Int. Ed., 2005, 44, 2922; (b) A. M. Piggott and
P. Karuso, Anal. Chem., 2007, 79, 8769; (c) L. Yi, H. Li, L. Sun,
L. Liu, C. Zhang and Z. Xi, Angew. Chem., Int. Ed., 2009, 48, 4034.
9 (a) Q. Zhou and T. M. Swager, J. Am. Chem. Soc., 1995, 117, 12593;
(b) J. S. Yang and T. M. Swager, J. Am. Chem. Soc., 1998, 120, 11864;
(c) L. H. Chen, D. W. McBranch, H. L. Wang, R. Helgeson, F. Wudl
and D. G. Whitten, Proc. Natl. Acad. Sci. U. S. A., 1999, 96, 12287.
10 (a) T. M. Swager, Acc. Chem. Res., 1998, 31, 201; (b) U. H. F. Bunz,
Chem. Rev., 2000, 100, 1605; (c) D. T. McQuade, A. E. Pullen and
T. M. Swager, Chem. Rev., 2000, 100, 2537; (d) B. Liu and
G. C. Bazan, Chem. Mater., 2004, 16, 4467.
11 (a) K. E. Achyuthan, T. S. Bergstedt, L. Chen, R. M. Jones,
S. Kumaraswamy, S. A. Kushon, K. D. Ley, L. Lu, D. McBranch,
H. Mukundan, F. Rininsland, X. Shi, W. Xia and D. G. Whitten,
J. Mater. Chem., 2005, 15, 2648; (b) S. W. Thomas, G. D. Joly and
T. M. Swager, Chem. Rev., 2007, 107, 1339; (c) H. A. Ho, A. Najari
and M. Leclerc, Acc. Chem. Res., 2008, 41, 168; (d) Y. Liu,
K. Ogawa and K. S. Schanze, J. Photochem. Photobiol., C, 2009,
10, 173.
It is also of great interest to examine the fluorescence response
of PPE-(COOK)4/Cu2+ to other proteins, since there usually exist
the non-specific interactions between the charged CPEs and
proteins.27 In order to accomplish this goal, ten commonly used
proteins/enzymes have been tested in this work, in which the
experimental procedures were almost the same as the GR activity
assay with the exception of GR being replaced by one of these
proteins. As can be seen from Fig. 7, none of these proteins
showed a notable influence on the emission of PPE-(COOK)4/
Cu2+ (with or without the presence of GSSG and NADPH)
except for a slight increase in the case of trypsin, nor did these
species markedly affect the fluorescence of PPE-(COOK)4 itself
in PBS (Fig. S6, ESI†). These results have successfully addressed
the efficacy of the proposed GR activity assay to be used in
certain complicated systems.
12 (a) C. Y. Tan, M. R. Pinto and K. S. Schanze, Chem. Commun., 2002,
446; (b) M. Liu, P. D. Kaur, H. Waldeck, C. H. Xue and H. Y. Liu,
Langmuir, 2005, 21, 1687; (c) M. Stork, B. S. Gaylord, A. J. Heeger
and G. C. Bazan, Adv. Mater., 2002, 14, 361.
Conclusions
In summary, an anionic CPE that bears four carboxylate groups
on each repeat unit has been designed and synthesized by inte-
grating the two synthetic strategies for conjugated PPE and
dendritic PAMAM. It is also expected that a set of CPEs with
more pendant groups will possibly be obtained by increasing the
‘‘generation number’’, as described for PAMAM dendrimer.
Owing to the increased carboxylate groups, the resulting PPE-
(COOK)4 has been greatly improved in many aspects, e.g., good
water solubility, slight aggregation, and relatively high quantum
yields. More excitingly, it has further been demonstrated to show
not only high sensitivity but remarkable specificity toward Cu2+,
which makes it an excellent fluorescent probe for Cu2+.
13 (a) I. B. Kim, A. Dunkhorst, J. Gilbert and U. H. F. Bunz,
Macromolecules, 2005, 38, 4560; (b) I. B. Kim, R. Phillips and
U. H. F. Bunz, Macromolecules, 2007, 40, 5290; (c) Q. L. Xu,
L. L. An, M. H. Yu and S. Wang, Macromol. Rapid Commun.,
2008, 29, 390.
14 (a) I. B. Kim and U. H. F. Bunz, J. Am. Chem. Soc., 2006, 128, 2818;
(b) X. F. Liu, Y. L. Tang, L. H. Wang, J. Zhang, S. P. Song, C. Fan
and S. Wang, Adv. Mater., 2007, 19, 1471; (c) Z. Fang, K. Y. Pu and
B. Liu, Macromolecules, 2008, 41, 8380.
15 (a) N. DiCesare, M. R. Pinto, K. S. Schanze and J. R. Lakowicz,
Langmuir, 2002, 18, 7785; (b) F. He, Y. L. Tang, M. H. Yu,
S. Wang, Y. L. Li and D. B. Zhu, Adv. Funct. Mater., 2006, 16, 91;
(c) F. He, F. Feng, S. Wang, Y. L. Li and D. B. Zhu, J. Mater.
Chem., 2007, 17, 3702; (d) T. Zhang, H. L. Fan, G. L. Liu, J. Jiang,
J. G. Zhou and Q. H. Jin, Chem. Commun., 2008, 5414.
10906 | J. Mater. Chem., 2010, 20, 10901–10907
This journal is ª The Royal Society of Chemistry 2010

View Article Online
16 (a) H. A. Ho and M. Leclerc, J. Am. Chem. Soc., 2004, 126, 1384; (b)
M. B. Aberem, A. Najari, H. A. Ho, J. F. Gravel, P. Nobert,
D. Boudreau and M. Leclerc, Adv. Mater., 2006, 18, 2703.
17 (a) C. C. You, O. R. Miranda, B. Gider, P. S. Ghosh, I. B. Kim,
B. Erdogan, S. A. Krovi, U. H. F. Bunz and V. M. Rotello, Nat.
Nanotechnol., 2007, 2, 318; (b) O. R. Miranda, C. C. You,
R. Phillips, I. B. Kim, P. S. Ghosh, U. H. F. Bunz and
V. M. Rotello, J. Am. Chem. Soc., 2007, 129, 9856; (c) J. Wang and
B. Liu, Chem. Commun., 2009, 2284.
18 (a) S. Kumaraswamy, T. Bergstedt, X. B. Shi, F. Rininsland,
S. Kushon, W. S. Xia, K. Ley, K. Achyuthan, D. McBranch and
D. Whitten, Proc. Natl. Acad. Sci. U. S. A., 2004, 101, 7511; (b)
F. Rininsland, W. S. Xia, S. Wittenburg, X. B. Shi, C. Stankewicz,
K. Achyuthan, D. McBranch and D. Whitten, Proc. Natl. Acad.
Sci. U. S. A., 2004, 101, 15295; (c) J. H. Wosnick, C. M. Mello and
T. M. Swager, J. Am. Chem. Soc., 2005, 127, 3400; (d) L. L. An,
Y. L. Tang, F. D. Feng, F. He and S. Wang, J. Mater. Chem.,
2007, 17, 4147.
G. C. Bazan, J. Am. Chem. Soc., 2003, 125, 896; (c) B. Liu and
G. C. Bazan, Proc. Natl. Acad. Sci. U. S. A., 2005, 102, 589.
21 (a) H. A. Ho, M. Boissinot, M. G. Bergeron, G. Corbeil, K. Dore,
D. Boudreau and M. Leclerc, Angew. Chem., Int. Ed., 2002, 41,
1548; (b) S. A. Kushon, K. D. Ley, K. Bradford, R. M. Jones,
D. McBranch and D. Whitten, Langmuir, 2002, 18, 7245; (c)
H. A. Ho, K. Dore, M. Bissoinot, M. G. Bergeron, R. M. Tanguay,
D. Boudreau and M. Leclerc, J. Am. Chem. Soc., 2005, 127, 12673;
(d) A. Najari, H. A. Ho, J. F. Gravel, P. Nobert, D. Boudreau and
M. Leclerc, Anal. Chem., 2006, 78, 7896.
22 (a) Y. S. Wang and B. Liu, Anal. Chem., 2007, 79, 7214; (b)
X. R. Duan, S. Wang and Z. P. Li, Chem. Commun., 2008, 1302; (c)
F. He, F. Feng, X. R. Duan, S. Wang, Y. L. Li and D. B. Zhu,
Anal. Chem., 2008, 80, 2239.
23 (a) S. Wang and G. C. Bazan, Adv. Mater., 2003, 15, 1425; (b) B. Liu,
S. Baudrey, L. Jaeger and G. C. Bazan, J. Am. Chem. Soc., 2004, 126,
4076.
24 (a)T. Zhang,H. L. Fan, J. G. Zhou, G. L. Liu, G. D. FengandQ. H. Jin,
Macromolecules, 2006, 39, 7839; (b) T. Zhang, H. L. Fan, J. G. Zhou
and Q. H. Jin, J. Polym. Sci., Part A: Polym. Chem., 2009, 47, 3056.
25 H. A. Dieck and R. F. Heck, J. Organomet. Chem., 1975, 93, 259.
26 P. A. Karplus, R. L. Krauth-Siegel, R. H. Schirmer and G. E. Schulz,
Eur. J. Biochem., 1988, 171, 193.
27 (a) S. J. Dwight, B. S. Gaylord, J. W. Hong and G. C. Bazan, J. Am.
Chem. Soc., 2004, 126, 16850; (b) I. B. Kim, A. Dunkhorst and
U. H. F. Bunz, Langmuir, 2005, 21, 7985.
19 (a) M. R. Pinto and K. S. Schanze, Proc. Natl. Acad. Sci. U. S. A.,
2004, 101, 7505; (b) X. Y. Zhao, Y. Liu and K. S. Schanze, Chem.
Commun., 2007, 2914; (c) Y. Liu, K. Ogawa and K. S. Schanze,
Anal. Chem., 2008, 80, 150; (d) Y. Liu and K. S. Schanze, Anal.
Chem., 2008, 80, 8605; (e) Y. Liu and K. S. Schanze, Anal. Chem.,
2009, 81, 231.
20 (a) B. S. Gaylord, A. J. Heeger and G. C. Bazan, Proc. Natl. Acad. Sci.
U. S. A., 2002, 99, 10954; (b) B. S. Gaylord, A. J. Heeger and
This journal is ª The Royal Society of Chemistry 2010
J. Mater. Chem., 2010, 20, 10901–10907 | 10907