Feature Article
Water-Soluble Conjugated Polymers forFluorescent-Enzyme Assays
Fude Feng, Libing Liu, Qiong Yang, Shu Wang*
Enzyme assays are receiving more and more research and application interest because of therapidly increasing demands of clinical diagnosis, environmental analysis, drug discovery, andmolecular biology. Water-soluble light-harvesting conjugated polymers (CPs) coordinate theaction of a large number of absorbing units to afford an amplified fluorescence signal, whichmakes them useful as optical platforms in highlysensitive chemical and biological sensors. ThisFeature Article highlights recent developmentsof water-soluble CPs for fluorescent assays ofenzymes. Different signal transduction mechan-isms, such as electron transfer, fluorescenceresonance energy transfer (FRET), and aggregationor conformation changes of CPs, are employed inthese assays according to the dissimilar nature ofenzymes. Potential challenges and future researchdirections in these approaches based on CPs arealso discussed.
Introduction
Conjugated polymers (CPs) are characterized by a deloca-
lized electronic structure and coordinate the action of a
large number of absorbing units, which exhibit unique
optoelectrical properties. They have beenwidely utilized as
electronic and optical components in devices, such as light-
emitting diodes (LEDs), field effect transistors (FETs), and
photovoltaic cells in the past few decades.[1–8] Water-
soluble CPs are CPs decorated with pendant water-soluble
ionic groups such as sulfonate (�SO�3 ), carboxylate (�CO�
2 ),
phosphonate (�PO2�3 ), and ammonium (�NRþ
3 ) groups. In
the presence of oppositely charged energy acceptors, the
S. Wang, F. Feng, L. Liu, Q. YangBeijing National Laboratory for Molecular Sciences, KeyLaboratory of Organic Solids, Institute of Chemistry, ChineseAcademy of Sciences, Beijing 100190, P. R. ChinaFax: 86-10-62636680; E-mail: [email protected]
Macromol. Rapid Commun. 2010, 31, 1405–1421
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excitation energy transfers along the whole backbone of
water-soluble CPs to acceptor sites over long distances,
which results in the amplified fluorescence signal.[9] This
amplification mechanism can be used to significantly
increase the sensitivity of biological detection. Further-
more, pendant ionic groups confer CPs a simple tool to tune
their interactions with biomacromolecules and control the
average distance between optical chromophores.[10] In
recent years, researchers have presented great interest in
water-soluble CPs for their applications in chemical and
biological sensors with high sensitivity.[11–18] Diverse
water-soluble CPs have been designed for the purposes of
detecting metal ions and small molecules, and also for
bioassays of proteins and nucleic acids.[19–41]
Enzymeassaysare receivingmoreandmore researchand
application interest because of the rapidly increasing
demands of clinical diagnosis, environmental analysis,
drug discovery, and molecular biology. In particular, some
kinds of enzymes, such as esterases, proteases, and kinases
DOI: 10.1002/marc.201000020 1405
F. Feng, L. Liu, Q. Yang, S. Wang
Fude Feng received his B.S. degree from Departmentof Chemistry at Tsinghua University in 2001 and hisPh.D. degree from Institute of Chemistry, ChineseAcademy of Sciences under the guidance of ProfessorShu Wang in 2009. His Ph.D. thesis focuses on designand synthesis of new water-soluble conjugated poly-mers for DNA methylation and enzyme detection.
Shu Wang received his B.S. degree from Departmentof Chemistry, Hebei University, in 1994 and his Ph.D.degree in organic chemistry at Department of Chem-istry, Peking University, in 1999. Following two yearsof postdoctoral research at the Institute of Chemistry,Chinese Academy of Sciences, he moved to the Insti-tute of Polymers and Organic Solids, University ofCalifornia at Santa Barbara to continue his postdoc-toral research. In 2004, he was appointed Professor atthe Institute of Chemistry, Chinese Academy ofSciences. His current research interests includedesign, synthesis, and properties of light-harvestingconjugated polymers, biosensors, and chemicalbiology.
1406
not only play an essential role in normal life processes, but
are also found to be tightly associated with diseases.[42–45]
Detectionsensitivity isoneof themost importantaspects in
enzyme biosensor development,with the ultimate goal the
trace detection of enzymes from biological fluids. This
Feature Article aims to highlight applications of water-
soluble CPs in enzyme assaysmainly based on fluorescence
techniques. Six types of enzymes as detection targets are
described: protease, kinase, oxidase, esterase, transferase,
and nuclease. The enzyme assays based on CPs have three
unique features. First, different from small fluorescent
molecules, CPs impart the enzyme assay high sensitivity.
Second, the water-soluble CPs can form complexes with
oppositely charged enzyme substrates through electro-
static and/or hydrophobic interactions to avoid labelling
the CPs by covalent linkages, which should significantly
reduce the synthetic complexity. Third, the high density of
pendant charges of CPs enormously enhances the electro-
static attraction or repulsion between CPs and enzyme
substrates, which is favorable to extend the application
window of CPs for wide-spectrum enzyme detections.
The Design and Preparation of CPs and theSignal Transduction Mechanism
The chemical structures of water-soluble CPs are designed
with anionic or cationic pendant groups according to the
charge properties required by researchers, based on various
CP backbones. Of the various primary CP backbones,
polyfluorenes (PFs), poly(phenylene vinylene)s (PPVs),
poly(phenylene ethynylene)s (PPEs), polythiophenes (PTs)
and their related structures have been widely used in
fluorescent assays of enzymes[11–18] (see Figure 1 for their
chemical structures).WudlandSchanzehavewell reviewed
the comprehensive synthetic methods for CPs.[46,47] In
general, the polymerization methods for carbon–
carbon bond formation reactions in CP preparation are
suitable for the synthesis of water-soluble CPs as well,
except for somenecessarymodificationsofbranchedchains
before or after polymerization. Since structural defects in
the CP backbone that result from the polymerization
process can influence the electronic delocalization, new
synthetic techniques have been inspired.[48] The most
popular polymerization process performed under mild
conditions to yield defect-free CPs includes Suzuki, Stille,
and Yamamoto coupling reactions for PFs,[49–52] Wittig–
Horner and Heck reactions for PPVs,[53,54] Sonogashira
coupling and alkyne metathesis reactions for PPEs,[55,56]
and electropolymerization, metal-catalyzed polycondensa-
tion, and chemical oxidative reactions for PTs.[57–59]
Recently, a valuable microwave-assisted method has been
reported to produce CPs within just ten minutes in high
yield and reduce the amount of side reactions.[60] The
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molecular weight of a CP can be determined by gel
permeation chromatography (GPC), but in some cases it
is not available because of the poor solubility of CPs in
organic solvents such as N,N-dimethylformamide (DMF)
and tetrahydrofuran (THF). Dialysis againstwater or buffer
solutions to cut-off low molecular weight polymers
provides an alteration to characterize the CP size, mean-
while purifying the polymers.
In the present CP-based enzyme assays, three types of
signal transductionmechanisms dominate: electron trans-
fer, fluorescence resonance energy transfer (FRET), and
aggregation or conformation changes of CPs. Fluorescent
superquenching of CPs by traces of quencher through
trapping energy and/or electron migrating along the
backbone is one important feature of CPs.[61,62] The normal
quenching behavior follows the Stern–Volmer equation:
F0=F ¼ 1þ KSV½Q� (1)
where F0 denotes the initial fluorescence intensity of
CPs, F denotes the fluorescence intensity of CPs in the
presence of quencher, [Q] denotes the quencher concen-
tration and KSV is the Stern–Volmer constant.[63] In a
superquenching system, because the dynamic quenching
and static quenching may exist simultaneously,
Equation (1) is untenable except at an extremely low
concentration of quencher. A concept of ‘‘sphere-of-action’’
is useful to interpret the superlinear behavior of the
Stern–Volmer curve that appears upon increasing the
quencher concentration, and a modified form of
Equation (1) that describes the coexistence of static
DOI: 10.1002/marc.201000020
Water-Soluble Conjugated Polymers for Fluorescent-Enzyme Assays
Figure 1. Examples of water-soluble CPs in enzyme assay applications.
quenching and dynamic quenching is shown in
Equation (2):
Macrom
� 2010
F0=F ¼ ð1þ KSSV½Q�Þ expðvN½Q�Þ
¼ ð1þ KSSV½Q�Þ expðaV ½Q�Þ (2)
where v is the volume of the sphere-of-action, N is
Avogadro’s constant, KSSV is the static quenching constant,
V is defined as nN, and a is the coefficient for charge-
induced enhancement of the local quencher concentra-
tion.[64] Superquenching behaviors greatly lower the
background signals, and are frequently employed for the
enzyme-catalyzed cleavage of a linker between analyte
and quencher to turn on the fluorescence of CPs.
The quenchers can be designed as energy acceptors such
that their emission spectra overlap well with the absorp-
tion spectra of the CPs to allow for efficient FRET from the
CPs to acceptors. FRET is a long-range energy transfer, and
arises from dipole–dipole interactions based on Forster
theory.[65] When exciting the CP backbone, the acceptor
emits fluorescence much more strongly than when being
directly excited at its maximum absorption wavelength,
which leads to significant signal amplification.[9] Further-
more, the FRET technique provides a ratiometric measure-
ment that is not so susceptible to the environment as the
quenching methodology, making it easier for quantitative
and robust bioassays.
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The aggregation or conformation
change of CPs is another mechanism in
the fluorescence assay of enzymes. In
these methods, the addition of enzyme
substrates to CPs results in interchain
interactions between the CPs, which
leads to fluorescence quenching because
of p-stacking of the backbones of the CPs.Upon adding enzymes, the aggregates of
CPs are broken up or their conformations
are recovered, the CPs show a ‘‘turn-on’’
fluorescent response.[12,66,67] Of course,
the fluorescence behavior of water-solu-
ble CPs in aqueous solution is compli-
cated in comparison to small organic
molecules. The possible formation of
polymer aggregates in water could cause
a lower emission intensity. The addition
of surfactant can enhance CP emission
intensities as aggregates are broken up
and the effect of self-quenching is dimin-
ished. Bovine serum albumin (BSA) was
reported to interact with CPs in a non-
specific manner and enhance CP fluores-
cence with a simultaneous blue-shift
because of its surfactant effect.[68]
Protease Assays Using CPs
In protease-targeted assays based on CP platforms, Schanze
and co-workers, Whitten and co-workers, and Swager and
co-workers,[14–16] respectively, developed distinctive and
mutually complementary approaches. Pinto and Schanze
established a model of ‘‘turn-on’’ and ‘‘turn-off’’ responses
ofanionicCP-based sensors forassayingproteaseactivity in
homogeneousmedia.[69] In the turn-on approach, as shown
in Figure 2, a cationic substrate peptide labeled with a
quencher p-nitroanilide (p-NA) bound to anionic sulfo-
nated poly(phenylene-ethynylene) (PPESO3) and quenched
its fluorescence. In the presence of target protease or
thrombin that catalyzed the hydrolysis of peptide bonds,
thep-NAunitbecameremovedfromthepolymer,which led
to the recovery of polymer fluorescence. By this means,
protease was detectedwith a high sensitivity increasing to
greater than two orders of magnitude compared to
traditional assays based on absorption spectroscopy. In
the turn-off approach, carboxylate-substituted PPE poly-
mer (PPECO2) fluorescence was not affected by weakly
fluorescent Rho-Arg2 that binds to PPECO2, but quenched
by the papain-catalyzed hydrolysis product Rho-Arg
through an energy transfer mechanism. The protease-
catalyzed hydrolysis process was monitored in a real-time
manner by reading the emission intensities of conjugated
www.mrc-journal.de 1407
F. Feng, L. Liu, Q. Yang, S. Wang
Figure 2. a) Structures for polymers and quencher substrates. b) Mechanism of the turn-on and turn-off CP-based enzyme sensors. Reproduced with permission from Ref. [69].Copyright 2004, National Academy of Sciences, USA.
1408
polyelectrolytes (CPE) during incubation. The method is
very useful to determine enzymatic kinetics within a short
time (second scale) with small amounts of proteases and
substrates because of an instant sensitive response in
fluorescence emission. More importantly, the strategy is
valuable for reference in enzyme assays that involve
enzymatic cleavage. Very recently, Liu and co-workers have
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extended this sensing mechanism to
assay trypsin by a FRET technique.[70]
A problem frequently encountered in
homogeneous enzyme assays is that self-
quenching of water-soluble CPs through
inter- and intrachain interactions may
interfere with fluorescence measure-
ments, particularly at higher CP concen-
trations. Whitten and co-workers devel-
oped a smart technique that employed
microsphere-based CP sensors for pro-
tease assays (demonstrated in
Figure3).[71] The targetprotease triggered
thecleavageofaquencher-linkedpeptide
substrate and then interacted through
biotin-avidin recognition with anionic
PPE-coated microspheres on which PPE
maintained a high quantum yield. This
resulted in a strong response of the PPE
fluorescence signal as distant quenchers
did not affect the PPE fluorescence.
Devoid of protease preincubation, a
superquenching phenomenon occurred
and a very low background signal was
present. Based on the relationship
between fluorescence intensities and
substrate concentrations, enzymekinetic
parameters for three different proteases
(EK, CASP-3/7, BSEC) were determined.
This method also takes advantages of a
high-throughput screening (HTS) techni-
que for drug screening by studying
enzyme inhibition. As compared to
homogeneous assays, the method pro-
vides a solution that overcomes draw-
backs such as self-quenching and poor
tolerance to solvents. However, improve-
ments should be executed because CP-
coated microspheres can halt the activ-
ities of some enzymes by irreversible
adsorption of enzymes through strong
hydrophobic interactions.
A label-free, homogeneous, and sensi-
tive fluorescence turn-on approach was
designed by our group to rapidly detect
protease using serine-functionalized
polythiophene (POWT)[72] (Figure 4).
The fluorescence of a POWT solution that contained BSA
as a substrate was efficiently quenched by Cu2þ ions
through a coordinate interaction with serine moieties.
Upon adding trypsin to the solution, the BSA was cleaved
intoaminoacidorpeptide fragments that are strongerCu2þ
chelators and formed more stable complexes with Cu2þ
ions. Thus theCu2þ ionswere displaced from the POWTand
DOI: 10.1002/marc.201000020
Water-Soluble Conjugated Polymers for Fluorescent-Enzyme Assays
Figure 3. Schematic representation of the QTL assay for protease and their inhibitors,and the chemical structures of fluorescent CPs used. Reproduced with permission fromref.[71]. Copyright 2004, National Academy of Sciences, USA.
the fluorescence was recovered. By triggering the turn-on
signal of POWT, it is possible to detect the trypsin in real
time. The turn-on response as a readout signal is able to
effectively reduce background noise and increase detection
sensitivity. Because of the simplicity, high sensitivity, and
rapid response, this new enzyme assay shows great
potential for protease detection in the future.
Another label-free fluorescence approach based on
intramolecular FRET was designed by our group to assay
hyaluronidase[73] (Figure 5). Cationic water-soluble PFs
containing 2,1,3-benzothiadiazole (BT) units (PFP-BT 1–3)
have been synthesized and characterized. These polymers
demonstrate intramolecular/intermolecular energy trans-
fer from the fluorene units to the BT sites[66,67] when
oppositely charged hyaluronan is added because of the
formation of electrostatic complexes, which leads to an
emission color shift from blue to green or brown. Upon
adding hyaluronidase, the hyaluronan was cleaved into
Figure 4. Schematic representations of the trypsin assay based on PT. Reproduced withpermission from ref.[72]. Copyright 2009, American Chemical Society.
Macromol. Rapid Commun. 2010, 31, 1405–1421
� 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
fragments. The relatively weak electro-
static interactions of the hyaluronan
fragments with PFs resulted in separa-
tion of the main chains and energy
transfer from the fluorene units to the
BT sites was inefficient, as such the
polymers recovered their blue emission.
The complexes of CPs/hyaluronan can be
utilized as probes for sensitive and facile
fluorescence assays for hyaluronidase.
The new assay method interfaces with
the aggregation and light harvesting
properties of CPs. Similar to the assay
mechanism for hyaluronidase, trypsin
activity could be probed by anionic PFs
that contain BT units using a peptide
substrate composed of six arginine resi-
dues (Arg6 peptide).[66] The fluorescence
enhancement of the polymer even
reached 22-fold after trypsin cleaved
the Arg6 peptide.
BSA can enhance the CP fluorescence by breaking up its
aggregates. Jin et al. haveprepareda complex of anionic PPE
withBSA thatwas successfully utilized toassay trypsin and
pepsin activities.[68] Ho and Leclerc have developed a new
enzyme detection strategy that takes advantage of an
aptamer–enzyme binding-induced conformation transi-
tion of a cationic PT to result in its color changes.[74] This
method provides a convenient method for thrombin
detection with high sensitivity and selectivity. Recently,
Wand and Liu have used this strategy to trigger FRET from
an anionic CP to a dye-labeled aptamer, which provides a
convenient method for lysozyme detection in biological
media.[75]
Different from methods employing electrostatic inter-
actions between a CP probe and quencher-linked substrate,
Swager and co-workers synthesized PPEs with a pendant
quencher-modified peptide by covalent linkages as shown
in Figure 6.[76] The high density of
hydrophilic oligo(ethylene glycol) moi-
eties, instead of ionic groups, improves
the water solubility of polymers, yet not
sufficiently to eliminate hydrophobic
interactions between the protease and
polymer.HighconcentrationsofTritonX-
100were introduced to allow cleavage of
the peptide by the target trypsin, which
results in a turn-on response of
PPE fluorescence. One significant advan-
tage over other methods based on elec-
trostatic complex-based detection plat-
forms lies in that the ionic strength of the
buffer solution does not affect the assay
results.
www.mrc-journal.de 1409
F. Feng, L. Liu, Q. Yang, S. Wang
Figure 5. The assay strategy of HAase and the chemical structures of cationic PFP-BT 1–3and hyaluronan. Reproduced with permission from ref.[73]. Copyright 2009, ScienceChina Press Co., Ltd.
1410
Kinase and Phosphatase Assays Using CPs
A protein kinase is an enzyme that modifies other proteins
by chemically adding phosphate groups to them (phos-
phorylation). On the contrary, phosphatase is an enzyme
that removes phosphate groups of other proteins (depho-
sphorylation). Bothof themregulate themajorityof cellular
pathways and are involved in signal transduction.
Like detection platforms based on gold nanoparticles,
metal ions are also able to mediate pulling together
polymers and peptides. Whitten and co-workers employed
the fluorescent superquenching principle based on PPE-
coated microspheres to monitor the enzymatic process of
kinase and phosphatase by the incorporation of di- or
trivalent metal ions and a quencher–tether–ligand (QTL)
sensor.[77] As indicated in Figure 7, Ga3þ ions were
investigated and found capable of binding carboxylic and
phosphate groups. Anionic PPE-coated microspheres could
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bind Ga3þ by pendant carboxylic groups
to generate a positively charged surface,
and the polymer fluorescence could be
efficiently quenched by the presence of a
rhodamine-labeled phosphorylated pep-
tide (rho-phosphopeptide) that bound to
Ga3þ ions, while the polymer fluores-
cence remained unaffected in the
absence of phosphorylation. A protein
kinase A (PKA) phosphorylated rhoda-
mine-labeled peptide gave a turn-off
fluorescence response of PPE, and in the
reverse process the phosphatase cata-
lyzed the dephosporylation of rho-phos-
phopeptide, which led to a turn-on
response of polymer fluorescence. A
further approach for a kinase activity
assay, for instance, kinase PKCa, utilized
an unmodified peptide as substrate and
rho-phosphopeptide as quencher. The
heterogeneous assays exhibit a high
repeatability and a high tolerance to
the testing environment. Furthermore, a
high sensitivity was obtained in these
assays because of the superquenching
behavior of the CP that offers very low
background noise. It is noted that the
enzyme activity might be influenced
more or less by complicated factors such
as physical adsorption.
A label free, simple, and real-time
protocol to assay ATP-dependent hexo-
kinases (HK) recently has beendeveloped
by our group (Figure 8).[78] Cationic
polythiophene (CPT) formed a complex
with ATP through electrostatic interac-
tions. The spatial constraints within the CPT/ATP complex
force the CPT to adopt a more planar conformation, which
leads to an absorption maximum at 537nm. Phosphoryla-
tion of glucose to G-6-P by HK converted ATP into ADP that
contains less negative charges. The weak electrostatic
binding of CPT to ADP caused CPT to return to the unbound
state, where it adopted a random-coil conformation and
exhibited anabsorptionmaximumat a shorterwavelength
(457nm). Enzymatic action thereby causes the solution
color to change from pink-red to yellow and it can be
monitoredbyUV–vis spectraorbysimplevisual inspection.
A non-labeling optical platform that takes advantage of
aggregation triggered by ions has also been developed by
our group to assay phosphatase (Figure 9).[66] An oppositely
charged enzyme substrate could induce aggregation of
cationicalyy charged PFs that contain BT units (PFP-BT 1)
through electrostatic interactions and allow for energy
transfer from the fluorene units to BT sites as shown in
DOI: 10.1002/marc.201000020
Water-Soluble Conjugated Polymers for Fluorescent-Enzyme Assays
Figure 6. Mechanism of the fluorescence turn-on assay for protease and chemical structures for the protease substrate. Adapted withpermission from ref.[76]. Copyright 2005, American Chemical Society.
Figure 5. The emission of fluorene units in cationic PFP-BT 1
at 410nm was significantly quenched in the presence of
ATPwhich carried a high density of negative charges,while
it was slightly lowered by addition of ADP or AMP. The
aggregationwas indicated by a color changeunderUV light
as the BT sites emitted green light when FRET took place. A
turn-on fluorescence response at 410nm occurred after
degradationofATPbyalkalinephosphatase (ALP) todestroy
aggregation. An organic solvent such as DMF was indis-
pensable to optimize the enzymatic reaction in order to
diminish the possible hydrophobic interactions between
the enzyme and polymer and also to break up the
aggregates after hydrolysis. Under optimal conditions,
fluorescence of PFP-BT 1 could be enhanced by 120% after
hydrolysis of ATP by ALP. Since ATP participates in a great
population of biological reactions as anenergy supplier, the
platform is highly valuable to monitor the enzymatic
process. Importantly, the approach deserves sufficient
attention for the utilization of non-labeling modification
of substrates, which provides an objective insight into
enzyme functions. Further improvements would be neces-
sary to reduce thehighdependenceupon solvents and ionic
strength of the reaction buffer.
Recently, Liu and Schanze have also developed a new
method for an assay of ALP based on PPECO2.[79] The
fluorescence of the anionic PPECO2 was efficiently
quenched by Cu2þ ions through an electron transfer
mechanism. Upon the addition of pyrophosphate (PPi) into
the solution of a PPECO2/Cu2þ complex, the fluorescence of
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� 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
PPECO2 was recovered since the PPi can effectively
sequester Cu2þ and remove it from the PPECO2. Using
the PPECO2/Cu2þ system as the signal transducer, a real-
time fluorescence turn-off assay for ALP using PPi as the
substrate is realized. The assay offers a straightforward and
rapid detection of ALP activity with the enzyme present in
the nanomolar concentration range, operating either in an
end point or real-time format. Later on, they extended the
PPECO2/Cu2þ systemtoassayadenylatekinase ina turn-off
manner.[80]
Esterase Assays Using CPs
An assay of acetylcholinesterase (AChE) activity is
another successful example based on superquenching
of fluorescent CPs (Figure 10).[81] Considering that detec-
tion methods for AChE activity and inhibition have
not been substantially developed in the past decades,
the sensitive and quantitative technique represents
remarkable value because of the tight correlation
betweenAChE andAlzheimer’s disease (AD) formation.[82]
ACh-Dabcyl, a Dabcyl (quencher)-modified substrate of
AChE, could heavily quench anionic PEP� SO�3 fluores-
cence in aqueous solution upon binding to the polymer
through electrostatic interactions, which leads to an
extremely low background signal. AChE recognized and
catalyzed the hydrolysis of ACh-Dabcyl to release the
Dabcyl group from the anionic polymer, which triggered
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F. Feng, L. Liu, Q. Yang, S. Wang
Figure 7. a) Structure of anionic CP. b) Scheme for kinase and phosphatase assays. c) Scheme for kinase assays using unlabeled peptidesubstrate. Reproduced with permission from ref.[77]. Copyright 2004, National Academy of Sciences, USA.
1412
a turn-onfluorescence responseof PEP� SO�3 . Thepolymer
fluorescence could be enhanced more than 130-fold.
Charge reversal on the quencher caused the quencher
to escape from the polymer after AChE-catalyzed
hydrolysis of the substrate, and stopped the quenching
instantly without any adverse influence upon polymer
fluorescence. This feature was designed to simplify the
detection platform and increase detection sensitivity. By
virtue of the continuous assay, the enzyme reaction
kinetics was studied to give the Km value of AChE
within just 100 seconds. To ensure the reliability of the
data obtained from the superquenching system, another
water-soluble polymer PFP-COOH with a far lower KSV
valuewas used to assay AChE activity in the same fashion
and provided a similar Km value as that obtained from
PFP� SO�3 PFP-SO
�3 . These data suggest that both polymers
were appropriate for kinetics determination of AChE. The
method affords the high sensitivity for the AChE assay
with a limit of detection (LOD) of 0.05 units �mL�1 that is
comparable to that of most sensitive chemiluminescent
techniques.
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Our recent work shows that the AChE assay can be
realized using a fluorescent microgel as a transduction
platform.[83] We prepared a novel fluorescent microgel
that contained poly(N-isopropylacrylamide) and cationic
conjugated PF (PFP-NIPAm) with a size of 100 nm
(Figure 11). The PFP-NIPAm microgel itself exhibited blue
emission under UV light with excitation at 365 nm.
Addition of ACh-Dabcyl to the cationic PFP-NIPAm
microgel did not lead to complex formation because of
electrostatic repulsion, therefore, the fluorescence of PFP-
NIPAm was not quenched by the Dabcyl. Upon adding
AChE, ACh-Dabcyl was catalytically hydrolyzed to pro-
duce choline and a negatively charged residue that
contained theDabcylmoiety (AD�). Owing to electrostatic
attraction, the PFP-NIPAm/AD� complex formed and the
Dabcyl moiety resided in close proximity to the PFP-
NIPAm, therefore, the fluorescence of PFP-NIPAm was
efficiently quenched. In light of the quenchedfluorescence
intensity of PFP-NIPAm, the AchE can be detected in a
continuous and real-time manner. The increase of AChE
amount accelerated the cleavage reaction rate and
DOI: 10.1002/marc.201000020
Water-Soluble Conjugated Polymers for Fluorescent-Enzyme Assays
Figure 8. Schematic representation of the assay for HK catalyzed ATP-dependentglucose phosphorylation using CPT. Reproduced from ref.[78].
resulted in a high level of fluorescence quenching. The
microgel can be reused following simple washing steps,
and the detection media can be used from homogeneous
solution to gel phase, which made it possible to detect the
enzymes in practice.
A fluorescence turn-off assay for phospholipase C (PLC)
has been reported by Schanze and co-workers based on the
reversible interactions between the natural substrate
(phosphatidylcholine) and anionic CPs.[84] The emission
intensity of the polymer in aqueous solution was sig-
nificantly increasedby theadditionof thephospholipid as a
result of complex formation. Upon adding PLC to the
polymer–lipid complex, the phosphatidylcholine was
hydrolyzed by PLC to release the complex, which leads to
quenching of the polymer emission. The assay provides a
convenient, rapid, and real-time platform for a PLC activity
assay. Whitten and co-workers have also developed a
heterogeneous phospholipase A2 (PLA2) assay based on
silica microspheres coated with cationic CPs (Figure 12).[85]
In this assay, fluorescence recovery was taken as the
readout signal and the system shows great potential to
assay enzymes in a real-time manner using a multi-well
plate reader or flow cytometry.
Oxidase Assays Using CPs
Oxidase is an enzyme that catalyzes the transfer of
electrons from the reductant to the oxidant. Tyrosinase
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� 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
is a copper-containing protein, and
accumulated tyrosinase is considered
as a marker for melanoma cancer
cells.[86] Our group synthesized a
new water-soluble oligo(phenylenevi-
nylene) that contained a tyrosine
moiety (OPV-Tyr) for tyrosinase assay
(Figure 13).[87] Upon additions of
tyrosinase, the tyrosine moiety was
oxidated into quinone with quenching
ability, and the OPV-Tyr demonstrated
intramolecular electron transfer from
the phenylenevinylene unit to the
quinone site, followed by the fluores-
cence quenching of OPV-Tyr. Thus, the
OPV-Tyr canact as afluorescentprobe to
optically detect tyrosinase activity.
Except for in aqueous buffer solution,
the tyrosinase activity can also be
detected in agarose gel that mimics
the environment of the cell.
Very recently, we have found that the
tyrosinase can also be used to design
fluorescent enzyme-coupled systems for
enzyme detection by combining with
water-soluble CPs.[88] To demonstrate the
concept, b-galactosidase and tyrosinase are chosen as
the target and reporter enzymes, respectively. b-
Galactosidase is widely used as a marker enzyme to
identify cell types, to examine transcription regulation, or
studygeneexpression.[89] Theanionic PFP� SO�3 can forma
complex with the cationic b-galactosidase substrate
through electrostatic interactions, where the PFP� SO�3
emits strong fluorescence upon excitation at 376nm. Upon
adding b-galactosidase to the solution that contained the
complex and tyrosinase, the substrate is hydrolyzed to b-
galactose and a phenol derivative, followed by conversion
by the tyrosinase into quinone. The fluorescence of
PFP� SO�3 is efficiently quenched by the quinone by an
electron transfer process. In light of the fluorescence
quenching of PFP� SO�3 , the b-galactosidase activity can
be monitored in a continuous and real-time manner. In
principle, this sensing mechanism will extend the applica-
tion window of CPs for wide-spectrum enzyme detections.
This ‘‘mix-and-detect’’ approach could be expanded to a
high-throughput method.
Our group’s work shows that glucose oxidase (GOx) can
be detected through an indirect pathway employing a CP-
based H2O2 sensor.[90,91] The boronate-protected fluores-
cein (peroxyfluor-1) is convalently linked to the side chain
of water-soluble PF (PF-FB) (Figure 14a). The peroxyfluor-1
exists as a lactone formthat is colorless andnonfluorescent.
The FRET fromthefluoreneunits (donor) to theperoxyfluor-
1 (acceptor) is absent and only blue donor emission is
www.mrc-journal.de 1413
F. Feng, L. Liu, Q. Yang, S. Wang
Figure 9. a) Emission spectra of PFP-BT 1/ATP as a function of theALP enzyme digestion time. b) Emission intensity of PFP-BT 1/ATPat 410nm as a function of the ALP enzyme digestion time. Theinsert shows the dependence of themaximum emission intensityof PFP-BT 1 on the concentrations of ALP. The excitation wave-length is 370nm. Reproduced with permission from ref.[66]. Copy-right 2007, Royal Society of Chemistry.
Figure 10. Schematic representation of activity assays of AChEusing anionic CP.
1414Macromol. Rapid Commun. 2010, 31, 1405–1421
� 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
observed upon excitation of the fluorene units. In the
presence of H2O2, the peroxyfluor-1 can specifically react
with H2O2 to deprotect the boronate protecting group
and to generate fluorescent fluorescein (Fl). In this case,
efficient intramolecular FRET from the fluorene units to
fluorescein is observed. By triggering the shift in emission
color and the ratio change of blue to green emission
intensities, it is possible to assayH2O2and its concentration
change in both buffer solution and in a blood sample.
Because GOx can catalyze the oxidation of glucose in the
presence of oxygen to generate H2O2 (Figure 14b), glucose
and GOx can be detected rapidly and conveniently
(Figure 14c).
Transferase Assays Using CPs
Adenosine deaminase (ADA) is a deaminating enzyme that
canconvert theadenosineanddeoxyadenosine into inosine
and deoxyinosine, respectively. Both an inherited ADA
deficiency and ADA plethora may cause diseases. We
developed amagnetically assisted fluorescence ratiometric
technique for ADA assays with high sensitivity using
water-soluble cationic conjugated polymer (CCP)
(Figure 15).[92] The assay contains three elements: a
biotin-labeled aptamer of adenosine (biotin-aptamer), a
single-stranded DNA-tagged fluorescein at the terminus
(ssDNA-Fl) as a signaling probe, and a CCP. The specific
binding of adenosine to the biotin-aptamer unhybridized
the biotin-aptamer and ssDNA-Fl and the ssDNA-Fl
was washed out after streptavidin-coated magnetic
beads were added and separated from the assay solution
under magnetic field. In this case, after the addition of
CCP to the magnetic beads solution, the FRET from CCP
to fluorescein was inefficient. Upon adding adenosine
deaminase, the adenosine was converted into inosine,
and the biotin-aptamer was hybridized with ssDNA-Fl
to form doubled-stranded DNA (biotin-dsDNA-Fl). The
ssDNA-Fl was attached to the magnetic beads at the
separation step, and the addition of CCP to the magnetic
bead solution led to efficient FRET from CCP to the
fluorescein. Thus the adenosine deaminase activity can
be monitored by fluorescence spectra in view of the
intensity decrease of CCP emission or the increase of
fluorescein emission in aqueous solutions. The assay
integrates surface-functionalized magnetic particles with
significant amplification of the detection signal of water-
soluble CCPs.
Nuclease Assays Using CPs
Nucleases can cleave DNA, which is involved in many
important biological processes, such as DNA replication,
DOI: 10.1002/marc.201000020
Water-Soluble Conjugated Polymers for Fluorescent-Enzyme Assays
Figure 11. The synthetic routine to the fluorescent microgel PFP-NIPAm. Reproducedwith permission from ref.[83]. Copyright 2009, AmericanChemical Society.
recombination, and repair.[93] In recent years, new techni-
ques based onCPshave beendeveloped for nuclease assays,
which offer a homogeneous, sensitive and simple assay in
comparison to the typical methods, such as gel electro-
phoresis, filter binding, high performance liquid chromato-
graphy (HPLC), and enzyme-linked immunosorbent assay
(ELISA).[94] Assemblies formed by cationic PT and analytes
display attractive optical properties in absorption spectra
dependant on polymer conformation.[12,95] When binding
DNA, cationic PT changes from a random-coil to a highly
conjugated and planar conformation through electrostatic
andhydrophobic interactions,whichresults inared-shifted
absorption wavelength.[28] We take advantage of this
phenomenon to utilize PMNT as an optical probe to assay
the hydrolysis process of ssDNA by ssDNA-specific nucle-
ase, S1 (Figure 16).[96] Short DNA fragments, in particular
those shorter than ten bases, bound to PMNT very weakly
andcouldnot formahelixwithPMNT.As thedigestion time
increased, the PMNT absorption gradually decreased at
394nm and increased at 520nm, arising from the decreas-
ingamountsofuncleavedDNA.Asa result of the significant
red-shift of the PMNT absorption, a dramatic color change
wasvisibleby thenakedeye froman initial yellowcolor toa
final pink-red. Consequently, the color change allowed
visual sensing of enzymatic hydrolysis of DNA instead of
Macromol. Rapid Commun. 2010, 31, 1405–1421
� 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
determination by UV–vis spectroscopy. The strategy was
further supported by inhibition assays and other non-
specific enzyme digestion experiments. Although the
technique is not applicable to the dsDNA system, the rapid
and direct response and label-free sensing clearly exhibits
advantages over other detection platforms.
Wehavedeveloped another optical platformemploying
the FRET technique for nuclease assays including both
ssDNA and dsDNA digestion (Figure 17).[97] Cationic CCP1
acted as an optical probe. The formation of CCP1/Fl-DNA
assemblies, whether Fl-ssDNA or Fl-dsDNA, allowed for
efficient FRET in aqueous solution. When ionic strength of
the buffer solution was high, the FRET efficiency for short
Fl-DNAsequences sharplydeclinedbecauseof the strongly
weakened electrostatic interactions, while the FRET
efficiency remained high for long Fl-DNA sequences. As
a result, the energy acceptor fluorescein, located on the
longDNA sequence achieved significant optical amplifica-
tion by even more than 10-fold upon exciting CCP1 at
380 nm. Small Fl-DNA fragments only slightly affected the
FRET through very weak interactions with the polymer. A
ratiometric methodology was introduced to exclude
nonspecific effects and the FRET ratio, defined as I424nm/
I528nm, was correlated to digestion level. S1
nuclease, BamHI, and EcoRI endonuclease were chosen
www.mrc-journal.de 1415
F. Feng, L. Liu, Q. Yang, S. Wang
Figure 12. Phospholipase A2 assay mechanism based on silica microspheres coated with cationic CPs using flow cytometry (a,b,c) andchemical structures of cationic CPs (MSPPE), quencher AQS and DMPG lipid investigated in the assay. Adapted with permission from ref.[85].Copyright 2008, American Chemical Society.
1416
as model enzymes to elucidate the method for assaying
nuclease activity. The CP-based qualitative analysis is
general since the Fl-DNA substrate can be single stranded,
double stranded, and hairpin-shaped as long as the
recognition sequence is located near the 50-terminus
Macromol. Rapid Commun. 2010, 31, 1405–1421
� 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
where fluorescein is labeled. The sensitivity provided by
optical amplification is rather high for S1 assays to afford a
LOD at the picomolar scale because S1 nuclease can cut
DNA thoroughly to present an extremely low background
signal at 528 nm.
DOI: 10.1002/marc.201000020
Water-Soluble Conjugated Polymers for Fluorescent-Enzyme Assays
Figure 13. a) Schematic representation of tyrosinase assays. b) Emission spectra of OPV-Tyr in phosphate buffer solution (50� 10�3 M, pH 7.4)as a function of the incubating time of tyrosinase. c) Emission intensity of OPV-Tyr at 467 nm as a function of the incubating time oftyrosinase. The excitation wavelength is 330nm. Adapted with permission from ref.[87]. Copyright 2008, American Chemical Society.
Figure 14. a) Schematic representation of the H2O2 assays. b) Catalyzed oxidation of glucose by GOx in the presence of oxygen to generateH2O2. c) Emission spectra of PF-FB/GOx in the absence and presence of glucose. Adaptedwith permission from ref.[91]. Copyright 2007, RoyalSociety of Chemistry.
Macromol. Rapid Commun. 2010, 31, 1405–1421
� 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.mrc-journal.de 1417
F. Feng, L. Liu, Q. Yang, S. Wang
Figure 15. a) Schematic for the CCP-based magnetically assisted assay for adenosinedeaminase. b) Mechanism of the deamination of adenosine by adenosine deaminase,the chemical structures of CCP and the sequences of aptamer and signaling ssDNA-Fl.Reproduced with permission from ref.[92]. Copyright � 2009 Science in China Press.
1418
A further approach was exploited to study methyltrans-
feraseactivitybasedontheresultsofnucleaseassays.[97] The
methylation of cytosine or adenine bymethyltransferase at
a recognition sequence is able to keep the DNA hydrolysis
from the endonuclease, which favors the DNAmethylation
analysis. The FRET from CCP1 to fluorescein was blocked
after hydrolysis of Fl-DNA by EcoRI, butwas turned on if Fl-
DNA was pretreated by EcoRI-related methyltransferase
(M.EcoRI). Compared to electrophoresis analysis, which is
commonly used in nuclease andmethyltransferase assays,
themethod is very simple since isolation steps are avoided.
The method also imparts high sensitivity and DNA can be
probed at or below the nanomolar level. The drawback lies
in the impracticability of a continuous assay.
Very recently, we have designed an energy transfer
cascade using the assembly of cationic CPs with negatively
charged branched DNA respectively labeled at the 50-
terminus with fluorescein, Tex Red, and Cy5 dyes
(Figure 18).[98] The multi-step FRET process regulates the
Macromol. Rapid Commun. 2010, 31, 1405–1421
� 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
fluorescence intensities of the CP and
three dyes. Different logic gate types can
be operated by observation of different
dye emission wavelengths with
nucleases as inputs. The logic signals
give rise to a new method for the
simultaneous detection of multiplex
nucleases.
A simple method for nuclease detec-
tion using CPs and a DNA/intercalating
dye complex was described by Ren et al.
(Figure 19).[99] The method relies on the
large signal amplification through effi-
cient FRET from the CP to the intercalat-
ing dye mediated by DNA. The discrimi-
nationofDNAbeforeorafterdigestionby
nuclease denotes the universal applica-
tion of the approach, in which either
dsDNAor ssDNAsubstrates couldbeused
for detecting nuclease activity. This
method can be extended to most
nucleases by simply changing the sub-
strate DNA. Compared with previous
studies where a chromophore-labeled
DNA substrate is needed, the present
method is label-free, rapid, and highly
sensitive. In addition, this assay is easy to
implement for visual detection with the
assistance of a UV transilluminator.
Conclusion
Taking advantage of the unique signal
amplification of water-soluble CPs, it is
possible toassayvariousenzymeswithhighsensitivityand
selectivity. Although a great deal of progress has already
been made in this field, major challenges remain before
these techniques can be widely utilized in commercial
applications. They are still in their infancy in comparison
with other commercial standardized techniques such as
spectrophotometric analysis, andarenotyet asuniversal as
gold nanoparticles that have been widely utilized in
bioapplications. This problem results from the complexity
of the CP-based system since various signal transduction
mechanisms may coexist and function in an unbalanced
and incalculable fashion. For example, the self-quenching
of CPs frequently exists to different extents in homo-
geneous aqueous assay solutions. Water-soluble CPs are
generally prepared with inequable molecular weights and
varying quantum yields, which affects the reproducibility
of experimental reports. There still remains a great
challenge to solve the limitations in the biocompatibility
of CPs, which exhibits disadvantages in comparison with
DOI: 10.1002/marc.201000020
Water-Soluble Conjugated Polymers for Fluorescent-Enzyme Assays
Figure 16. A) Schematic representation of the assay for nuclease.B) Chemical structures of PMNT and ssDNAs with different baselength. Reproduced with permission from ref.[96]. Copyright2006, American Chemical Society.
Figure 17. The schematic representations of the assays fornucleases. A) Non-restriction nucleases for ssDNA cleaving.B) Restriction nucleases for dsDNA cleaving. C) Chemical structureof CCP1, sequences of DNA-1, 2, and 3, and their specific nucleases.Arrows are noted as the cleavage sites of dsDNA by restrictionendonucleases. Reproduced from ref.[97].
quantum dots, gold nanoparticles, and even small dye
molecules that are used in enzyme assays in vivo and in
vitro. Furthermore, how to effectively lower the back-
ground signal and avoid an undesired influence upon
enzyme activity from the polymer are also key questions in
designingtheenzymeassaysystemsbasedonCPplatforms.
Although various enzymes have been assayed up to date,
the types of enzymes are still limited because many
Macromol. Rapid Commun. 2010, 31, 1405–1421
� 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
reaction types are difficult to assay by combining CPs with
fluorophore-labeled enzymatic substrates. Multiple
enzymes coupling assays are waiting to be extended for
wide-spectrum enzyme assays. Since many enzymes are
targets for active drugs, drug screening is an important
application of CP-based enzyme assays in the future.[100]
Despite these remaining challenges, a very bright future of
CPs forenzymeassays isexpected. Therearegoodpotentials
www.mrc-journal.de 1419
F. Feng, L. Liu, Q. Yang, S. Wang
Figure 19. The schematic representations of the assays fornucleases. A) Restriction nucleases for dsDNA cleaving. B) Non-restriction nucleases for ssDNA cleaving. Reproduced with per-mission from ref.[99]. Copyright 2010, American Chemical Society.
Figure 18. Schematic representation of the multiplex detection ofnucleases. Reproduced with permission from ref.[98].
1420Macromol. Rapid Commun. 2010, 31, 1405–1421
� 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
for CP-based approaches to be incorporated into routine
enzyme assay protocols in the near future.
Acknowledgements: This work was supported by the NationalNatural Science Foundation of China (No. 20725308 and 20721061,TRR61) and theMajor Research Plan of China (No. 2006CB932102).
Received: January 7, 2010; Revised: February 25, 2010; Publishedonline: May 20, 2010; DOI: 10.1002/marc.201000020
Keywords: conjugated polymers; enzyme assays; fluorescence;FRET; probes; superquenching
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