Electrochimica Acta 51 (2006) 6025–6037

Review article

Electrochemical sensors based on conducting polymer—polypyrrole

A. Ramanavičius a,b,∗ , A. Ramanavičienė a,b , A. Malinauskas c
a
Department of Analytical and Environmental Chemistry, Vilnius University, Naugarduko 24, 03225 Vilnius, Lithuania
b Laboratory of Immunoanalysis and Nanotechnology, Institute of Immunology of Vilnius University, Molėt˛u pl. 29, 08409 Vilnius, Lithuania
c Department of Organic Chemistry, Institute of Chemistry, Goštauto 9, 01108 Vilnius, Lithuania

Received 26 July 2005; received in revised form 16 November 2005; accepted 22 November 2005
Available online 6 May 2006

Abstract
Conducting polymers can be exploited as an excellent tool for the preparation of nanocomposites with nano-scaled biomolecules. Polypyrrole
(Ppy) is one of the most extensively used conducting polymers in design of bioanalytical sensors. In this review article significant attention is
paid to immobilization of biologically active molecules within Ppy during electrochemical deposition of this polymer. Such unique properties of
this polymer as prevention of some undesirable electrochemical interactions and facilitation of electron transfer from some redox enzymes are
discussed. Recent advances in application of polypyrrole in immunosensors and DNA sensors are presented. Some new electrochemical target
DNA and target protein detection methods based on changes of semiconducting properties of electrochemically generated Ppy doped by affinity
agents are introduced. Recent progress and problems in development of molecularly imprinted polypyrrole are considered.
© 2006 Elsevier Ltd. All rights reserved.

Keywords: Conducting polymers; Polypyrrole; Biosensor; DNA sensor; Immunosensor; Molecularly imprinted polymers; Bioelectrochemistry; Nanotechnology;
Nanobiotechnology

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6025
2. Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6026
2.1. Electrochemical polymerization of polypyrrole versus chemical polymerization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6026
2.2. Polypyrrole as versatile immobilization matrix in design of biosensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6027
2.3. Catalytic biosensors based on polypyrrole . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6028
2.4. Immunosensors based on polypyrrole . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6030
2.5. Polypyrrole in the design of DNA sensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6032
2.6. Application of polypyrrole in molecular imprinting technology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6033
3. Conclusions and future developments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6034
Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6034
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6034

1. Introduction mers can be exploited as an excellent tool for the preparation

of nanocomposites with entrapped nano-scaled biomolecules,
Nanotechnology is rapidly evolving to open new materials mainly proteins and single stranded DNA oligomers. Some con-
useful in solving challenging bioanalytical problems, includ- ducting polymers doped and/or covalently or not covalently
ing specificity, stability and sensitivity. Here conducting poly- modified by bionanomaterials mentioned exhibit unique cat-
alytic [1] or affinity [2] properties that can be easily applied
in the design of bioanalytical sensors (biosensors).
∗ Corresponding author. Tel.: +370 5 2330987; fax: +370 5 2330987. Polypyrrole is one of the most extensively used conduct-
E-mail address: arunas@imi.lt (A. Ramanavičius). ing polymers in design of bioanalytical sensors [3] as well as

0013-4686/$ – see front matter © 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.electacta.2005.11.052
6026 A. Ramanavičius et al. / Electrochimica Acta 51 (2006) 6025–6037
for other purposes. Since 1990 up to June 2005 period solely
in the Journal Electrochimica Acta over 300 papers appeared
on various properties and applications of polypyrrole. Versatil-
ity of this polymer is determined by a number of properties:
redox activity [4], ability to form nanowires with room tem-
perature conductivity ranging from 10−4 to 10−2 S cm−1 [5],
ion-exchange and ion discrimination capacities [6,7], electro-
chromic effect depending on electrochemical polymerization
conditions and charge/discharge processes [8], strong absorp-
tive properties towards gases [9], proteins [10], DNA [11],
catalytic activity [12–14], corrosion protection properties [15],
etc. Most of these properties are depending on the synthesis
procedure as well as on the dopant nature [2]. Polypyrrole
might be electrochemically generated and deposited on the con-
ducting surfaces. This technique is successfully exploited for
development of various types of electrochemical sensors and
biosensors. Here several major directions are straightforward:
(i) catalytic sensors based on immobilized enzymes [1,16,17];
(ii) immunosensors based on immobilized affinity exhibiting
proteins [18]; (iii) DNA sensors based on covalently immo-
bilized and/or entrapped ssDNA [19–21]; (iv) affinity sensors
based on molecularly imprinted polymers [22]. Versatility of this
polymer is determined by the following: its biocompatibility;
capability to transduce energy arising from interaction of analyte
and analyte-recognizing-site into electrical signals that are eas-
ily monitored; capability to protect electrodes from interfering
materials; easy ways for electrochemical deposition on the sur-
face of any type of electrodes. Nowadays this polymer becomes
one of the major tools for nanobiotechnological applications
[23].
The aim of this study is to review major advances and appli-
cations of this polymer in design of catalytical biosensors,
immunosensors, DNA sensors and molecularly imprinted poly-
mer based sensors.
2. Discussion
2.1. Electrochemical polymerization of polypyrrole versus
chemical polymerization
Polypyrrole was firstly synthesized in 1912 [24]. Polypyr-
role synthesized by conventional chemical methods is insoluble
in common solvents because of strong inter-chain interactions
[25]. Two major ways are applied for polypyrrole synthesis
which are based on induction of polymerization by different
factors: (i) chemical initiation by oxidative agents [24,26]; (ii)
photo induced synthesis [27]; (iii) electrochemical activation
by anodic current [28]. All polymerization initiation methods
mentioned have particular application, e.g. chemical initiation
by oxidative agents might be successfully applied if a great
amount of polypyrrole is needed for application in the design
of chromatography columns [29] or for some other purposes.
By using chemical [26] or even biochemical [30] methods it
is easy to prepare Ppy particles of different and/or controlled
size ranging from several nanometers up to several micrometers
and/or containing various inclusions. Moreover, by chemical
methods it is possible to uniformly perform overoxidation of
this polymer, what is on special interest of affinity chromatogra-
phy since molecularly imprinted Ppy might be produced, which
might exhibit selectivity to molecules ranging from the small
organics [31–33] to high molecular weight biomolecules [22].
Photo induced Ppy synthesis is attractive in photolithographic
application of this polymer, since it allows alterations in syn-
thesized Ppy morphology by change of excitation light wave
length [34] and theoretically it might be applied for the design
of electronic chips. However, because of slow light induced
polymerization rate this polymerization type is still not very
often applied if compared with chemical or electrochemical
polymerization.
By using chemically induced polymerization the Ppy is
mainly produced in the bulk solution and just some amount of
synthesized polypyrrole is covering the surface of introduced
materials. It means that chemically induced polymerization is
not very efficient with respect to deposition of Ppy over some
surfaces. Moreover, Ppy is almost insoluble in usual solvents,
except some cases where it is doped with proper agents increas-
ing solubility of this polymer [35] and it means that deposition
(e.g. by solvent evaporation) of this polymer from the solution
containing dissolved polymer is possible at the stage where the
polymer is still in the form of colloid particles, before its precipi-
tation [30]. However, the major obstacle for use of this deposition
method for designing of Ppy based sensors is a poor adher-
ence of this deposit to the surface, contrary to the film obtained
by electrochemical polymerization. But all these disadvantages
might be avoided if electrochemical polymerization is applied. It
allows deposition of Ppy over electrodes deposited in the electro-
chemical cell. That is the reason why electrochemical polymer-
ization has found an application as a general deposition method
if thin Ppy layers are requested. By using this method thick-
ness and morphology of deposited layer might be controlled by
application of well-defined potential and known current passing
through the electrochemical cell [36]. Electrochemical depo-
sition of Ppy might be performed from various solvents (e.g.
acetonitrile, water, etc.). From the point of view of nanostructur-
ing of this polymer it is really very important that Ppy synthesis
might be performed from water solution at neutral pH, since
it opens the ways for entrapment and/or doping of polypyrrole
by various biomaterials like small organic molecules, proteins,
DNA and even living cells. However, if buffers with low buffer-
ing capacitance are used as polymerization solution, a potential
problem is the local production of a great amount of protons
in the course of the polymerization which may affect the prop-
erties of the biomolecules to be entrapped inside the Ppy film.
In particular cases overoxidized Ppy might be synthesized and
entrapped molecules and/or dopants might be extracted from
the Ppy structure. In such cases so called molecularly imprinted
polymers might be designed. Moreover, electrochemical poly-
merization is applied for deposition of polypyrrole layers inside
geometrically complicated electrochemical cells [37] and there
is almost no doubt that this polymerization method might be
extremely useful for deposition of Ppy layers inside microfluidic
devices. Furthermore, electrochemically synthesized Ppy has
some attractive features, such as good conductivity and very high
adherence of these films to the mostly for biosensor design used
 A. Ramanavičius et al. / Electrochimica Acta 51 (2006) 6025–6037
substrates leading towards sufficient stability of biosensors, even
in a neutral pH region. On the other hand, the electrochemical
properties of Ppy strongly depend on the redox state of this poly-
mer. At positive potentials an overoxidation of Ppy is occurring
what is leading towards lowering of Ppy conductivity and makes
easier leakage of anionic molecules if they were included into
polymeric backbone. Overoxidation of Ppy appears at lover pos-
itive potentials in water and/or oxygen-containing environment
and in this case it is leading towards partial destruction of poly-
meric backbone and generation of oxygen-containing (carboxyl,
carbonyl and hydroxyl) groups. Overoxidized Ppy has been used
in many electroanalytical applications that utilize its permse-
lectivity and is often used as discrimination membrane which
significantly increases selectivity of electrochemical biosensors
[38,39]. The capability of electrochemical polypyrrole synthesis
is significantly extended, since some different electrochemical
techniques might be applied for deposition of Ppy over the
electrodes: constant potential electrodeposition, galvanostatic
deposition, cyclic voltammetry, and potential pulse techniques
[40].
According to our experience based on application of conduct-
ing polymers in biosensor design the potential pulse technique
is the most suitable for nanostructuring of Ppy by entrapment
of biologically active materials within backbone of this poly-
mer. Potential pulse techniques enable to increase concentration
of entrapped biologically active material within nano-thin lay-
ers of polypyrrole [40] because various potentials might be
applied in step manner. Higher potential steps are applied for
initiation of Ppy polymerization and lower or negative potential
steps are used for attraction of higher amount of biomaterial,
which is entrapped into polymeric backbone during polymer-
ization step that is initiated by potential in the range of +0.6
to +1.2 V versus Ag/AgCl. The number of potential steps, the
profile of potential applied and duration of each step might
be set up individually depending on the application of deter-
mined requirements. All factors mentioned enable to prepare
large variation of nanostructured polymeric layers with differ-
ent analytical characteristics even if the same bulk solution
is used for polymerization. In general, electrochemical poly-
merization is more versatile if compared with chemical one
in terms of possible variations and control of polymerization
conditions. Moreover, combination of electrochemical tech-
niques with some chemical surface modification techniques
opens new opportunities for development of new nanobiostruc-
tural assemblies based on this polymer. More in detail, it was
demonstrated that the surface of electrochemically deposited
Ppy after some additional electrochemical/chemical functional-
ization might be covalently modified by enzymes [41]. Those
structures were applied in bio-catalytic biosensor design, and
it was demonstrated that Ppy layers modified by the same
enzyme exhibit significantly different selectivity towards var-
ious substrates if different Ppy modification approaches are
applied.
However, there are some particular cases where chemical
methods have some advantages if compared to electrochemi-
cal methods. Chemical methods are still mostly used if large
extent of Ppy or appropriate Ppy structures e.g. nanoparticles
6027
or Ppy coated nanoparticles of other materials are needed.
Nanocomposites of chemically synthesized polypyrrole are
mainly applied for affinity chromatography purposes [33], but
electrochemical methods are mainly used for construction of
chemical sensors, biosensors and actuators. So, in general, both
Ppy synthesis methods are finding particular application areas
for various technological purposes.
2.2. Polypyrrole as versatile immobilization matrix in
design of biosensors
Most important considerations during the creation of any
type of electrochemical biosensors are: (i) the immobilization of
the bio-catalyst; (ii) application of appropriate electrochemical
technique (e.g. potentiometric, amperometric and impedimet-
ric techniques are mainly applied for analytical signal regis-
tration in design of electrochemical sensors and biosensors);
(iii) establishment of efficient electron transfer if amperometric
detection is applied. Consequently, immobilization of biolog-
ically active material is of pivotal importance in the creation
of biosensors [42], since it allows application of the same bio-
logically active material for a number of analysis cycles. The
requirements for successful biomaterial immobilization are: (i)
biological recognition properties and/or catalytic properties of
biomaterial should remain after immobilization; (ii) the bioma-
terial should be well fixed on/within the substrate, otherwise the
biosensor will lose its activity; (iii) improve or at least minimally
decrease selectivity of constructed biosensor or bioanalytical
system; (iv) improve electron transfer if amperometric measure-
ments are applied as signal transduction system. To solve the
majority and sometimes all these tasks, conducting polymers
can be considered as a very effective substrate for biomaterial
immobilization. Among other conducting polymers, polyani-
line is often used as immobilizing substrate for biomolecules
[39] and sometimes as efficient electrocatalysts. However, the
necessity to detect bio-analytes at neutral pH range leads to
electro-inactivity of the deposited films, discouraging the use
of polyaniline and polythiophene as biosensing materials. As
opposed to polyaniline, polypyrrole might be easily deposited
from neutral pH aqueous solutions containing pyrrole monomer.
It makes this polymer very attractive and at present it is one of the
most extensively studied materials useful for immobilization of
different biomolecules and even living cells. On the other hand,
Ppy is often used in catalytic and affinity biosensors because of
good biocompatibility and the easy ways for immobilization of
biomolecules [43]. Biomaterials might be immobilized by vari-
ous methods: (i) adsorption on electrochemically or chemically
formed Ppy surface [44]; (ii) entrapment during electrochemical
deposition of polypyrrole [17,28,36,37,40]; (iii) self entrapped
if biomaterial is able to initiate polypyrrole synthesis [30]. As
it was mentioned in previous paragraph, by chemical initiation
it is easy to produce large extent of polypyrrole modified with
biomaterials, however, this method is not well suitable for the
formation of well defined layered structures such as usually are
needed for sensor design. On the contrary, the electrochemical
Ppy polymerization allows the formation of uniform films, the
thickness and morphology of such films might be controlled
6028 A. Ramanavičius et al. / Electrochimica Acta 51 (2006) 6025–6037
by regulation of passing current and/or potential applied. Here
the application of pulsed potential techniques allows preconcen-
tration of biologically active molecules (e.g. DNA, enzymes,
etc.) by applying of proper potential between the pulses initiat-
ing polymerization of polypyrrole [45]. In majority of cases the
pulse technique allows at least to avoid a strong diminution of
biologically active compound concentration near the electrode
surface which takes place at the steady-state diffusion regime
and it strongly enhances amount of inside the film incorporated
biologically active compound if compared to the steady-state
polymerization. Moreover, it was shown that Ppy is able very
effectively discriminate cations and anions, since permeability
and permselectivity of Ppy depends on the counter ion incor-
porated during polymerization as well as on the ions present
in the sample [7]. In particular cases anions (e.g. phosphate)
doped electrochemically deposited Ppy if properly doped by
some anions might be not permeable for anions what is very
useful for electrochemical biosensors since the majority of elec-
trochemically interfering materials present in biological samples
are anions [38,39]. It was also demonstrated that Ppy protects
electrodes from fouling by proteins and another biological sub-
stances present in the real samples as blood serum and urine
[46]. It was shown that various nanocomposites that might be
designed using combinatorial methods by polymerization of a
number of electrochemically polymerizable compounds are use-
ful for biosensor design [47].
The stability of Ppy based biosensors is sufficient and mainly
determined by degradation of Ppy in the water surrounding
if biosensor is applied for continuous measurements. In con-
clusion, from the point of view of electrochemical biosensing
Ppy has a number of very attractive characteristics: (i) it might
be synthesized electrochemically and modified by enzymes in
several different ways that gives different analytical charac-
teristics for constructed biosensors; (ii) it protects electrodes
from fouling and interfering materials such as electroactive
anions; (iii) it is biocompatible and, hence, causes minimal
and reversible disturbance to the working environment; (iv) in
some particular cases it might be exploited as redox media-
tor able to transfer electrons from the redox enzymes towards
electrodes.
2.3. Catalytic biosensors based on polypyrrole
Catalytic biosensors are described as compact analytical
devices, incorporating a bio-catalytic element or integrated
within a transducer system [48]. The detection of analyte in
this kind of biosensors is based on specific catalytic conver-
sion of the analyte of interest by a bio-catalyst immobilized on
the suitable signal transducer. The specific interaction of ana-
lyte with bio-recognition element results in a change of one or
more physicochemical properties (e.g. electron transfer, capac-
ity, optical properties) which can be detected and measured via
signal transduction and registered by registration devices. Elec-
trochemical catalytic biosensors are on special interest since they
can be applied for detection of analytes in non-transparent sam-
ples and the majority of electrochemical catalytic sensors are
relatively cheap and easy in application.
Fig. 1. Generalized scheme of electrochemical biosensors based on glucose
oxidase. Mox , oxidized form of redox mediator; Mred , reduced form of redox
mediator.
Thus, as it was mentioned previously, the redox enzymes are
mainly used in the design of catalytic biosensors. This class of
enzymes can be divided into several major subtypes that differ
with respect to cofactors involved into catalytic reaction. Cofac-
tors mainly effect the signal transduction process and should be
taken into account during catalytic biosensor construction and all
these biosensors have some specific properties mainly related to
the used enzymes. If enzymes are applied in the design of amper-
ometric biosensors efficient electron transfer route is crucial for
registration of analytical signal and the most successful sensors
are so called “reagent less” biosensors that are operating without
addition of any other soluble materials essential for analytical
signal registration.
Successful application of polypyrrole modified by enzymes
in the design of catalytic biosensors started by entrapment
of glucose oxidase from Aspergilus niger within polypyrrole
[49,50], later polyaniline was also successfully modified with
this enzyme [51,52] and employed for glucose sensing. Now,
FAD-dependent oxidases [53–57], NAD+ -dependent dehydro-
genases [58–62], PQQ-dependent dehydrogenases [16,63–67],
peroxidases [68–71] and some multicofactor enzymes [72–74]
are mostly used in the design of catalytic biosensors. The embed-
ment of enzymes within a conducting polymer film prevent the
enzyme from being leached out, while at the same time main-
taining accessibility of the catalytic sites due to the permeability
of the film to analytes [75]. Pulse technique for the electrochem-
ical deposition of polymer films on electrode surfaces enabled
the increase in the concentration of entrapped enzyme within
thin layers of Ppy [40]. Further, the enzyme activity is usually
detected by characterization of final reaction products or redox
mediators using amperometric or potentiometric methods.
In the case of application of FAD-dependent oxidases oxi-
dation of enzymatically produced H2 O2 is only possible at
high electrode potentials (Fig. 1). Some suitable redox medi-
ators able to facilitate the electron transfer between the active
site of the enzyme and electrode can be applied in the design
of electrochemical catalytic biosensors. So-far described oxi-
dases entrapped within conducting polymer-based biosensors
are requiring soluble redox mediators or conducting polymer
backbone should be enhanced by redox species [76].
The most successful biosensors based on oxidases entrapped
within polypyrrole were reported when redox polymers were
constructed on the basis of pyrrole which was copolymerized
with pyrrole substituted by redox mediators. Thus, for fixing of
 A. Ramanavičius et al. / Electrochimica Acta 51 (2006) 6025–6037
Fig. 2. Generalized scheme of electrochemical biosensors based on NAD+ -
dependent glucose dehydrogenase. Mox , oxidized form of redox mediator; Mred ,
reduced form of redox mediator.
redox mediators several main strategies might be applied, e.g.: (i)
osmium bipyridine complex based redox species were attached
to pyrrole and after copolymerization of this compound with pyr-
role several different redox polymers able to transfer electrons
from redox center of glucose oxidase were designed [64,77]; (ii)
PQQ-dependent glucose dehydrogenase was wired by ferrocene
derivatives [78]; (iii) modification of polymeric layers with some
soluble mediators that are almost freely diffusing within the film
[67,63,65,74].
In the case of application of oxidases the sensor response
was dependent on the availability of molecular oxygen; it is a
significant drawback because this concentration is not constant
and might significantly differ from sample to sample. This draw-
back might be avoided by application of dehydrogenases since
these enzymes do not require any oxygen as electron acceptor.
Dehydrogenases that are suitable for application in ampero-
metric biosensors might be divided into two major subclasses:
NAD+ -dependent dehydrogenases, and PQQ-dependent dehy-
drogenases. In the case of the NAD+ -dependent dehydrogenases
(Fig. 2) one may successfully circumvent the problems imposed
by molecular oxygen [79].
However, in the case of NAD+ -dependent dehydrogenase
application the coenzyme should be added to the analyte solution
which is only possible in specifically designed flow-injection
systems [80], or should be entrapped within conducting polymer
backbone [81] or graphite paste electrode matrix [82]. Therefore,
intensive attention was focused on finding new enzymes with
improved characteristics, such as exhibit the PQQ-dependent
dehydrogenases (Fig. 3) [83]. The use of PQQ-dependent
enzymes is more promising, since these enzymes are oxygen
independent, and their PQQ-cofactor in some cases is tightly
bound within the enzyme’s active site [84]. Significant advan-
tage was achieved when polypyrrole based on osmium-complex
modified redox polymer with entrapped PQQ-dependent glu-
cose dehydrogenase was applied for glucose sensing indepen-
dent on oxygen concentration fluctuations [64].
Some multicofactor enzymes, like PQQ and heme-c based
alcohol dehydrogenase were found to be able to transfer elec-
trons directly to some conducting surfaces including polypyrrole
(Fig. 4). Such enzymes might be easily applied in the design of
amperometric biosensors based on polypyrrole because appli-
6029
Fig. 3. Generalized scheme of electrochemical biosensors based on PQQ-
dependent glucose dehydrogenase. Mox , oxidized form of redox mediator; Mred ,
reduced form of redox mediator.
cation of any redox mediators is not essential in this case. It was
shown that PQQ-dependent alcohol dehydrogenase (QH-ADH)
covalently attached to the backbone of polypyrrole retains its
catalytic activity [41]. Moreover, it was demonstrated that QH-
ADH entrapped within polypyrrole exhibit direct electron trans-
fer to this polymer [72]. It might be predicted that Ppy and heme-
c-containing dehydrogenases based polymeric configurations
might be promising in the design of biofuel cells [85] and other
bioelectronic devices [86]. However, there is just a very lim-
ited number of such enzymes able to directly transfer electrons
toward backbone of conducting polymers. On the other hand,
the highest currency densities in biosensors based on PQQ and
heme-c based alcohol dehyrogenase were achieved when they
were deposited over electropolymerized 4-ferrocenylphenol, N-
(4-hydroxybenzylidene)-4-ferrocenylaniline, and 2-ferrocenyl-
4-nitrophenol [78].
Biosensors with different selectivity and activity character-
istics might be designed, since several different conceptions
might be used for immobilization of the same enzymes: (i)
adsorption of enzymes over electrochemically deposited Ppy
layer [44,87,88]; (ii) covalent attachment of enzymes after intro-
duction of amino groups into Ppy backbone and formation of
amide bounding due to interaction with carbodiimide activated
carboxylic groups of enzyme [46,41,89]; here two major dif-
ferent Ppy functionalization ways might be applied one based
Fig. 4. Generalized scheme of electrochemical biosensors based on direct elec-
tron transfer able PQQ-dependent alcohol dehydrogenase. H, heme-c moiety.
6030 A. Ramanavičius et al. / Electrochimica Acta 51 (2006) 6025–6037
on application of amino group modified pyrrole monomers for
copolymerization with unmodified pyrrole monomers, next on
introduction of functional groups following after preparation
of Ppy film; (iii) entrapment within backbone of polypyrrole
[17,40,59,64,89]. Entrapment of enzymes within polypyrrole
seems the most promising for construction of catalytic biosen-
sors, since this method allows to entrap significant amount of
redox enzymes that are able to convert high amount of ana-
lyte into the products and this process causes high changes of
electrochemical signals. In some cases after enzyme entrapment
during electrochemical deposition of Ppy over electrodes it was
detected that enzyme reactivation phase is very actual before sen-
sor might be applied for exact analyte determination. In several
cases this phase was 15–30 h long depending on the thickness of
Ppy layer formed [17,74]. The existence of this effect is deter-
mined by swelling of Ppy because there are some evidences
that immediately after the electrochemical deposition enzyme is
trapped within very dense polymeric structure which increases
steric hindrances for the substrate and deforms native structure
of the enzyme. After some swelling period water permeable
cavities become larger and substrate has more possibilities for
diffusion, as well as enzyme has more space to become native
conformation which possesses highest activity if compared with
other—not natural conformations. It was also demonstrated that
even after swelling period Ppy is able to accept electrons from
redox centers of some redox enzymes and transfer those elec-
trons to the metal electrodes [17]. It seems that polypyrrole is
an inherently biocompatible material. The sufficient water con-
tent ensures that the surface energy of this material is such that
it causes minimum disturbance to the biologically active com-
pound. In recent years, many catalytic biosensor configurations
and transducers have been designed allowing us to detect the
analyte in very low concentrations and with high precision. How-
ever, for practical applications, a few main problems remain to
be solved: (i) for one-way biosensors the production must be
so reproducible that calibration-free measurements can be per-
formed; (ii) long-term stability in water containing environment
should be increased.
2.4. Immunosensors based on polypyrrole
Immunosensors are the subject of increasing interest
mainly because of their potential application as an alterna-
tive immunoassay technique in areas such as clinical diagnos-
tics and environmental control. Enzyme-linked immunosorbent
assay (ELISA) is one of the most frequently used methods
for immunoassay, because of its good sensitivity, selectivity,
and ease in use. Although spectrometric methods are widely
used for the detection of enzymatic products resulting from
the Ag–Ab reactions in ELISA, the electrochemical methods
can provide capabilities of monitoring, free from color and tur-
bid interferences and which are relatively inexpensive, that the
spectrophotometric methods cannot compete with them [90].
Electrochemical affinity sensors acting on the principles similar
to ELISA usually are cheaper and faster in use if compared to
traditional ELISA. Moreover by immunosensors samples with-
out any analyte enrichment can be analyzed. In many cases
the purification and/or sample pretreatment step is not needed,
which is normally essential for standard analytical methods
such as mass spectrometry, gas chromatography and high per-
formance liquid chromatography. This factor is important for
many applications, especially in clinical diagnostics, where dif-
ferent analytes in whole blood, serum or urine containing a lot
of different substances, such as proteins, amino acids, sugars,
hormones, etc., are analyzed. Also immunosensors have con-
siderable advantages over standard methods with respect to time
and sensitivity [33].
Major indispensable condition during the development of
affinity sensors is immobilization of analyte binding reagent.
This task is often solved by application of conducting polymer,
polypyrrole, since Ppy is the mostly used conducting polymer
in affinity sensors because of the best biocompatibility, due to
efficient polymerization at neutral pH and very easy ways for
immobilization of various biologically active compounds. On
the other hand, it seems that this polymer is capable to trans-
fer energy as electrochemical transducer [20]. Affinity sensors
mainly rely on immobilized biomolecules or artificially formed
structures able to interact non-covalently with analyte as inter-
action partner and to form multimolecular complexes. Among
such non-covalently interacting materials are DNA, antibod-
ies, various proteins and molecularly imprinted polymers. The
development of immunosensors would lead to alternatives or
at least improvement in the existing immunoassay techniques.
Most related methods to immunoassay are immunosensors; they
are mainly applied in areas where both high selectivity and
high sensitivity are required [91]. Immunosensor is a device
that is able to detect the interaction between an antibody (Ab)
and an antigen (Ag) [92]. One of the binding able materials in
immunosensors is usually immobilized and at least one must be
found in sample as analyte. The conversion of the binding event
into a measurable signal, the regenerability and the reusabil-
ity are among major topics and challenges in immunosensor
development research. The conducting polymers and especially
polypyrrole can be considered as effective material for immo-
bilization of biomaterials and for transducing/amplification of
analytical signal in design of immunosensing devices [72,93].
Electrochemical modification of electrodes by conducting poly-
mers doped with biologically active compound included within
polymeric backbone is a simple step that is often used for cre-
ation of different immunosensors.
In the design of immunosensors antibodies [94,95], lig-
ands/receptors [96] and antigens [130] are applied as biological
materials able non-covalently to bind analyte. Antibodies are
considered to be well-suited and mostly used recognition ele-
ments for construction of immunosensors. The high specificity
and affinity of an antibody for corresponding antigen allows
a selective binding of the analyte which is present in nano-
to pico-molar range in the presence of hundreds of other sub-
stances, even if they exceed the analyte concentration by 2–3
orders of magnitude [97]. At the time, antibodies can be gen-
erated against almost all analytes, even if the analyte is non-
immunogenic. Moreover, recombinant antibody technology has
now been developed to the level that allows the expression of
single chain fragments in E. coli in large quantities [98].
 A. Ramanavičius et al. / Electrochimica Acta 51 (2006) 6025–6037
In many designs of electrochemical immunosensors sec-
ondary antibodies able to recognize analyte complex with immo-
bilized receptor were applied. Since antibodies are usually
not electrochemically active within the desired potential range
redox-active compounds and/or enzymes (mainly horseradish
peroxidase) [99–102] that are able to generate electrochem-
ical signal can be applied as labels for indication. Labeled
immunosensor format belongs to indirect analytical signal detec-
tion methods. In indirect electrochemical immunoassays the
binding reaction is visualized indirectly via an auxiliary reaction
by a labeling compound. Amperometric transducers in indirect
electrochemical immunoassay are used much more frequently
than others. For amperometric immunoassay the labeling redox
compound should have the following properties: it should be
reversibly electroactive; it should not cause electrode foul-
ing; chemical groups for coupling should be available [103].
Species such as nitrophenol, H2 O2 , and NH3 that can be deter-
mined electrochemically are the substrates or enzymatic reaction
products of alkaline phosphatase, horseradish peroxidase, and
urease, generally labeled on immunoreagents. Among these,
because amonia is electrochemically inactive at low potentials
and can only be detected by an ammonia gas-sensing elec-
trode, potentiometry is the only choice for the urease-labeled
immunoassay [104]. Indirect electrochemical immunoassay can
be divided into two major types: non-amplified and amplified.
In non-amplified redox-labeled electrochemical immunoassays
the indication of one antigen or antibody molecule will generate
one signal equivalent. Since the sensitivity of an amperometric
sensor for the redox compound is in the lower micromolar range,
this kind of assay makes sense only if the concentration of the
analyte to be determined is also in that range [105]. For more sen-
sitive immunoassays amplification principles are necessary. One
way to amplify the amperometrical signal is preconcentration
step. During this step concentration of redox-active compound
is increasing many times and only after some time (1–5 min) the
measurement starts. However, there are often some difficulties
to regenerate the sensor before each measurement.
The major disadvantage of indirect immunosensors is the
necessity to apply additional immunochemicals labeled by elec-
trochemical labels; it makes this method more expensive, time
consuming since additional procedures mainly based on incuba-
tion with labeled antibodies are essential for indirect detection
of analyte of interest. On the one hand, such procedures increase
sensitivity, but on the other hand they often decrease selectiv-
ity since usually broad-range-selectivity exhibiting secondary
antibodies are applied. In this respect so called ‘label-free’
immunosensors are more attractive, since such sensors allow
measurement without any additional hazardous reagents even
in real-time [48]. Label-free conversion of the binding event
into a measurable signal in particular at a low concentration of
the analyte, is one of the major challenges in biosensorics [48].
This topic is quite well solved in surface plasmon resonance
sensors what was reviewed previously [18]. Electrochemical
label-free analyte detection methods are developing not so fast
but they are very useful if colored and/or not transparent sam-
ples are under investigation or detection of analyte should be
performed in the body of patient. The majority of label-free
6031
electrochemical immunoassays are based on changes in charge
densities or conductivities for transduction and do not need any
auxiliary electrochemical reaction. If conducting polymers are
applied for immobilization of affinity towards analyte exhibit-
ing reagents after formation of immobilized receptor and analyte
complex changes in capacitance/resistance are registered [106].
Here potentiometric [107], capacitive [102] and amperomet-
ric [99–101] transducers have been used for electrochemical
immunoassays that indicate the binding of analyte directly.
Amperometric techniques have been used to monitor bind-
ing of analyte in real-time without using a labeled compound. A
polymer-modified antibody electrode has been used in combina-
tion with pulsed amperometric detection. The current obtained
at the immunochemical/polypyrrole based electrodes occurs via
the following steps: diffusion of ions to the electrode; charge
transfer at the porous polypyrrole membrane interface; migra-
tion through the polymer membrane; adsorption–desorption of
the analyte at the immunochemical/polypyrrole interface with
solution. The slow rate of adsorption–desorption process in the
last step is considered to be the rate-determining step. This
step can be controlled through the appropriate choice of elec-
trical potential [108]. Pulsed amperometric detection (PAD)
immunoassay techniques are such techniques where sensor can
be used for analyte detection in static or flow injection mode by
applying pulsed potentials between the sensor surface (or work-
ing electrode) and the reference electrode. The current obtained
can be directly related to the concentration of the analyte in
solution [109].
Besides amperometric transducers, capacitive transducers
have been used for the real-time and label-free measurement of
the Ag–Ab reaction. They are based on the principle that for elec-
trolytic capacitors the capacitance depends on the thickness and
dielectric behavior of a polymeric layer before and after inter-
action with analyte [110]. In some particular cases conductivity
measurements as one of transduction principle might be applied
in the design of electrochemical immunosensors. Conductivity
measurements have been adapted for immunoassay based on
ion concentration increased by the action of enzymatic label.
An enzyme immunoassay based on conductivity measurements
has been reported, in which urease was used as the secondary
antibody label. The enzyme retains the activity under conditions
of low ionic strength, so a low background conductance could be
employed [111]. However, conductivity measurements are dif-
ficult due to the variable ionic background of clinical samples
and the relatively small conductivity changes that are observed
in such high ionic strength solutions. The second comparative
‘blank’ electrode must be used, but variable drift at two separate
electrodes poses a universal drawback [112].
The inherent speed, accuracy and precision of electrochemi-
cal measurements have stimulated efforts towards the develop-
ment of both competitive and non-competitive electrochemical
immunoassay formats [113]. Sensors employing enzyme labels
with amperometric detection have been frequently reported with
drugs [114], hormones [115–118] and proteins [119] as target
analytes, and the detection of trace amounts in the sub-attomole
(<10–18 mol/l) range has been achieved [120]. In such detec-
tion scheme, the enzyme label is registered via formed/degraded
6032 A. Ramanavičius et al. / Electrochimica Acta 51 (2006) 6025–6037
electrochemically active product [121], and the function of
enzyme-labeled immunosensors is similar to that of catalytic
biosensors based on enzymes covalently attached to the sur-
face of polypyrrole. Glucose oxidase, horseradish peroxidase,
microperoxidase, ␤-galactosidase, alkaline phosphatase and
glucose-6-phosphatase dehydrogenase, all have been employed
in this mode with separation of free from bound label [122–124].
The NADH generated by glucose-6-phophatase dehydrogenase
reaction can be readily oxidized by mediators such as 2,6-
dichlorphenolindophenol [125] and 1,4-benziquinone [126], the
oxidation of those is followed amperometrically.
Several immunosensors were applied for continuous mea-
surements [106,110,127]. The major problem to use the
immunosensor for continuous measurements is stability of
Ag–Ab complexes. To overcome this problem for dissociation
of Ag–Ab complex buffers with extreme pH values, glycine
buffers or extreme salt concentrations are usually used. Flow
injection mode applied together with pulsed-amerometric detec-
tion immunoassay techniques is among such techniques which
can be successfully applied for continuous measurements in
flow throw electrochemical cell [106,110,127]. Electrochemical
label free immunosensors based on polypyrrole were developed
[106,128]. In a novel sampling strategy, antibody-based elec-
trodes were used for the repeat, intermittent on-line monitoring
of tissue corticosteroids in experimental animals. Here, in a com-
petitive assay, sample steroid competed with enzyme-labeled
corticosteroid for the antibody immobilized on a platinum elec-
trode surface. Amperometric detection of the enzyme product
was used to follow the reaction, and to measure enzyme activity
related to the analyte concentration in the sample [129]. How-
ever, for continuous or quasi-continuous measurements of an
analyte the problem of regeneration should be solved, because
extreme pH values, extreme salt concentrations or other fac-
tors which are usually used for dissociation of Ag–Ab com-
plexes lead to destruction of polymeric immobilization matrix
or distortion of sequence analytical signals. The most immediate
potential applications of immunosensors are medical diagnostics
including the determination of infections [130,131], environ-
mental analysis, food and beverages control.
2.5. Polypyrrole in the design of DNA sensors
DNA is a unique biomolecule, which is served in known liv-
ing species as genetic information storage. Number of genome
projects is providing massive amounts of genetic information
that should revolutionize the understanding of living nature.
Because genome variations between species and some groups
of individuals are straightforward and might be clearly distin-
guished it makes specific DNA sequences very attractive as
universal analyte. Nowadays the detection of appropriated DNA
sequences is important for diagnosis of genetic or infectious dis-
eases, environmental testing for bacterial contamination, rapid
detection of biological warfare agents, forensic investigations
and scientific explorations in genomics and proteomics.
Moreover, DNA might be determined as important nanoma-
terial, which is involved in a number of nanotechnological and/or
bioelectronic applications [132]. There were several demon-
strations that DNA in combination with conducting polymers
might be applied for the formation of unique nanostructures like
polypyrrole nanotubes [133] and polyaniline nanovires [134].
On the other hand, DNA in combination with polypyrrole was
described in a number of DNA sensors, like in the case of
immunosensors, electrochemically labeled and label-free DNA
sensors are designed. Early works on electrochemical DNA
sensors were mainly based on the application of electrochemi-
cal labels for indication of immobilized single-stranded DNA
(ssDNA) interaction with target DNA present in the sample
[135,136]. Such approaches were mainly based on measuring
changes in the peak currents of redox labels, if the DNA duplex
formed during hybridization is exposed to the solution of the
indicator [137]. For this purpose cationic methal complexes
such as tris(2,2 -bipyridine)ruthenium (III) ([Ru(bpy)3 ]3+ ) [138]
or tris(1,10-phenanthroline)cobalt(III) ([Co(phen)3 ]3+ ) [139],
aromatic compounds such as dye Hoechst 33258 [140],
daunomycin [141] and naphthalene diimine with covalently
attached two fereocene moieties [142] or enzymatic labels (e.g.
horseradish peroxidase) based redox indicators [143] might be
applied.
However, the most advantageous are label-free DNA
sensors. Such systems are mainly based on monitoring changes
in electronic or interfacial properties accompanying DNA
hybridization [19]. It means that the key factor concerning
the development of such electrochemical label-free DNA
sensors is the achievement of efficient interface between the
nucleic acid system and the electronic transducer. Conducting
polymer molecular interfaces are particularly suitable for
modulating DNA interactions, for inducing electrical signals
accrued from such interactions, and for localizing DNA probes
onto extremely small surfaces [19]. Among other conducting
polymers polypyrrole is most frequently used in the design of
electrochemical DNA sensors mainly for immobilization of
ssDNA [144]. As it was demonstrated in several studies, doping
of Ppy by ssDNA is a very simple procedure if electrochemical
deposition of this polymer in the presence of short ssDNA
oligonucleotides is applied [145]. In this way the formed
polymeric layer exhibits high affinity to complementary ssDNA
strands. Ppy/DNA sensors are based on various ssDNA immo-
bilization strategies such as adsorption [11], direct covalent
binding [146], entrapment in a polymer matrix [20,21,137,147]
or indirect binding by the use of intermediate systems like
biotine-avidine clips [148]. The most distinct electrochemical
signals are generated after DNA hybridization. Two different
ways of ssDNA entrapment might be distinguished: (i) entrap-
ment in the presence of other counter ions [21,144,147,149];
(ii) doping of Ppy film by ssDNA if ssDNA is present as
single ion in electrochemical-polymerization solution and is
served as counter ion [20,153]. In this way polypyrrole bearing
[21,144,147] and doped [20,153] with single-stranded DNA
was applied for the development of DNA sensors. On the other
hand, polypyrrole is capable to transfer energy as an electro-
chemical transducer and direct label-free, electrical detection of
DNA hybridization has also been accomplished by monitoring
changes in the impedance and capacity/resistance of conducting
polymer molecular interfaces [21]. Electrochemical impedance
 A. Ramanavičius et al. / Electrochimica Acta 51 (2006) 6025–6037
technique seems most informative in label-free DNA detection
since this method brings the highest number of information on
target DNA interaction with immobilized ssDNA [150,151].
Significant differences in impedance between electrochem-
ical systems containing single-stranded DNA immobilized
within polypyrrole and double-stranded DNA (dsDNA) can
be converted into electrical signals that are easily monitored
[152]. However, this method can be successfully replaced by
other less sophisticated electrochemical techniques like cyclic
votammetry [144], pulsed amperometric detection [21] or basic
amperometry [153]. Garnier et al. have reported a study in which
nucleic acid probes were linked to the polypyrrole surface and
cyclic voltammetry investigations were performed. This study
demonstrated a potential shift and wave broadening in the cyclic
voltammograms registered after interaction with target DNA
[144]. Cyclic voltammetry was applied for biosensing of DNA
hybridization by polypyrrole functionalized with ferrocenyl
groups [154]. Our group has showed that pulsed amperometric
detection might be applied for detection of target DNA if ssDNA
entrapped within Ppy is deposited over working electrode [21].
Investigations of Wang’s group illustrated that short term
current peaks provoked by the hybridization event at constant
potential might be registered if short complementary poly-A,
poly-G, poly-T and poly-C oligonucleotides are interacting with
complementary ones. However, if not-complementary DNA is
present in the sample the distinctly opposite current peaks were
observed [153]. High stability of Ppy/DNA films allows detec-
tion of target DNA in flow-trough systems what is a significant
advantage for continuous routine measurements [155]. On the
other hand, it was demonstrated that pyrrole–DNA might be eas-
ily addressed towards appropriated electrode what is extremely
useful for designing electrochemical-DNA arrays [156].
In some polypyrrole based DNA sensors, quartz crys-
tal microbalances or fluorescence methods were successfully
applied and such sensors were used for multiple determination
of target DNA [148]. It might be predicted that new DNA sensors
will be developed where intrinsic electron transfer properties of
DNA [157] and conducting polymers will be combined together.
Next promising direction which is offered by nanotechnology
is application of DNA in combination with nanoparticles [158].
Here polypyrrole based nanoparticles [30] might be applied,
since previously described DNA sorption on polypyrrole-silica
nanocomposites was very efficient [159]. Carbon nanotubes
modified with Ppy/ssDNA seem very promising for electro-
chemical DNA sensor design [150,151,160].
2.6. Application of polypyrrole in molecular imprinting
technology
Indispensable condition during the development of previ-
ously described affinity sensors is that the bind-able reagents
should be immobilized. However, given to poor chemical and
physical stability of biomolecules even if they are well immo-
bilized, molecularly imprinted polymers and artificial receptor
based sensors have been gaining importance as a possible alter-
native to other affinity sensors (e.g. immunosensors, DNA sen-
sors, etc.) which are based on immobilized biomolecules [161].
6033
The preparation of molecularly imprinted polymers requires
polymerization around a print species using monomers that
are selected for their capacity to form specific and definable
interactions with the print species. Within polymer entrapped
molecules can be removed by solvent extraction and the molec-
ularly imprinted polymer is ready for use. Cavities are formed
in the polymer matrix with ‘images’ of the size and shape of the
imprinted molecules. Furthermore, chemical functionalities of
the monomer residues become spatially positioned around the
cavity in accord with complementarity to the chemical structure
of the imprint molecule. Molecularly imprinted polymers are
very promising during the development of synthetic recognition
systems and are of great interest to workers in the field of sensor
technology. Molecular imprinting is increasingly becoming rec-
ognized as a versatile technique for the preparation of artificial
receptors based on molecularly imprinted conducting polymers
(MIPs) containing tailor-made recognition sites. MIP is another
class of substances of great interest in the field of chemical sen-
sor technology [162]. Moreover, these sensors are able to detect
low molecular mass organic molecules [31–33,163,164]. It is the
reason why the development of synthetic recognition systems is
of great interest to workers in the field of sensor technology
[165]. These highly stable synthetic polymers possess molecu-
lar recognition properties due to cavities formed in the polymer
matrix that are complementary to the analyte (ligand) both in
shape and in positioning of functional groups [166]. Some of
these polymers have shown very high selectivity and affinity
constants fully comparable to natural recognition systems such
as antibodies [167–169]. In general, molecular imprinting is a
technology for the manufacture of synthetic polymers with pre-
determined molecular recognition properties [170]. The prepara-
tion of molecularly imprinted polymers requires polymerization
around the print species using monomers that are selected for
their capacity to form specific and definable interactions with the
imprinted species [171]. Furthermore, chemical functionalities
of the monomer residues become spatially positioned around
the cavity in a pattern which is complementary to the chemical
structure of the print molecule [172]. These imprints consti-
tute a permanent memory for the print species and enable the
imprinted polymer to rebind the print molecule from a mixture
of closely related compounds selectively [173]. Finally, the print
molecules are removed by solvent extraction and the molecularly
imprinted polymer is ready for use. Some of these polymers have
been shown to be useful in sensor applications, exhibiting tol-
erance towards acid, base, high temperature and organic phases
[174]. It was found that the manufacture of composites con-
sisting of molecularly imprinted conducting polymers results
in obtaining materials that exhibit both predetermined selective
molecular recognition and electrical conductivity [175]. This
type of materials is of special interest for use in the field of
sensor technology [176]. Here overoxidized polypyrrole is most
frequently applied.
Overoxidized polypyrrole exhibits an improved selectivity,
which is attributed to the removal of positive charges from
Ppy films due to introduction of oxygen functionality, such
as carbonyl groups. The preparation of molecularly imprinted
polypyrrole requires polymerization around printed species.
6034 A. Ramanavičius et al. / Electrochimica Acta 51 (2006) 6025–6037
Then within Ppy entrapped molecules are removed by sol-
vent and the molecularly imprinted polymer is ready for use
[29]. Such polymer possesses nano-pores and nano-cavities that
are complementary to removed dopant. Furthermore, chemi-
cal functionalities of the monomer residues become spatially
positioned around the cavity in a pattern that is complemen-
tary to the chemical structure of the print molecule. These
imprints constitute a permanent memory for the print species
and enable the imprinted polymer to selectively rebind the print
molecule from a mixture of closely related compounds. Sen-
sors based on molecularly imprinted Ppy for serotonin and
1-naphthalensulfonate [177], amino acids [31,175], caffeine
[32,163], atropine [178], sacharide [179], glycoproteins [22]
were reported. Both chemically and electrochemically synthe-
sized Ppy can be applied in the development of molecularly
imprinted polymers. The electrochemical properties of Ppy
strongly depend on their redox state. At positive potentials
overoxidation of polypyrrole occurs. It leads to the partial degra-
dation of polypyrrole polymeric backbone and introduction of
carboxylic, carbonilic and hydroxilic groups into polymeric
backbone that determines semi-permeability as well as abil-
ity to recognize imprinted molecules [38]. The best results are
achieved if during electrochemical deposition overoxidized Ppy
is imprinted by small molecular weight molecules [31–33,176].
Moreover, attempts to imprint Ppy by large molecular weight
rigid structure possessing proteins were reported as well as, in
this case viral envelope proteins possessing rigid structure were
imprinted within overoxidized polypyrrole [22].
The conversion of the binding event into a measurable signal
in particular at a low concentration of the analyte, the prevention
or elimination of non-specific interactions, the regenerability,
and reusability among other topics are the major challenges
in molecularly imprinted polymer based biosensors. In such
sensors differences in capacitance and/or resistance arising in
electrochemical system during interaction of affinity agents can
be converted into signals that are easily monitored [180]. Here
pulsed amperometric detection can be applied as basic electro-
chemical detection method [21,22].
3. Conclusions and future developments
Polypyrrole is the most extensively used among other con-
ducting polymers for the construction of different types of bio-
analytical sensors. The background presented illustrates that
polypyrrole is a very attractive, versatile material, suitable for
preparation of various catalytic and affinity sensors and biosen-
sors. Both electrochemically and chemically induced pyrrole
polymerization methods has potential application in the devel-
opment of analyte-recognizing/converting layers.
In several studies it was shown that Ppy is able to transduce
analytical signal generated by some redox enzymes directly if
redox center of enzyme is deeply buried in the protein globule.
Polypyrrole modified by covalently attached redox groups might
be applied to facilitate electron transfer. From the other side, the
presented overview shows that polypyrrole might be applied
as immobilization matrix in the design of various affinity sen-
sors like immunosensors, DNA sensors and sensors based on
molecularly imprinted polymers. The use of polypyrrole in con-
junction with bioaffinity reagents has provident to be a powerful
route that has expanded the range of applications of electro-
chemical detection and its future development is expected to
continue. The interaction between the proteins, mainly nega-
tively charged at neutral pH, and the delocalized positive charges
along the polypyrrole chains induces changes in capacitance of
this nanostructured material. Consequently, such interactions,
evidenced from electrochemical measurement are the basis of
bioaffinity signal. The use of a wide range of counterions will
provide significant change in affinity at the Ppy ion-exchange
sites. The application of nanoelectrode (e.g. carbon nanotubes)
technologies already established in “electronic-nose” devices
will be beneficial to polypyrrole based immunosensors. Fur-
ther exploitation of this technology to immobilize bioaffinity
reagents with the polymer matrix may enable the design of
smaller, more compact and portable biosensing systems.
Current achievements show that electrochemical affinity sen-
sor based on molecularly imprinted polypyrrole could have a
great potential for direct electrochemical sensing. It is straight
forward that in the future molecularly imprinted polymer based
sensors will require deliberate control of the molecular structure
at the surface of the electrode to exhibit higher affinity to ana-
lyte. As the surface microstructure becomes more complicated,
more chemical methods of molecular structure construction will
be required. These methods will use “molecular technology”
instead of bulk technology mainly used at present time. In addi-
tion, for the construction of more complex nanostructures, some
degree of molecular self-assembly will be needed and conduct-
ing polymers become more complex, versatile and will find the
new applications. New nanotechnological approaches to over-
come these challenges are still in their infancy and application of
conducting polymers, in particular cases polypyrrole, will find
proper place in future molecular technology.
Acknowledgement
This work was partially financially supported by Lithuanian
State Science and Studies Foundation project number C 03047
and COST program D33.
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