ARTICLE IN PRESS

Biomaterials 28 (2007) 791805

www.elsevier.com/locate/biomaterials

Review

Biomolecular immobilization on conducting polymers

for biosensing applications
Tarushee Ahujaa, Irfan Ahmad Mira, Devendra Kumara, Rajeshb,
a
Department of Applied Chemistry, Delhi College of Engineering, University of Delhi, Bawana Road, Delhi-110042, India
b
Intellectual Property Management Division, Council of Scientific & Industrial Research, 14, Satsang Vihar Marg,
Special Institutional Area, New Delhi-110067, India
Received 6 July 2006; accepted 27 September 2006
Available online 20 October 2006

Abstract

A detail study on different aspects of biomolecule immobilization techniques on conducting polymers (CP) for applications in
biosensors is described. Comparative studies are conducted in between the different mode of biomolecule immobilization techniques, viz.
physical, covalent and electrochemical immobilization onto the conducting polymer lms for the fabrication of electrochemical
biosensors for clinical, food and environmental monitoring applications. This review focuses on the current status of biomolecule
immobilization techniques on CP and their applications in the development of amperometric biosensors.
r 2006 Elsevier Ltd. All rights reserved.

Keywords: Conducting polymers; Biosensors; Electrochemical polymerization; Physical adsorption; Covalent immobilization; Electrochemical
immobilization

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 791
1.1. Historical background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 792
1.2. Conducting polymers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 792
1.3. Biosensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 792
2. Electrochemical synthesis of conducting polymers and their application in biosensors . . . . . . . . . . . . . . . . . . . . . . . . . . . 793
3. Methods of immobilization. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 794
3.1. Physical adsorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 795
3.2. Covalent immobilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 795
3.3. Electrochemical immobilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 796
4. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 800
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 800

1. Introduction of nancial investment, the published literature and the

During the last two decades, research and development
in the eld of sensors has expanded exponentially in terms
Corresponding author. Tel.: +911126560722; fax: +911126968819.
E-mail address: rajesh_csir@yahoo.com (Rajesh).
number of active researchers. Sensors provide information
on our physical, chemical and biological environment.
Since the chemical and physical properties of polymers may
be tailored by the chemist for particular needs, they gained
importance in the construction of sensor devices [1]. A new
class of polymers known as intrinsically conducting

0142-9612/$ - see front matter r 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biomaterials.2006.09.046
 ARTICLE IN PRESS
792 T. Ahuja et al. / Biomaterials 28 (2007) 791805
polymer or electro active conjugated polymers (extended
p-conjugation along the polymer backbone) has recently
emerged [28]. These conducting polymers (CP) are
of great scientic and technological importance
because of their electrical, electronic, magnetic and optical
properties [9].
The CP belonging to polyenes and polyaromatics such as
polyacetylene, polyaniline (PANI), polypyrrole (Ppy),
polythiophene, poly (p-phenylene), poly (phenylene viny-
lene) classes have been studied extensively. Among these
PANI is the oldest conductive polymer known which was
prepared by Letheby in 1862 [8].
CPs can be synthesized either by chemical or electro-
chemical oxidation, the latter approach being often
preferred as it results in polymeric lms deposited on
anode surface, which can be removed to give free standing
lms. Also, in electrochemical synthesis, reactions can be
carried out at room temperature [2]. A number of
polymerization techniques of CPs have been reported in
literature [8,1016].
Electrochemically polymerized CPs have received con-
siderable attention over the last two decades. The remark-
able switching capability of these materials between
conducting oxidized (doped) and insulating reduced (un-
doped) state is the basis of many applications [17].
The surge of interest in these CPs has been utilized in a
number of practical applications recently, like electronic
displays, telecommunication, electrochemical storing sys-
tems, molecular electronics & sensors, etc. [1821].
The entrapment of enzymes in CP lms provides a
controlled method of localizing biologically active mole-
cules in dened area on the electrodes [22].
CPs have the ability to efciently transfer electric charge
produced by the biochemical reactions to electronic circuit
[23]. Moreover, CPs can be deposited over dened areas of
electrode. This unique property along with possibility to
entrap enzymes during electrochemical polymerization has
been widely exploited for fabrication of amperometric
biosensors [2427].
1.1. Historical background
The electrochemical synthesis of CPs has its roots in the
early attempts of DallOlio et al. in 1968. They prepared
pyrrole black, a polymer in the form of a powdery
insoluble precipitate, by electrochemical oxidation of
pyrrole monomer in aqueous sulfuric acid on a platinum
electrode. The resulting polymer had a conductivity of
approx. 8 S cm 1 [4].
In 1979, Diaz et al. revived the electrochemical approach
for the synthesis of CPs. They synthesized continuous and
freestanding lms of conducting PPy using platinum
electrodes. These lms showed much higher electrical
conductivity (100 S cm 1) and excellent air stability [28].
Various review articles have been published on electro-
chemical synthesis of CPs [4,11,2934].
1.2. Conducting polymers
CPs show unusual electrochemical properties like high
electrical conductivity, low ionization potential, high
electronic afnities, and optical properties. These proper-
ties are only because of conjugated p electron backbones in
CPs. There must be high degree of overlapping of the
polymer molecular orbital, which permits the formation of
a delocated molecular wave function and partial occupa-
tion of the molecular orbital if there is to be free movement
of electrons through out the lattice [2].
CPs exhibit intrinsic electronic conductivity ranging
from about 10 14 to 102 S cm 1 due to extension of the
doped state [35]. In the neutral (undoped) state these
materials are only semi-conducting and electronic con-
ductivity only appears when the material is doped with
small sized ions (e.g. when electrons or holes are injected
into the super orbital). This kind of doping is normally
produced by chemical or electrochemical oxidation of the
monomer, in which the polymer chains acquire positive
charges and the electro neutrality of the resulting material
is preserved by the incorporation of small counter ions
from the electrolyte solution.
In recent years, there has been growing interest in CPs
because of their wide range of potential application in the
areas such as rechargeable batteries [36,37], gas separation
membranes, EMI shielding [38], electro chromic display
devices [39], etc.
1.3. Biosensors
A biosensor is a chemical sensing device in which a
biologically derived recognition entity is coupled to a
transducer, to allow the quantitative development of some
complex biochemical parameter [33].
There are three so-called generations of biosensors:
(i) First generation biosensors where the normal product
of the reaction diffuses to the transducer and causes
the electrical response.
Analyte Enzyme
H2O2 +
(oxidized) 2H
Enzyme
O2 2e-
(reduced)
Analyte
Electrode
(oxidized)
Product
(ii) Second generation biosensors which involve specic
mediators between the reaction and the transducer in
order to generate improved response. The second
generation biosensors involve two steps: rst, there is a
redox reaction between enzyme and substrate that is
reoxidized by the mediator, and in second step
eventually the mediator is oxidized by the electrode.
 ARTICLE IN PRESS
T. Ahuja et al. / Biomaterials 28 (2007) 791805 793

(iii) Third-generation biosensors arise from the self-con-
tained nature of the sensor where the reaction itself
causes the response and no product or mediator
diffusion is directly involved. Third-generation sensors
are accompanied by co-immobilization of enzyme and
mediator at an electrode surface making the biorecog-
nition component an integral part of the electrode
transducer i.e. direct electrical contact of enzyme to
electrode. Since neither mediator nor enzyme must be
added, this design facilitates repeated measurements.
electrodes for aqueous system are SCE, NCE, silver
electrode and mercurous sulfate.
Those compounds, which possess relatively low anodic
oxidation potential and are susceptible to electrophilic
substitution reaction, can give CPs by electrochemical
techniques.
H
H
N
N S

X X
X
Polypyrrole Poly(Thiophene) Poly(Aniline)
Analyte Electron
Enzyme mediator
(oxidized) (reduced)

Enzyme Electron e-
Product (reduced) mediator
(oxidized)
X
N
H X
Poly(Acetylene) Poly(Indole)

The major challenge when using CPs in the design of

biosensor with electrochemical transducer is to understand Mechanism of electro polymerization of CP can be best
the mechanism of the electron transfer in conguration explained by using PPy as an example [4,41].
that usually runs parallel to the charging of a double layer -2e-
at the electrode surface and the mass transport processes at anodic oxidation at the
the polymer interface [40]. electrode surface .
2 2
Over the last decade, biosensors have attracted a lot of N ing
N+
pl
global attention and plenty of active research throughout ou
a lc H
the world is being dedicated to the development of new and H
ra dic
al -
novel biosensors. Biosensors have found a lot of applica- H ra dic H

tions in every sphere of life. They nd their use in N

+
N
diagnosis, food technology, biotechnology, genetic engi- 2 -2H + 2
neering, environmental monitoring and a number of N
aromatization
N

similar areas. H H

H H H
+ -e-
2. Electrochemical synthesis of conducting polymers and N N N
their application in biosensors -2H
+

N N
Electrochemical polymerization is normally carried out
H H
in a single or dual compartment cell by adopting a N+ electrochemical
standard three-electrode conguration in a typical electro- propagation
chemical bath consisting of a monomer and a supporting H H H

electrolyte both dissolved in an appropriate solvent. N N

Generally, electrochemical polymerization can be carried
out either potentiostatically (to obtain thin lms) or
galvanostatically (to obtain thick lms) [33].
A standard three-electrode system used comprises of a
working electrode, a counter electrode and a reference
electrode. The function of working electrode is to support
the polymer lm. Metals like gold, platinum, titanium,
nickel, palladium, and carbon are used. Glass coated with
tin oxide or indium tin oxide can also be employed.
Counter electrode supplies current required by the
working electrode. Metallic foil of platinum, gold, nickel
is generally used as counter electrode. Common reference
N
n
H
The rst step involves the formation of pyrrole radical
cation by electrode surface. It is followed by dimerization
by deprotonation. The dimer oxidizes slightly more easily
than the monomer and thus gets reoxidized to allow further
coupling reaction to proceed.
The oxidation potential of the polymer is always lower
than that of monomer. The polymer is electrochemically
 ARTICLE IN PRESS
794 T. Ahuja et al. / Biomaterials 28 (2007) 791805
ionized to a conducting state and the overall electrical
neutrality is maintained by incorporation of the counter
ion from the supporting electrolyte. This is essential
because precipitation of the unoxidized, insulating polymer
would stop the reaction.
During the process of electrochemical polymerization
negatively charged molecules are present in the electrolytic
solution such as anions. Polymers and enzymes can be
embedded into the positively charged backbone as dopants
[42,43]. This promotes an attractive route to trap func-
tional molecules within organic polymers during their
electro generation of electrode, and offers a new means to
fabricate a range of novel composite material with novel
properties.
In electrochemical polymerization, doping and proces-
sing takes place simultaneously. Also uniform doping is
accomplished. It is also possible to produce freestanding
lms, homogenous and coherent lms even at room
temperature. Frietas et al. reported the synthesis of novel
conducting polymer: the poly [(3-pyrrolyl) octanic acid] by
electrochemical polymerization. Characterization by FTIR
spectroscopy provided evidence that the carboxylic term-
inal group is kept in the polymer-modied electrode, which
is essential for biomolecule immobilization in biosensors
[11].
CPs have an organized molecular structure on metal
substrates, which permit them to function as a three
dimensional matrix for the immobilization of active
catalyst and preserve the activity for long duration. This
property of CPs together with their functionality as a
membrane has provided opportunities to investigate the
development of sensors [44,45].
CP serves as the immobilizing matrices for biomolecules
and provides a suitable environment for their immobiliza-
tion [27,4652]. The redox properties of these materials
especially PPy, have been intensively studied in recent
years, with much emphasis on their synthesis and
characterization. PPy is presently considered as the most
promising CP for the development of advanced sensor
devices due to its relative stability, ease of preparation, and
good conductivity [10,53].
The chemical structure of inherently CP and the facile
methods of assembly available make them compatible with
many of the chemistries found in nature, thus facilitating
interaction with biological micro molecules [13]. This leads
to the application of CP in biosensors [50,54].
The use of CPs in the areas of bioanalytical sciences is of
great interest since their compatibility opens up the
possibility of using them in vivo biosensor application
[55], for continuous monitoring of drugs or metabolites in
biological uids [56], or as a means of opening the eld to a
variety of new analytes. Amine et al. have discussed the
general application of various biosensors with emphasis on
food and environmental applications. Further, the general
approaches used for enzyme immobilization, enzyme
catalysis and inhibition mechanism have been reported
[57]. Geetha et al. discussed the application of conducting
PPy as a biosensor for some biomolecules and drug
delivery systems [58].
Malhotra et al. have reported recent advances in
biosensors and their application in food analysis, environ-
mental control, clinical detection, drug and agricultural
industries, etc. [59]. Adhikari et al. discussed different
materials for sensor applications and reported the use of
polymers either in sensing mechanism or in immobilizing
the component responsible for sensing the analyte [1].
Conjugated polymers have been used to facilitate
electron transfer mediators, such as ferrocene. If incorpo-
rated into the conjugated polymers, plays an important
role in the sensitivity and response of amperometric
enzyme based biosensors [60,61]. Fast electron transfer of
polymer/thin lms bearing the mediators is desirable to
enhance biosensor sensitivity [62,63]. A promising ap-
proach for the development of electrochemical biosensors
by direct transfer was reported by Freir et al. [64].
Recent achievements in the eld of analytical application
of conducting polymer have been reviewed by Trojanowicz
[40]. The lms prepared by electrochemical co-deposition
of enzymes on CP or conductive substrates have been used
to fabricate biosensors [27,6570]. Among the different
biomolecules, the entrapment within PPy matrices has been
mainly focused on enzyme molecules, particularly oxidases.
Immobilization of coenzyme, antibodies, cells and biologi-
cal tissues illustrates the wide potentialities of this method
for biomolecule retention [7174]. S.F. DSouza reviewed
the use of microbial cells as biological sensing elements in
biosensors and discussed some of the recent developments
in the eld [67].
3. Methods of immobilization
With the advance in the development of biosensors,
several problems surfaced relating to functioning of
enzyme system like loss of enzyme (especially expensive
enzymes), maintenance of enzyme stability and shelf life of
the biosensors. In addition to this, there grew a need to
reduce the time of enzymatic response and offer disposable
devices, which can easily be used in stationary or in, ow
systems [57]. In order to overcome these problems, several
immobilization procedures have been developed. The
procedure of biomolecule immobilization on conductive
surfaces remains a key step for the performance of the
resulting electrochemical devices. For efcient deposition
of a biomolecule, it must satisfy a few pre-requisites, that:
 there must be an efcient and stable immobilization of
the biological macromolecules on transducer surfaces,
 it must retain its biological properties completely
 it should be compatible and chemically inert towards
host structure,
 it should be accessible when immobilized [75].
Immobilized enzyme has many operational advantages
over free enzyme such as reusability, enhanced stability,
 ARTICLE IN PRESS
T. Ahuja et al. / Biomaterials 28 (2007) 791805
continuous operational mode, rapid termination of reac-
tion, easy separation of biocatalyst from product, and
reduced cost of operation [65]. Immobilization of biomo-
lecules can be carried out using many different procedures,
like physical adsorption, covalent linking and electro
immobilization, while retaining the biological recognition
properties of the biomolecules.
3.1. Physical adsorption
In physical adsorption process, the biomolecule gets
adsorbed in the polymer/solution interface due to static
interactions between the polycationic matrix of the
oxidated polymer and the total negative enzyme charge,
provided pH is higher than the isoelectric point (pI)
of the enzyme [34]. But this process has some known
disadvantages like binding forces can change with
pH, adsorption is limited to one monolayer on the polymer
surface hence amount of enzyme incorporated is very
small [76]. Also, since biomaterial is immobilized on the
outer layer of conducting polymer, it gets leached out into
sample solution during measurements. This decreases the
lifetime stability of enzyme electrode [77]. This method has
been used for the preparation of many biosensors [7885].
Immobilization by physical adsorption also involves
cross-linking. Cross-linking gives sensors with short
response times but poor stability because enzyme is directly
exposed to the bulk solution and partly denaturized by the
cross-linking. Immobilization of acetylcholine esterase
(AChE) by cross-linking involving glutaraldehyde signi-
cantly increased the attachment of enzyme to the transdu-
cer and hence led to a more direct electron exchange. Here
AChE was co immobilized with choline oxidase (ChO) on
to a Pt surface using a solution of gluteraldehyde [57].
Immobilization of AChE in nylon net using gluteraldehyde
has been reported by Gulla et al. [86]. The method involves
immobilization by sandwiching the enzyme layer between
two cellophane membranes kept in close contact with a
gold electrode. Milagres et al. [87] immobilized salicylate
hydroxylase by cross-linking. Similarly, immobilization of
tyrosinase [88] and lactate dehydroganase [89] has also
been reported in literature.
A dual electrode amperometric biosensor for simulta-
neous determination of acetylcholine and choline was
fabricated by Guerrieri et al. by electrosynthesising poly
(pyrrole)/poly (2-napthol) bilayer membrane as an effective
anti interference layer. This device is potentially useful for
the analysis of real matrices, (e.g. brain homogenates &
CSF) without the use of a chromatographic step typically
required to separate acetylcholine and choline [90]. Rigid
conducting carbon polymer composites were reported to be
ideal for construction of electrochemical sensors by
Cespedes et al. A great number of biological materials
can be immobilized by blending them with these compo-
sites. This technique is very attractive for the mass
fabrication of amperometric biosensors [91].
795
A cholinesterase potentiometric biosensor based on a
glassy electrode modied with processible PANI has
been developed and explored for the detection of organo-
phosphorous and carbamic pesticides by Ivanov et al.
Acetyl and butyryl-cholinesterase from various sources
were immobilized on the surface of PANI modied
electrode by cross linking with gluteraldehyde. The
detection limits of the pesticides investigated were found
lower than those obtained with other similar cholinesterase
[92].
Brahim et al. utilized PPy-hydrogel composites for the
construction of clinically important biosensors. They
reported the use of poly (2-hydroxyethyl methacrylate) in
which PPy and various oxidoreductase enzymes were
physically entrapped to function as a viable matrix. The
optimized glucose oxidase (GOx) biosensor displayed a
wide linear glucose response range (5.0  10 52.0 
10 2 M) and a response time of 3540 s [93].
As per the reported literature, conguration of a
biosensor plays a pivotal role in performance of a
biosensor device. Shan et al. studied the immobilization
of enzymes with clay. It was reported that if the enzyme/
clay ratio was decreased from 1 to 0.125, a sharp decrease
in sensitivity of biosensor was observed. They also studied
the inuence of thickness of the clay lm on the biosensor
performance and observed that higher sensitivity to
cyanide and lower detection limits with thinner coatings
[94]. Another group used a lower amount of immobilized
acetylcholine-esterase to measure lower concentration of
paraoxan [95].
3.2. Covalent immobilization
In order to achieve an increased lifetime stability of
enzyme electrode, it is necessary that there should be a
strong and an efcient bonding between the enzyme and
the immobilizing material. Hence, covalent linking of
biomolecules on transducer is an efcient method of
immobilization. It comes with an advantage of low
diffusional resistance and such a sensor shows good
stability under adverse conditions. Covalent linking of a
biomolecule to polymer matrix is accomplished in a two-
step process i.e. synthesis of functionalized polymer
followed by covalent immobilization. Since, immobiliza-
tion takes place only on the outer surface of the polymer, it
permits selection of optimum reaction conditions for each
step [34]. Guilbault bound GOx via acyl azide derivatives
of polyacrylamide for glucose detection [96]. Ramanathan
et al. used GOx bound to poly (o-amino benzoic acid) as
glucose biosensor [97].
Rahman et al. recently fabricated a biosensor by
covalent immobilization of pyruvate oxidase on the
nanoparticle-comprised poly (terthiophene carboxylic
acid), poly-TTCA on glassy carbon electrode for ampero-
metric detection of phosphate ions [98]. A linear response
for the detection of phosphate ions was observed between
1.0 and 100 mM with a detection limit of about 0.3 mM.
 ARTICLE IN PRESS
796 T. Ahuja et al. / Biomaterials 28 (2007) 791805

Rajesh et al. have developed an amperometric phenol
biosensor by covalent immobilization of tyrosinase onto an
electrochemically prepared novel copolymer poly (N-3-
aminopropyl pyrrole-co-pyrrole) lm. The covalent linkage
of enzyme and porous morphology of the polymer lm lead
to high enzyme loading and an increased lifetime stability
of the enzyme electrode [17]. An amperometric biosensor
based on the use of pH sensitive, redox active dissolved
hematin molecule was also developed by the same group
and has been used for the quantitative determination of
urea in aqueous solution. This biosensor had a 95%
steady-state current value and 80% of the enzyme activity
was retained for 2 months [77]. Similarly, they have also
reported other techniques for the fabrication of biosensors
and enhancement of related properties [99102].
Oennerfjord et al. immobilized tyrosinase covalently on
the surface of carbodiimide-activated graphite and used in
a ow injection system [103]. Another technique of
covalent linking involves integrating enzymes in mono-
mers, which can be electrochemically oxidized to generate
polymers. By virtue of this process greater amount of
enzymes get immobilized [34]. Immobilization of GOx by
covalent bonding has been reported by many research
groups [104110].
enzyme is spatially controlled irrespective of geometry,
shape and dimension of the electrode [114]; the lm
thickness can be precisely controlled through, e.g. the
charge involved in the deposition step and it can be used to
build up multiplayer/multienzyme structures; it allows the
reproducible and precise formation of a polymer coating
over surfaces whatever their size and geometry may be
[75,115,116]. The fabrication of biosensor by entrapment of
biomolecules in electro-polymerized lms is the most
popular approach. Since, the pioneering work by Umana
and Waller [27] and Bartlett et al. [117] numerous papers
have been published on this approach [2,41,50,54,118125].
Immobilization by enzyme entrapment involves electro-
chemical oxidation of the monomer in the presence of the
enzyme to form a polymer, which incorporates the
homogenously distributed enzyme molecules during its
growth process. This method is the most straightforward
method of immobilization with some inherent advantages
like simplicity and reproducibility of the one step prepara-
tion procedure and the possibility of immobilizing of
mediators, co-enzymes or the other enzymes simulta-
neously simply by adding them to the polymerization
solution [34].
GOx _

H H
3.3. Electrochemical immobilization
N N
+
Although, the conventional procedures for deposition of N N
H H
N N
+
N N

biomolecules on to the transducers like, physical adsorp- H H H H

tion, covalent linkage, entrapment and cross-linking have
been used extensively, they suffer from a low reproduci-
bility and have a poor spatially controlled deposition. In
recent years, the focus of immobilization has been shifted
towards the entrapment of biomolecules in the layers of
electrochemically synthesized polymers. Aizawa and
Yabuki had pioneered the application of PPy and PANI
to the immobilization of enzymes. Since, then hundreds of
papers have been published and patents have been granted
on this subject [24,35,111,112].
Serge Cosnier reviewed different electrochemical proce-
dures for biomolecule immobilization based on electro
polymerized lms and describes the adsorption of amphi-
philic monomers and biomolecule before the polymeriza-
tion step. Examples of organic phase enzyme electrode and
electrical wiring of immobilized enzymes are presented.
Furthermore, the construction of controlled architectures
based on spatially segregated multilayers, exhibiting
complimentary biological activities is described [54].
The electrochemical formation of polymer layers of
controlled thickness and enzyme activity in galvanostatic
or potentiostatic conditions offers numerous advantages
over conventional procedures for the design of biosensors,
such as complete coverage of the active surface, greater
control over lm thickness and greater reproducibility. The
major advantages of the electrochemical immobilization
techniques are: it is a one step, fast than in all immobiliza-
tion procedures [113]; the distribution of the immobilized
_
GOx
The mechanism of incorporation of the enzyme into
electro synthesized polymer lms has been an object of
various speculations. It has been suggested that electro-
static interaction plays the major role in enzyme incorpora-
tion and this assumption has been adequately supported by
indirect experimental evidence [126]. This is not the case for
non-ionic polymer lms where pre-adsorption of enzyme
onto the electrode surface seems to be the important step in
determining the enzyme loading [47,48,127]. In this respect,
enzyme/polymer interaction is of paramount importance to
improve the fundamental knowledge about the biological
interface of the biosensor. Incorporation of the enzyme
results in changes in the apparent optical properties and
density of the polymeric lms, suggesting a mutual
stabilization of polymer and the enzyme. Grifth et al.
have reported the amount of GOx entrapped in conducting
and non-conducting electro polymerized lm (on golf
surface) at different adsorption time and enzyme concen-
trations in the growing solution [128]. Although, biomo-
lecules are only physically conned near transducer, it has
a high diffusion barrier [96]. Entrapment of GOx during
the growth of PANI lms at low pH due to its high
tolerance has been extensively reported [125,129137].
Compagnone et al. used thiophene derivative polymers
synthesized in organic solvents for entrapment [138], while
 ARTICLE IN PRESS
T. Ahuja et al. / Biomaterials 28 (2007) 791805
Gambhir et al. entrapped urease and glutamate dehydro-
genase in PPy/polyvinyl sulfonate lms for urea detection
[84]. Similarly, incorporation of a number of other such
enzymes has been studied and reported in literature
[73,85,139148].
This process involves application of an appropriate
potential to the working electrode soaked in aqueous
solution containing both biomolecule and electro poly-
merizable monomer. Biomolecules present in the immedi-
ate vicinity of the electrode surface are thus physically
incorporated into the growing polymer. This, reagentless
electrochemical approach is easily applicable to a wide
variety of biological macromolecules [75]. Ramanathan
[149] has immobilized GOx on different conducting
matrices such as PPy, PANI, polyammino benzoic acid
(PAB) and studied the response characteristics, lifetime
detail, etc. [2].
Amongst all CP PPy has been extensively used for
immobilization, as it has relatively stable electrical
conductivity and can be electro synthesized under biocom-
patible conditions [35,150153]. But there are few problems
with sensors based on this polymer, like the lm is
degraded by hydrogen peroxide. Covalent attachment of
mediating components onto pyrrole monomers can avoid
these problems but the process is technically complex and
there is no guarantee that the modied monomer will
polymerize [115]. Recently, many reports have been
published on the immobilization of enzymes into the PANI
lm [81,154,155]. Metheby et al. developed an ampero-
metric biosensor by insito deposition of horseradish
peroxide (HRP) enzyme on PANI doped Pt disk electrode.
HRP was electrostatically immobilized onto the surface of
the PANI lm and voltametry was used to monitor the
electro catalytic reduction of H2O2 under diffusion-
controlled conditions. The linear range of this biosensor
was from 2.5  10 4 to 5  10 3 M [156].
Chun-Xiang Li et al. reported a sensitive H2O2 ampero-
metric sensor based on HRP-labelled nano-Au colloids.
Nano-Au colloids were immobilized by the thiol group of
cysteamine, which was associated with the carboxyl groups
of poly (2,6-pyridinedicarboxylic acid). With the aid of
hydroquinone the sensor displayed excellent electrocataly-
tical response to the reduction of H2O2 in the range of
3.0  10 72  10 3 M. The detection limit was 1.0  10 7 M
[157].
Earlier Nakabayashi et al. reported an amperometric
biosensor for sensing H2O2 based on electron transfer
between HRP and ferrocene as a mediator [158]. Similarly,
Thanachasai et al. developed novel H2O2 sensors based on
peroxide carrying poly {pyrrole-co-[4-(3-pyrrolyl) butane-
sulfonate]}copolymer [159].
A novel method of electro polymerization of GOx in PPy
was reported by Wolowacz et al. [160]. GOx was
chemically derivatized by coupling N-(2-carboxyethyl)
pyrrole to surface lysyl residues with a carbodiimide-
promoted reaction. The modied enzyme displays almost
identical properties to the native enzyme except that the pH
797
was slightly more acidic and stability was 6 fold higher at
pH 7.0 and 60 1C. This new procedure incorporated more
enzyme activity into the conducting polymer than the
previously reported physical entrapment technique at
equivalent enzyme concentrations in the polymerization
media. The covalently entrapped enzyme was 171 fold
more stable than the soluble native enzyme.
Yasuzawa et al. discussed the properties of glucose
sensors based on the immobilization of GOx in N
substituted PPy lm [161]. Sensors were prepared by
electro polymerization of 3-(1-pyrrolyl)propionic acid in
the presence of the enzyme following the treatment with
water soluble carbodiimide to provide covalent bonding
between GOx and PPy derivatives. The treatment with
water-soluble carbodiimide improves the response stability
of the enzyme immobilized electrodes.
The entrapment of enzymes in CP provides a facile
means for ensuring proximity between the active site of the
enzyme and conducting surface of the electrode with
considerable potential for biosensor construction. This
method provides a facile and controllable method for the
deposition of biologically active molecules to dened areas
on the electrodes [2]. This well-controlled procedure of
enzyme immobilization by electro polymerization is of
great signicance in fabrication of micro-sensors in the
preparation of multilayer devices [162,163] and multi-
enzyme biosensor [126,163167]. Vedrine et al. constructed
an amperometric enzyme biosensor using tyrosinase for the
determination of phenolic compounds and herbicides. The
enzyme was entrapped in a conducting polymer, poly 3, 4
ethylene dioxythiophene (PEDT) electrochemically gener-
ated on a glassy carbon electrode. Detection of herbicides
was obtained from the inhibition of tyrosinase electrode
responses [168]. Mulchandani et al. have reported an
electrochemically deposited ferrocene modied phenylene
diamine lm on a glassy carbon electrode and used it for
the entrapment of peroxide enzyme, thereby utilizing the
assembly as a biosensor [169].
The preparation of a cholesterol amperometric biosensor
using a platinized Pt electrode as a support for the electro
polymerization of a PPy lm, in which cholesterol oxidase
and ferrocene monocarboxylic acid were co-entrapped has
been described by Vidal et al. All the biosensor preparation
steps (platinized and electro polymerization) and the
cholesterol determination take place in the same ow
system. The presence of the mediator enhances the
sensitivity and selectivity of the platinized biosensor
without modifying the dynamic parameters of the response
and the platinized layer improves the operational lifetime
of the mediated sensor [170].
Arslan et al. constructed and characterized poly acrylic
acid and poly (1-vinyl imidazole) based biosensor and
entrapped invertase in the interpenetrating polymer net-
work [171]. Retama et al. developed a glucose biosensor by
synthesizing semi-conducting PPy/polyacrylamide micro
particles and immobilized GOx into the aqueous phase of
the concentrated emulsion before starting polymerization.
 798

Table 1
Characteristics of biosensors based on different mode of biomolecule immobilization techniques on conducting polymers

Matrix (polymer) Biomolecule Analyte Range Response time Detection limit Shelf life References

Physical adsorption
9
Glassy carbonpolyaniline Cholinesterase Pesticide 5  10 [92]
2
p(HEMA) GOx Glucose 5  10 52  10 3540 s 25 mM [93]
3
Polyaniline/multiwall nanotube Choline oxidase Choline 1  10 62  10 M 3s 0.3 mM 1 month [177]
(MWNT)
Octadecyl aniline Urease Urea 020 mM 15 s 0.2 mM [182]
1
Nano Au-o-phenylene diamine/Prussian Antirubella Rubella 8.1  10 88  6 min 4.0108 lgccid 50 ml 6 days [183]
1
Blue 10 6 lgccid 50 ml
Polypyrrole polyvinyl sulfonate DNA OCP 0.125 ppm 30 s 4 months [184]
Polyaniline graphite oxide nano- DNA DNA [185]
composite in carbon paste electrode
Polypyrrole Trypsin N a -benzoyl arginine 0.050.5 mmol 0.05 mmol [186]
ethyl ester hydro
chloride
Poly(1,3-phenylene diamine) Lactate oxidase Lactate 115 mM 1 min 9 months [187]
5
Poly(pyrrolebiotin) Glucose oxidase Glucose Up to 2  10 M 0.2 mM [188]
Alkaline phosphatase Phenyl phosphate
Solgel Cholesterol oxidase Cholesterol 210 mM 50 s [189]
HRP
Tetraethyl orthosilicate

Covalent immobilization
Poly(N-3 aminopropyl pyrrole-co- Tyrosinase Phenol 1.35222.3 mM o10 s 4 months [17]
ARTICLE IN PRESS

pyrrole)
Poly(N-3 aminopropyl pyrrole-co- Urease Urea 6.3  10 6 2550 s 2 months [99]
4
pyrrole) 4.07  10 M
T. Ahuja et al. / Biomaterials 28 (2007) 791805

Poly(N(2-carboxyethyl)pyrrole) GOx Glucose [160]

1
Polyaniline DNA DNA 1  10 12 mol l [179]
Single wall carbon nanotube HAS HSA 1 nm [190]
1
Polyaniline Cholesterol oxidase, esterase, Cholesterol oleal & 2550 mg dl 240 s 25 mg dl 1 6 weeks [191]
peroxidase cholesterol
5 1
Polypyrrole Antilisteria Listeria 30 min 10 cell ml [192]
1 1
Poly 1,2-diamino benzene Glucose oxidase Glucose 0.55 mmol l 15 s 0.05 mmol l 90 days [193]
Polypyrrole DNA DNA [194]
Polyterthiophene DNA DNA [195]
7
Poly(2,6-pyridinedicarboxylic acid) HRP H2O2 3.0  10 7 10 s 1.0  10 M 23 weeks [196]
2.0  10 3 M
Poly(styrenesulfonate) Cholesterol oxidase Cholesterol 0.23.0 mM o20 s [197]
1 1
Modied ODT Cholesterol oxidase Cholesterol 50500 mg dl 50 mg dl 2 months [198]
Cystamine(SAM) Cholesterol oxidase Cholesterol 0.071.25 mM 3s 5 days [199]
7
Graphite-methacrylate Lysine oxidase Lysine 1.6  10 4 M 42 s 8.2  10 M [200]

Electrochemical immobilization
Polyaniline-co-uoro aniline Gox Glucose 0.522 mM 60 s 15 days [22]
Polypyrrole Alkaline phosphatase- Phosphate 2 mM [68]
polyphenol oxidase
Poly(N-methyl pyrrole) Gox Glucose 2s [127]
Polyaniline polythiophene Gox Glucose 0.0112 mm 0.01 mM 5 months [137]
Polyaniline HRP H2O2 2.5  10 45  10 3 m [156]
Ferocenyl carbon nanotubes/polypyrrole Gox Glucose [157]
3
Poly(3-aminophenol) HRP H2O2 0.620 mmol dm 140 days [158]
Poly{pyrrole-co-(4-(3- HRP H2O2 10 710 4 M 3 weeks [159]
pyrrolyl)butanesulfonate)}
1
Polythiophene Tyrosinase Phenol 5500 nM 10.5 mg l [168]
Polypyrrole Cholesterol oxidase Cholesterol Up to 1.3 mM 8.7 s 12.4 mM 10 days [170]
Phenol derivatives Gox Glucose o4 s [172]
Polypyrrole Tyrosinase Phenol 3.3220.350 mM 4075 s 0.82.450 mM 3 months [201]
PTAE poly(thiophen-3-ylacetic acid 1,3- DNA DNA 1n mole [202]
dioxo-1,3-dihydro-isoindol-2-yl ester)
Polypyrrole Tyrosinase phenol 9.984.7 mM 50 s 2.90 mM 12 weeks [203]
Poly(3,4-ethylene dioxythiophene) Gox Glucose 0.28 mM 25 s 4  10 5 M 1 month [204]
Polypyrrole Fructose dehydrogenase Fructose 10 mM10 mM 25 s [205]
3
Poly(N-methyl pyrrole) Gox Glucose 0.00.22 mol m 5s 2 months [206]
1
Polypyrrole Virostate anti AZT AZT 10 pg ml [207]
Poly(o-aminophenol) carbon nonotubes Gox Glucose Up to 5 mm 3s 0.01 mm 30 days [208]
Polysiloxane/polypyrrole Tyrosinase Catechol 35 days [209]
Polypyrrole Anti-BSA BSA 075 ppm [210]
4 5
Polypyrrole Anti-listeria Listeria 9  10 72  10 M 30 min 5  10 M 2 weeks [211]
Poly(N-methyl pyrrole) Uricase Uric acid 00.24 mM dm 3 40 days [212]
Poly(3-aminophenol) Gox Glucose [213]
2
Poly(HEMA)/polypyrrole Cholesterol oxidase Cholesterol 5  10 41.5  10 M 30 s 120 mM 12 months [214]
PANI-PVS HRP H2O2 [215]
7
Phosphatidyl choline Hb NO 1  10 7 1.0  10 mol l [216]
1
3  10 4 mol l
7
Poly(pyrrolebiotin) Polyphenol oxidase Catechol 34 s 2  10 M [217]
Polypyrrole Tyrosinase Phenol 4.5107.4 mM 80 s 4 months [218]

Glucose oxidaseGOx.
Dioxyribonucleic acidDNA.
p (2 hydroxyethyl methacrylate)p(HEMA).
Horseradish peroxidaseHRP.
Organochlorine pesticides OCP.
ARTICLE IN PRESS

Human serum albuminHAS.

Benzene sulfonic acidBSA.
T. Ahuja et al. / Biomaterials 28 (2007) 791805

AzidothymidineAZT.
HemoglobinHb.
Polyaniline-polyvinyl suphonatePANI-PVS.
799
 ARTICLE IN PRESS
800 T. Ahuja et al. / Biomaterials 28 (2007) 791805
The micro particles with immobilized enzymes were used
as biological component of amperometric glucose
biosensor [9].
Bartlett et al. electrochemically polymerized phenol and
its derivatives and used them to immobilize GOx at a
platinum electrode surface. The resulting lms were thin
and blocked the electrochemistry of ferro/ferricyanide to a
greater or lesser degree. The lms are active towards the
oxidation of glucose and thus can be used to detect
hydrogen peroxide generated by reaction between glucose
and oxygen catalyzed by GOx trapped within the lm. The
response times for different phenolic lms were all similar,
typically o4 s [172].
Boyukbayram et al. grafted copolymers of thiophene
capped polytetrahydrofuran and pyrrole using SDS as
supporting electrolyte to produce biosensor for determina-
tion of phenolic compound in red wine [65]. Nunes et al.
presented a comparison of several AChE immobilization
procedures carried out on tetracyanoquinone diamino
methane (TCNQ) modied graphite working electrode.
The biosensor produced by this procedure showed good
results, fast response, good reproducibility, wide working
range and excellent sensitivity [173]. Although, this method
of immobilization is mainly used for entrapment of
enzymes, a few examples are reported wherein immobiliza-
tion of other biomolecules has been done by electrochemi-
cal entrapment [7274,174,175].
Kaotit et al. developed a biosensor for evaluation of
atrazine which provides rapid and technically simple
system at concentration below the ppm level. This
technique involved immobilization of enzyme polyphenol
oxidase (PPO) during the anodic electro polymerization of
PPy [176].
With the emergence of nanotechnology, many researchers
tried to incorporate it in biosensor development. Qu et al.
prepared conducting PANI lms and assembled these lms
with multiwalled carbon nano tubes layer by layer on glassy
carbon electrodes. The assembly was used as an ampero-
metric biosensor for choline detection. This biosensor
displayed a rapid response and an expanded linear response
range as well as excellent reproducibility and stability [177].
Ramanavicius et al. have discussed self-encapsulation of
redox enzyme GOx within conducting polymer PPy
recently. GOx based nanoparticles coated by pyrrole were
developed, which exhibited detectable catalytic activity
with increased Km value. GOx/PPY nanoparticles were
applied as novel transducer in the design of amperometric
biosensors [178]. Recently, Zhu et al. applied PANI
nanowire modied electrode as DNA biosensor. The
nanowires of CP were directly synthesized through a
three-step electrochemical deposition procedure in Aniline
containing electrolyte solution by using the glassy carbon
electrode as working electrode. Such wires have been found
to be of great promise for sensitive electrochemical
biosensor applications [179].
Similarly, nanocomposites of poly (o-anisidine) contain-
ing TiO2 nanoparticles, carbon black and multiwalled
carbon nanotubes (MWCNT) were prepared by Carrora
et al. The synthesized materials were deposited in thin lms
and analysis of such lms demonstrated that materials
based on polymeric nanocomposites are best suited for
biosensor applications because they provide the best
electron transfer and assure the faster ion mass transfer
[180]. Sotiropoulou and Chaniotakis stabilized AChE
using nano porous carbon matrix. By the use of this
matrix, the enzyme was signicantly stabilized and its
detection unit was decreased [181].
However, it should be noted that electropolymerization
process requires high concentrations of monomer and
biomolecules [75]. On the basis of the numerous studies
reported before, the enzymatic immobilization is one of the
most important steps involved in the biosensor design. The
choice of the technique used for connecting the biological
component (enzyme) to the transducer is crucial, since the
stability, the longevity and the sensitivity largely depends
on enzymatic layer conguration [57]. Characteristics of
biosensors based on different mode of biomolecule
immobilization techniques on CP are shown in Table 1.
4. Conclusion
Immobilization of biological molecule on to the trans-
ducers is a fundamental step in the development of
biosensors, wherein the conducting polymer made trans-
ducers are extensively used for this purpose. Different
physical, chemical and electrochemical methods of im-
mobilization, together with the advantages and disadvan-
tages associated therein are discussed in detail. Advances in
the investigation of biomolecule immobilization techni-
ques, novel functionalized CP, sensing techniques and
related disciplines constantly lead to new principles and
enable improvements to be made in the methods and
devices previously developed.
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