water-soluble conjugated polymers for fluorescent-enzyme assays

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

� 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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



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.

ol. Rapid Commun. 2010, 31, 1405–1421

WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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


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



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


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.



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.



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



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


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



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



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.



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


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



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



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


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


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



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



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



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


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.


� 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.mrc-journal.de 1417


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



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


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



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



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


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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water-soluble conjugated polymers for fluorescent-enzyme assays

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