Electrochemical Sensors Based on Conducting Polymer—polypyrrole

Electrochimica Acta 51 (2006) 6025–6037

Review article

Electrochemical sensors based on conducting polymer—polypyrrole

A. Ramanavicius a,b,∗, A. Ramanaviciene a,b, A. Malinauskas c

a Department of Analytical and Environmental Chemistry, Vilnius University, Naugarduko 24, 03225 Vilnius, Lithuaniab Laboratory of Immunoanalysis and Nanotechnology, Institute of Immunology of Vilnius University, Moletu pl. 29, 08409 Vilnius, Lithuania

c Department of Organic Chemistry, Institute of Chemistry, Gostauto 9, 01108 Vilnius, Lithuania

Received 26 July 2005; received in revised form 16 November 2005; accepted 22 November 2005Available 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 ispaid to immobilization of biologically active molecules within Ppy during electrochemical deposition of this polymer. Such unique properties ofthis polymer as prevention of some undesirable electrochemical interactions and facilitation of electron transfer from some redox enzymes arediscussed. Recent advances in application of polypyrrole in immunosensors and DNA sensors are presented. Some new electrochemical targetDNA and target protein detection methods based on changes of semiconducting properties of electrochemically generated Ppy doped by affinityagents 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 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60252. Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6026

2.1. Electrochemical polymerization of polypyrrole versus chemical polymerization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60262.2. Polypyrrole as versatile immobilization matrix in design of biosensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60272.3. Catalytic biosensors based on polypyrrole . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60282.4. Immunosensors based on polypyrrole . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60302.5. Polypyrrole in the design of DNA sensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60322.6. Application of polypyrrole in molecular imprinting technology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6033

3. Conclusions and future developments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6034. ..

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Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. Introduction

Nanotechnology is rapidly evolving to open new materialsseful in solving challenging bioanalytical problems, includ-ng specificity, stability and sensitivity. Here conducting poly-

∗ Corresponding author. Tel.: +370 5 2330987; fax: +370 5 2330987.E-mail address: [email protected] (A. Ramanavicius).

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ers can be exploited as an excellent tool for the preparationf nanocomposites with entrapped nano-scaled biomolecules,ainly proteins and single stranded DNA oligomers. Some con-

ucting polymers doped and/or covalently or not covalentlyodified by bionanomaterials mentioned exhibit unique cat-

lytic [1] or affinity [2] properties that can be easily appliedn the design of bioanalytical sensors (biosensors).

Polypyrrole is one of the most extensively used conduct-ng polymers in design of bioanalytical sensors [3] as well as

mailto:[email protected]

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or other purposes. Since 1990 up to June 2005 period solelyn the Journal Electrochimica Acta over 300 papers appearedn various properties and applications of polypyrrole. Versatil-ty of this polymer is determined by a number of properties:edox activity [4], ability to form nanowires with room tem-erature conductivity ranging from 10−4 to 10−2 S cm−1 [5],on-exchange and ion discrimination capacities [6,7], electro-hromic effect depending on electrochemical polymerizationonditions and charge/discharge processes [8], strong absorp-ive properties towards gases [9], proteins [10], DNA [11],atalytic activity [12–14], corrosion protection properties [15],tc. Most of these properties are depending on the synthesisrocedure as well as on the dopant nature [2]. Polypyrroleight be electrochemically generated and deposited on the con-

ucting surfaces. This technique is successfully exploited forevelopment of various types of electrochemical sensors andiosensors. Here several major directions are straightforward:i) catalytic sensors based on immobilized enzymes [1,16,17];ii) immunosensors based on immobilized affinity exhibitingroteins [18]; (iii) DNA sensors based on covalently immo-ilized and/or entrapped ssDNA [19–21]; (iv) affinity sensorsased on molecularly imprinted polymers [22]. Versatility of thisolymer is determined by the following: its biocompatibility;apability to transduce energy arising from interaction of analytend analyte-recognizing-site into electrical signals that are eas-ly monitored; capability to protect electrodes from interfering

aterials; easy ways for electrochemical deposition on the sur-ace of any type of electrodes. Nowadays this polymer becomesne of the major tools for nanobiotechnological applications23].

The aim of this study is to review major advances and appli-ations of this polymer in design of catalytical biosensors,mmunosensors, DNA sensors and molecularly imprinted poly-

er based sensors.

. Discussion

.1. Electrochemical polymerization of polypyrrole versushemical polymerization

Polypyrrole was firstly synthesized in 1912 [24]. Polypyr-ole synthesized by conventional chemical methods is insolublen common solvents because of strong inter-chain interactions25]. Two major ways are applied for polypyrrole synthesishich are based on induction of polymerization by different

actors: (i) chemical initiation by oxidative agents [24,26]; (ii)hoto induced synthesis [27]; (iii) electrochemical activationy anodic current [28]. All polymerization initiation methodsentioned have particular application, e.g. chemical initiation

y oxidative agents might be successfully applied if a greatmount of polypyrrole is needed for application in the designf chromatography columns [29] or for some other purposes.y using chemical [26] or even biochemical [30] methods it

s easy to prepare Ppy particles of different and/or controlledize ranging from several nanometers up to several micrometersnd/or containing various inclusions. Moreover, by chemicalethods it is possible to uniformly perform overoxidation of

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ica Acta 51 (2006) 6025–6037

his polymer, what is on special interest of affinity chromatogra-hy since molecularly imprinted Ppy might be produced, whichight exhibit selectivity to molecules ranging from the small

rganics [31–33] to high molecular weight biomolecules [22].hoto induced Ppy synthesis is attractive in photolithographicpplication of this polymer, since it allows alterations in syn-hesized Ppy morphology by change of excitation light waveength [34] and theoretically it might be applied for the designf electronic chips. However, because of slow light inducedolymerization rate this polymerization type is still not veryften applied if compared with chemical or electrochemicalolymerization.

By using chemically induced polymerization the Ppy isainly produced in the bulk solution and just some amount of

ynthesized polypyrrole is covering the surface of introducedaterials. It means that chemically induced polymerization is

ot very efficient with respect to deposition of Ppy over someurfaces. Moreover, Ppy is almost insoluble in usual solvents,xcept some cases where it is doped with proper agents increas-ng solubility of this polymer [35] and it means that depositione.g. by solvent evaporation) of this polymer from the solutionontaining dissolved polymer is possible at the stage where theolymer is still in the form of colloid particles, before its precipi-ation [30]. However, the major obstacle for use of this depositionethod for designing of Ppy based sensors is a poor adher-

nce of this deposit to the surface, contrary to the film obtainedy electrochemical polymerization. But all these disadvantagesight be avoided if electrochemical polymerization is applied. It

llows deposition of Ppy over electrodes deposited in the electro-hemical cell. That is the reason why electrochemical polymer-zation has found an application as a general deposition methodf thin Ppy layers are requested. By using this method thick-ess and morphology of deposited layer might be controlled bypplication of well-defined potential and known current passinghrough the electrochemical cell [36]. Electrochemical depo-ition of Ppy might be performed from various solvents (e.g.cetonitrile, water, etc.). From the point of view of nanostructur-ng of this polymer it is really very important that Ppy synthesis

ight be performed from water solution at neutral pH, sincet opens the ways for entrapment and/or doping of polypyrroley various biomaterials like small organic molecules, proteins,NA and even living cells. However, if buffers with low buffer-

ng capacitance are used as polymerization solution, a potentialroblem is the local production of a great amount of protonsn the course of the polymerization which may affect the prop-rties of the biomolecules to be entrapped inside the Ppy film.n particular cases overoxidized Ppy might be synthesized andntrapped molecules and/or dopants might be extracted fromhe Ppy structure. In such cases so called molecularly imprintedolymers might be designed. Moreover, electrochemical poly-erization is applied for deposition of polypyrrole layers inside

eometrically complicated electrochemical cells [37] and theres almost no doubt that this polymerization method might be
xtremely useful for deposition of Ppy layers inside microfluidicevices. Furthermore, electrochemically synthesized Ppy hasome attractive features, such as good conductivity and very highdherence of these films to the mostly for biosensor design used

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ubstrates leading towards sufficient stability of biosensors, evenn a neutral pH region. On the other hand, the electrochemicalroperties of Ppy strongly depend on the redox state of this poly-er. At positive potentials an overoxidation of Ppy is occurringhat is leading towards lowering of Ppy conductivity and makes

asier leakage of anionic molecules if they were included intoolymeric backbone. Overoxidation of Ppy appears at lover pos-tive potentials in water and/or oxygen-containing environmentnd in this case it is leading towards partial destruction of poly-eric backbone and generation of oxygen-containing (carboxyl,

arbonyl and hydroxyl) groups. Overoxidized Ppy has been usedn many electroanalytical applications that utilize its permse-ectivity and is often used as discrimination membrane whichignificantly increases selectivity of electrochemical biosensors38,39]. The capability of electrochemical polypyrrole synthesiss significantly extended, since some different electrochemicalechniques might be applied for deposition of Ppy over thelectrodes: constant potential electrodeposition, galvanostaticeposition, cyclic voltammetry, and potential pulse techniques40].

According to our experience based on application of conduct-ng polymers in biosensor design the potential pulse techniques the most suitable for nanostructuring of Ppy by entrapmentf biologically active materials within backbone of this poly-er. Potential pulse techniques enable to increase concentration

f entrapped biologically active material within nano-thin lay-rs of polypyrrole [40] because various potentials might bepplied in step manner. Higher potential steps are applied fornitiation of Ppy polymerization and lower or negative potentialteps are used for attraction of higher amount of biomaterial,hich is entrapped into polymeric backbone during polymer-

zation step that is initiated by potential in the range of +0.6o +1.2 V versus Ag/AgCl. The number of potential steps, therofile of potential applied and duration of each step mighte set up individually depending on the application of deter-ined requirements. All factors mentioned enable to prepare

arge variation of nanostructured polymeric layers with differ-nt analytical characteristics even if the same bulk solutions used for polymerization. In general, electrochemical poly-

erization is more versatile if compared with chemical onen terms of possible variations and control of polymerizationonditions. Moreover, combination of electrochemical tech-iques with some chemical surface modification techniquespens new opportunities for development of new nanobiostruc-ural assemblies based on this polymer. More in detail, it wasemonstrated that the surface of electrochemically depositedpy after some additional electrochemical/chemical functional-

zation might be covalently modified by enzymes [41]. Thosetructures were applied in bio-catalytic biosensor design, andt was demonstrated that Ppy layers modified by the samenzyme exhibit significantly different selectivity towards var-ous substrates if different Ppy modification approaches arepplied.

However, there are some particular cases where chemicalethods have some advantages if compared to electrochemi-

al methods. Chemical methods are still mostly used if largextent of Ppy or appropriate Ppy structures e.g. nanoparticles

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ica Acta 51 (2006) 6025–6037 6027

r Ppy coated nanoparticles of other materials are needed.anocomposites of chemically synthesized polypyrrole areainly applied for affinity chromatography purposes [33], but

lectrochemical methods are mainly used for construction ofhemical sensors, biosensors and actuators. So, in general, bothpy synthesis methods are finding particular application areasor various technological purposes.

.2. Polypyrrole as versatile immobilization matrix inesign of biosensors

Most important considerations during the creation of anyype of electrochemical biosensors are: (i) the immobilization ofhe bio-catalyst; (ii) application of appropriate electrochemicalechnique (e.g. potentiometric, amperometric and impedimet-ic techniques are mainly applied for analytical signal regis-ration in design of electrochemical sensors and biosensors);iii) establishment of efficient electron transfer if amperometricetection is applied. Consequently, immobilization of biolog-cally active material is of pivotal importance in the creationf biosensors [42], since it allows application of the same bio-ogically active material for a number of analysis cycles. Theequirements for successful biomaterial immobilization are: (i)iological recognition properties and/or catalytic properties ofiomaterial should remain after immobilization; (ii) the bioma-erial should be well fixed on/within the substrate, otherwise theiosensor will lose its activity; (iii) improve or at least minimallyecrease selectivity of constructed biosensor or bioanalyticalystem; (iv) improve electron transfer if amperometric measure-ents are applied as signal transduction system. To solve theajority and sometimes all these tasks, conducting polymers

an be considered as a very effective substrate for biomaterialmmobilization. Among other conducting polymers, polyani-ine is often used as immobilizing substrate for biomolecules39] and sometimes as efficient electrocatalysts. However, theecessity to detect bio-analytes at neutral pH range leads tolectro-inactivity of the deposited films, discouraging the usef polyaniline and polythiophene as biosensing materials. Aspposed to polyaniline, polypyrrole might be easily depositedrom neutral pH aqueous solutions containing pyrrole monomer.t makes this polymer very attractive and at present it is one of theost extensively studied materials useful for immobilization of

ifferent biomolecules and even living cells. On the other hand,py is often used in catalytic and affinity biosensors because ofood biocompatibility and the easy ways for immobilization ofiomolecules [43]. Biomaterials might be immobilized by vari-us methods: (i) adsorption on electrochemically or chemicallyormed Ppy surface [44]; (ii) entrapment during electrochemicaleposition of polypyrrole [17,28,36,37,40]; (iii) self entrappedf biomaterial is able to initiate polypyrrole synthesis [30]. Ast was mentioned in previous paragraph, by chemical initiationt is easy to produce large extent of polypyrrole modified withiomaterials, however, this method is not well suitable for the
ormation of well defined layered structures such as usually areeeded for sensor design. On the contrary, the electrochemicalpy polymerization allows the formation of uniform films, the
hickness and morphology of such films might be controlled

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y regulation of passing current and/or potential applied. Herehe application of pulsed potential techniques allows preconcen-ration of biologically active molecules (e.g. DNA, enzymes,tc.) by applying of proper potential between the pulses initiat-ng polymerization of polypyrrole [45]. In majority of cases theulse technique allows at least to avoid a strong diminution ofiologically active compound concentration near the electrodeurface which takes place at the steady-state diffusion regimend it strongly enhances amount of inside the film incorporatediologically active compound if compared to the steady-stateolymerization. Moreover, it was shown that Ppy is able veryffectively discriminate cations and anions, since permeabilitynd permselectivity of Ppy depends on the counter ion incor-orated during polymerization as well as on the ions presentn the sample [7]. In particular cases anions (e.g. phosphate)oped electrochemically deposited Ppy if properly doped byome anions might be not permeable for anions what is veryseful for electrochemical biosensors since the majority of elec-rochemically interfering materials present in biological samplesre anions [38,39]. It was also demonstrated that Ppy protectslectrodes from fouling by proteins and another biological sub-tances present in the real samples as blood serum and urine46]. It was shown that various nanocomposites that might beesigned using combinatorial methods by polymerization of aumber of electrochemically polymerizable compounds are use-ul for biosensor design [47].

The stability of Ppy based biosensors is sufficient and mainlyetermined by degradation of Ppy in the water surroundingf biosensor is applied for continuous measurements. In con-lusion, from the point of view of electrochemical biosensingpy has a number of very attractive characteristics: (i) it mighte synthesized electrochemically and modified by enzymes ineveral different ways that gives different analytical charac-eristics for constructed biosensors; (ii) it protects electrodesrom fouling and interfering materials such as electroactivenions; (iii) it is biocompatible and, hence, causes minimalnd reversible disturbance to the working environment; (iv) inome particular cases it might be exploited as redox media-or able to transfer electrons from the redox enzymes towardslectrodes.

.3. Catalytic biosensors based on polypyrrole

Catalytic biosensors are described as compact analyticalevices, incorporating a bio-catalytic element or integratedithin a transducer system [48]. The detection of analyte in

his kind of biosensors is based on specific catalytic conver-ion of the analyte of interest by a bio-catalyst immobilized onhe suitable signal transducer. The specific interaction of ana-yte with bio-recognition element results in a change of one orore physicochemical properties (e.g. electron transfer, capac-

ty, optical properties) which can be detected and measured viaignal transduction and registered by registration devices. Elec-
rochemical catalytic biosensors are on special interest since theyan be applied for detection of analytes in non-transparent sam-les and the majority of electrochemical catalytic sensors areelatively cheap and easy in application.
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ig. 1. Generalized scheme of electrochemical biosensors based on glucosexidase. Mox, oxidized form of redox mediator; Mred, reduced form of redoxediator.

Thus, as it was mentioned previously, the redox enzymes areainly used in the design of catalytic biosensors. This class of

nzymes can be divided into several major subtypes that differith respect to cofactors involved into catalytic reaction. Cofac-

ors mainly effect the signal transduction process and should beaken into account during catalytic biosensor construction and allhese biosensors have some specific properties mainly related tohe used enzymes. If enzymes are applied in the design of amper-metric biosensors efficient electron transfer route is crucial foregistration of analytical signal and the most successful sensorsre so called “reagent less” biosensors that are operating withoutddition of any other soluble materials essential for analyticalignal registration.

Successful application of polypyrrole modified by enzymesn the design of catalytic biosensors started by entrapmentf glucose oxidase from Aspergilus niger within polypyrrole49,50], later polyaniline was also successfully modified withhis enzyme [51,52] and employed for glucose sensing. Now,AD-dependent oxidases [53–57], NAD+-dependent dehydro-enases [58–62], PQQ-dependent dehydrogenases [16,63–67],eroxidases [68–71] and some multicofactor enzymes [72–74]re mostly used in the design of catalytic biosensors. The embed-ent of enzymes within a conducting polymer film prevent the

nzyme from being leached out, while at the same time main-aining accessibility of the catalytic sites due to the permeabilityf the film to analytes [75]. Pulse technique for the electrochem-cal deposition of polymer films on electrode surfaces enabledhe increase in the concentration of entrapped enzyme withinhin layers of Ppy [40]. Further, the enzyme activity is usuallyetected by characterization of final reaction products or redoxediators using amperometric or potentiometric methods.In the case of application of FAD-dependent oxidases oxi-

ation of enzymatically produced H2O2 is only possible atigh electrode potentials (Fig. 1). Some suitable redox medi-tors able to facilitate the electron transfer between the activeite of the enzyme and electrode can be applied in the designf electrochemical catalytic biosensors. So-far described oxi-ases entrapped within conducting polymer-based biosensorsre requiring soluble redox mediators or conducting polymerackbone should be enhanced by redox species [76].

The most successful biosensors based on oxidases entrappedithin polypyrrole were reported when redox polymers were

onstructed on the basis of pyrrole which was copolymerizedith pyrrole substituted by redox mediators. Thus, for fixing of

A. Ramanavicius et al. / Electrochimica Acta 51 (2006) 6025–6037 6029

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ig. 2. Generalized scheme of electrochemical biosensors based on NAD+-ependent glucose dehydrogenase. Mox, oxidized form of redox mediator; Mred,educed form of redox mediator.

edox mediators several main strategies might be applied, e.g.: (i)smium bipyridine complex based redox species were attachedo pyrrole and after copolymerization of this compound with pyr-ole several different redox polymers able to transfer electronsrom redox center of glucose oxidase were designed [64,77]; (ii)QQ-dependent glucose dehydrogenase was wired by ferroceneerivatives [78]; (iii) modification of polymeric layers with someoluble mediators that are almost freely diffusing within the film67,63,65,74].

In the case of application of oxidases the sensor responseas dependent on the availability of molecular oxygen; it is a

ignificant drawback because this concentration is not constantnd might significantly differ from sample to sample. This draw-ack might be avoided by application of dehydrogenases sincehese enzymes do not require any oxygen as electron acceptor.ehydrogenases that are suitable for application in ampero-etric biosensors might be divided into two major subclasses:AD+-dependent dehydrogenases, and PQQ-dependent dehy-rogenases. In the case of the NAD+-dependent dehydrogenasesFig. 2) one may successfully circumvent the problems imposedy molecular oxygen [79].

However, in the case of NAD+-dependent dehydrogenasepplication the coenzyme should be added to the analyte solutionhich is only possible in specifically designed flow-injection

ystems [80], or should be entrapped within conducting polymerackbone [81] or graphite paste electrode matrix [82]. Therefore,ntensive attention was focused on finding new enzymes withmproved characteristics, such as exhibit the PQQ-dependentehydrogenases (Fig. 3) [83]. The use of PQQ-dependentnzymes is more promising, since these enzymes are oxygenndependent, and their PQQ-cofactor in some cases is tightlyound within the enzyme’s active site [84]. Significant advan-age was achieved when polypyrrole based on osmium-complex

odified redox polymer with entrapped PQQ-dependent glu-ose dehydrogenase was applied for glucose sensing indepen-ent on oxygen concentration fluctuations [64].

Some multicofactor enzymes, like PQQ and heme-c based
lcohol dehydrogenase were found to be able to transfer elec-rons directly to some conducting surfaces including polypyrroleFig. 4). Such enzymes might be easily applied in the design ofmperometric biosensors based on polypyrrole because appli-
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ig. 3. Generalized scheme of electrochemical biosensors based on PQQ-ependent glucose dehydrogenase. Mox, oxidized form of redox mediator; Mred,educed form of redox mediator.

ation of any redox mediators is not essential in this case. It washown that PQQ-dependent alcohol dehydrogenase (QH-ADH)ovalently attached to the backbone of polypyrrole retains itsatalytic activity [41]. Moreover, it was demonstrated that QH-DH entrapped within polypyrrole exhibit direct electron trans-

er to this polymer [72]. It might be predicted that Ppy and heme--containing dehydrogenases based polymeric configurationsight be promising in the design of biofuel cells [85] and other

ioelectronic devices [86]. However, there is just a very lim-ted number of such enzymes able to directly transfer electronsoward backbone of conducting polymers. On the other hand,he highest currency densities in biosensors based on PQQ andeme-c based alcohol dehyrogenase were achieved when theyere deposited over electropolymerized 4-ferrocenylphenol, N-

4-hydroxybenzylidene)-4-ferrocenylaniline, and 2-ferrocenyl--nitrophenol [78].

Biosensors with different selectivity and activity character-stics might be designed, since several different conceptions

ight be used for immobilization of the same enzymes: (i)dsorption of enzymes over electrochemically deposited Ppyayer [44,87,88]; (ii) covalent attachment of enzymes after intro-uction of amino groups into Ppy backbone and formation of

ig. 4. Generalized scheme of electrochemical biosensors based on direct elec-ron transfer able PQQ-dependent alcohol dehydrogenase. H, heme-c moiety.

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n application of amino group modified pyrrole monomers foropolymerization with unmodified pyrrole monomers, next onntroduction of functional groups following after preparationf Ppy film; (iii) entrapment within backbone of polypyrrole17,40,59,64,89]. Entrapment of enzymes within polypyrroleeems the most promising for construction of catalytic biosen-ors, since this method allows to entrap significant amount ofedox enzymes that are able to convert high amount of ana-yte into the products and this process causes high changes oflectrochemical signals. In some cases after enzyme entrapmenturing electrochemical deposition of Ppy over electrodes it wasetected that enzyme reactivation phase is very actual before sen-or might be applied for exact analyte determination. In severalases this phase was 15–30 h long depending on the thickness ofpy layer formed [17,74]. The existence of this effect is deter-ined by swelling of Ppy because there are some evidences

hat immediately after the electrochemical deposition enzyme israpped within very dense polymeric structure which increasesteric hindrances for the substrate and deforms native structuref the enzyme. After some swelling period water permeableavities become larger and substrate has more possibilities foriffusion, as well as enzyme has more space to become nativeonformation which possesses highest activity if compared withther—not natural conformations. It was also demonstrated thatven after swelling period Ppy is able to accept electrons fromedox centers of some redox enzymes and transfer those elec-rons to the metal electrodes [17]. It seems that polypyrrole isn inherently biocompatible material. The sufficient water con-ent ensures that the surface energy of this material is such thatt causes minimum disturbance to the biologically active com-ound. In recent years, many catalytic biosensor configurationsnd transducers have been designed allowing us to detect thenalyte in very low concentrations and with high precision. How-ver, for practical applications, a few main problems remain toe solved: (i) for one-way biosensors the production must beo reproducible that calibration-free measurements can be per-ormed; (ii) long-term stability in water containing environmenthould be increased.

.4. Immunosensors based on polypyrrole

Immunosensors are the subject of increasing interestainly because of their potential application as an alterna-

ive immunoassay technique in areas such as clinical diagnos-ics and environmental control. Enzyme-linked immunosorbentssay (ELISA) is one of the most frequently used methodsor immunoassay, because of its good sensitivity, selectivity,nd ease in use. Although spectrometric methods are widelysed for the detection of enzymatic products resulting fromhe Ag–Ab reactions in ELISA, the electrochemical methodsan provide capabilities of monitoring, free from color and tur-id interferences and which are relatively inexpensive, that thepectrophotometric methods cannot compete with them [90].
lectrochemical affinity sensors acting on the principles similar
o ELISA usually are cheaper and faster in use if compared toraditional ELISA. Moreover by immunosensors samples with-ut any analyte enrichment can be analyzed. In many cases

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ica Acta 51 (2006) 6025–6037

he purification and/or sample pretreatment step is not needed,hich is normally essential for standard analytical methods

uch as mass spectrometry, gas chromatography and high per-ormance liquid chromatography. This factor is important forany applications, especially in clinical diagnostics, where dif-

erent analytes in whole blood, serum or urine containing a lotf different substances, such as proteins, amino acids, sugars,ormones, etc., are analyzed. Also immunosensors have con-iderable advantages over standard methods with respect to timend sensitivity [33].

Major indispensable condition during the development offfinity sensors is immobilization of analyte binding reagent.his task is often solved by application of conducting polymer,olypyrrole, since Ppy is the mostly used conducting polymern affinity sensors because of the best biocompatibility, due tofficient polymerization at neutral pH and very easy ways formmobilization of various biologically active compounds. Onhe other hand, it seems that this polymer is capable to trans-er energy as electrochemical transducer [20]. Affinity sensorsainly rely on immobilized biomolecules or artificially formed

tructures able to interact non-covalently with analyte as inter-ction partner and to form multimolecular complexes. Amonguch non-covalently interacting materials are DNA, antibod-es, various proteins and molecularly imprinted polymers. Theevelopment of immunosensors would lead to alternatives ort least improvement in the existing immunoassay techniques.ost related methods to immunoassay are immunosensors; they

re mainly applied in areas where both high selectivity andigh sensitivity are required [91]. Immunosensor is a devicehat is able to detect the interaction between an antibody (Ab)nd an antigen (Ag) [92]. One of the binding able materials inmmunosensors is usually immobilized and at least one must beound in sample as analyte. The conversion of the binding eventnto a measurable signal, the regenerability and the reusabil-ty are among major topics and challenges in immunosensorevelopment research. The conducting polymers and especiallyolypyrrole can be considered as effective material for immo-ilization of biomaterials and for transducing/amplification ofnalytical signal in design of immunosensing devices [72,93].lectrochemical modification of electrodes by conducting poly-ers doped with biologically active compound included within

olymeric backbone is a simple step that is often used for cre-tion of different immunosensors.

In the design of immunosensors antibodies [94,95], lig-nds/receptors [96] and antigens [130] are applied as biologicalaterials able non-covalently to bind analyte. Antibodies are

onsidered to be well-suited and mostly used recognition ele-ents for construction of immunosensors. The high specificity

nd affinity of an antibody for corresponding antigen allowsselective binding of the analyte which is present in nano-

o pico-molar range in the presence of hundreds of other sub-tances, even if they exceed the analyte concentration by 2–3rders of magnitude [97]. At the time, antibodies can be gen-
rated against almost all analytes, even if the analyte is non-mmunogenic. Moreover, recombinant antibody technology hasow been developed to the level that allows the expression ofingle chain fragments in E. coli in large quantities [98].

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In many designs of electrochemical immunosensors sec-ndary antibodies able to recognize analyte complex with immo-ilized receptor were applied. Since antibodies are usuallyot electrochemically active within the desired potential rangeedox-active compounds and/or enzymes (mainly horseradisheroxidase) [99–102] that are able to generate electrochem-cal signal can be applied as labels for indication. Labeledmmunosensor format belongs to indirect analytical signal detec-ion methods. In indirect electrochemical immunoassays theinding reaction is visualized indirectly via an auxiliary reactiony a labeling compound. Amperometric transducers in indirectlectrochemical immunoassay are used much more frequentlyhan others. For amperometric immunoassay the labeling redoxompound should have the following properties: it should beeversibly electroactive; it should not cause electrode foul-ng; chemical groups for coupling should be available [103].pecies such as nitrophenol, H2O2, and NH3 that can be deter-ined electrochemically are the substrates or enzymatic reaction

roducts of alkaline phosphatase, horseradish peroxidase, andrease, generally labeled on immunoreagents. Among these,ecause amonia is electrochemically inactive at low potentialsnd can only be detected by an ammonia gas-sensing elec-rode, potentiometry is the only choice for the urease-labeledmmunoassay [104]. Indirect electrochemical immunoassay cane divided into two major types: non-amplified and amplified.n non-amplified redox-labeled electrochemical immunoassayshe indication of one antigen or antibody molecule will generatene signal equivalent. Since the sensitivity of an amperometricensor for the redox compound is in the lower micromolar range,his kind of assay makes sense only if the concentration of thenalyte to be determined is also in that range [105]. For more sen-itive immunoassays amplification principles are necessary. Oneay to amplify the amperometrical signal is preconcentration

tep. During this step concentration of redox-active compounds increasing many times and only after some time (1–5 min) the

easurement starts. However, there are often some difficultieso regenerate the sensor before each measurement.

The major disadvantage of indirect immunosensors is theecessity to apply additional immunochemicals labeled by elec-rochemical labels; it makes this method more expensive, timeonsuming since additional procedures mainly based on incuba-ion with labeled antibodies are essential for indirect detectionf analyte of interest. On the one hand, such procedures increaseensitivity, but on the other hand they often decrease selectiv-ty since usually broad-range-selectivity exhibiting secondaryntibodies are applied. In this respect so called ‘label-free’mmunosensors are more attractive, since such sensors allow

easurement without any additional hazardous reagents evenn real-time [48]. Label-free conversion of the binding eventnto a measurable signal in particular at a low concentration ofhe analyte, is one of the major challenges in biosensorics [48].his topic is quite well solved in surface plasmon resonanceensors what was reviewed previously [18]. Electrochemical
abel-free analyte detection methods are developing not so fastut they are very useful if colored and/or not transparent sam-les are under investigation or detection of analyte should beerformed in the body of patient. The majority of label-free
da(t

ica Acta 51 (2006) 6025–6037 6031

lectrochemical immunoassays are based on changes in chargeensities or conductivities for transduction and do not need anyuxiliary electrochemical reaction. If conducting polymers arepplied for immobilization of affinity towards analyte exhibit-ng reagents after formation of immobilized receptor and analyteomplex changes in capacitance/resistance are registered [106].ere potentiometric [107], capacitive [102] and amperomet-

ic [99–101] transducers have been used for electrochemicalmmunoassays that indicate the binding of analyte directly.

Amperometric techniques have been used to monitor bind-ng of analyte in real-time without using a labeled compound. Aolymer-modified antibody electrode has been used in combina-ion with pulsed amperometric detection. The current obtainedt the immunochemical/polypyrrole based electrodes occurs viahe following steps: diffusion of ions to the electrode; chargeransfer at the porous polypyrrole membrane interface; migra-ion through the polymer membrane; adsorption–desorption ofhe analyte at the immunochemical/polypyrrole interface witholution. The slow rate of adsorption–desorption process in theast step is considered to be the rate-determining step. Thistep can be controlled through the appropriate choice of elec-rical potential [108]. Pulsed amperometric detection (PAD)mmunoassay techniques are such techniques where sensor cane used for analyte detection in static or flow injection mode bypplying pulsed potentials between the sensor surface (or work-ng electrode) and the reference electrode. The current obtainedan be directly related to the concentration of the analyte inolution [109].

Besides amperometric transducers, capacitive transducersave been used for the real-time and label-free measurement ofhe Ag–Ab reaction. They are based on the principle that for elec-rolytic capacitors the capacitance depends on the thickness andielectric behavior of a polymeric layer before and after inter-ction with analyte [110]. In some particular cases conductivityeasurements as one of transduction principle might be applied

n the design of electrochemical immunosensors. Conductivityeasurements have been adapted for immunoassay based on

on concentration increased by the action of enzymatic label.n enzyme immunoassay based on conductivity measurementsas been reported, in which urease was used as the secondaryntibody label. The enzyme retains the activity under conditionsf low ionic strength, so a low background conductance could bemployed [111]. However, conductivity measurements are dif-cult due to the variable ionic background of clinical samplesnd the relatively small conductivity changes that are observedn such high ionic strength solutions. The second comparativeblank’ electrode must be used, but variable drift at two separatelectrodes poses a universal drawback [112].

The inherent speed, accuracy and precision of electrochemi-al measurements have stimulated efforts towards the develop-ent of both competitive and non-competitive electrochemical

mmunoassay formats [113]. Sensors employing enzyme labelsith amperometric detection have been frequently reported with
rugs [114], hormones [115–118] and proteins [119] as targetnalytes, and the detection of trace amounts in the sub-attomole<10–18 mol/l) range has been achieved [120]. In such detec-ion scheme, the enzyme label is registered via formed/degraded

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lectrochemically active product [121], and the function ofnzyme-labeled immunosensors is similar to that of catalyticiosensors based on enzymes covalently attached to the sur-ace of polypyrrole. Glucose oxidase, horseradish peroxidase,icroperoxidase, �-galactosidase, alkaline phosphatase and

lucose-6-phosphatase dehydrogenase, all have been employedn this mode with separation of free from bound label [122–124].he NADH generated by glucose-6-phophatase dehydrogenase

eaction can be readily oxidized by mediators such as 2,6-ichlorphenolindophenol [125] and 1,4-benziquinone [126], thexidation of those is followed amperometrically.

Several immunosensors were applied for continuous mea-urements [106,110,127]. The major problem to use themmunosensor for continuous measurements is stability ofg–Ab complexes. To overcome this problem for dissociationf Ag–Ab complex buffers with extreme pH values, glycineuffers or extreme salt concentrations are usually used. Flownjection mode applied together with pulsed-amerometric detec-ion immunoassay techniques is among such techniques whichan be successfully applied for continuous measurements inow throw electrochemical cell [106,110,127]. Electrochemical

abel free immunosensors based on polypyrrole were developed106,128]. In a novel sampling strategy, antibody-based elec-rodes were used for the repeat, intermittent on-line monitoringf tissue corticosteroids in experimental animals. Here, in a com-etitive assay, sample steroid competed with enzyme-labeledorticosteroid for the antibody immobilized on a platinum elec-rode surface. Amperometric detection of the enzyme productas used to follow the reaction, and to measure enzyme activity

elated to the analyte concentration in the sample [129]. How-ver, for continuous or quasi-continuous measurements of annalyte the problem of regeneration should be solved, becausextreme pH values, extreme salt concentrations or other fac-ors which are usually used for dissociation of Ag–Ab com-lexes lead to destruction of polymeric immobilization matrixr distortion of sequence analytical signals. The most immediateotential applications of immunosensors are medical diagnosticsncluding the determination of infections [130,131], environ-

ental analysis, food and beverages control.

.5. Polypyrrole in the design of DNA sensors

DNA is a unique biomolecule, which is served in known liv-ng species as genetic information storage. Number of genomerojects is providing massive amounts of genetic informationhat should revolutionize the understanding of living nature.ecause genome variations between species and some groupsf individuals are straightforward and might be clearly distin-uished it makes specific DNA sequences very attractive asniversal analyte. Nowadays the detection of appropriated DNAequences is important for diagnosis of genetic or infectious dis-ases, environmental testing for bacterial contamination, rapidetection of biological warfare agents, forensic investigations
nd scientific explorations in genomics and proteomics.
Moreover, DNA might be determined as important nanoma-erial, which is involved in a number of nanotechnological and/orioelectronic applications [132]. There were several demon-

cDcp

ica Acta 51 (2006) 6025–6037

trations that DNA in combination with conducting polymersight be applied for the formation of unique nanostructures like

olypyrrole nanotubes [133] and polyaniline nanovires [134].n the other hand, DNA in combination with polypyrrole wasescribed in a number of DNA sensors, like in the case ofmmunosensors, electrochemically labeled and label-free DNAensors are designed. Early works on electrochemical DNAensors were mainly based on the application of electrochemi-al labels for indication of immobilized single-stranded DNAssDNA) interaction with target DNA present in the sample135,136]. Such approaches were mainly based on measuringhanges in the peak currents of redox labels, if the DNA duplexormed during hybridization is exposed to the solution of thendicator [137]. For this purpose cationic methal complexesuch as tris(2,2′-bipyridine)ruthenium (III) ([Ru(bpy)3]3+) [138]r tris(1,10-phenanthroline)cobalt(III) ([Co(phen)3]3+) [139],romatic compounds such as dye Hoechst 33258 [140],aunomycin [141] and naphthalene diimine with covalentlyttached two fereocene moieties [142] or enzymatic labels (e.g.orseradish peroxidase) based redox indicators [143] might bepplied.

However, the most advantageous are label-free DNAensors. Such systems are mainly based on monitoring changesn electronic or interfacial properties accompanying DNAybridization [19]. It means that the key factor concerninghe development of such electrochemical label-free DNAensors is the achievement of efficient interface between theucleic acid system and the electronic transducer. Conductingolymer molecular interfaces are particularly suitable forodulating DNA interactions, for inducing electrical signals

ccrued from such interactions, and for localizing DNA probesnto extremely small surfaces [19]. Among other conductingolymers polypyrrole is most frequently used in the design oflectrochemical DNA sensors mainly for immobilization ofsDNA [144]. As it was demonstrated in several studies, dopingf Ppy by ssDNA is a very simple procedure if electrochemicaleposition of this polymer in the presence of short ssDNAligonucleotides is applied [145]. In this way the formedolymeric layer exhibits high affinity to complementary ssDNAtrands. Ppy/DNA sensors are based on various ssDNA immo-ilization strategies such as adsorption [11], direct covalentinding [146], entrapment in a polymer matrix [20,21,137,147]r indirect binding by the use of intermediate systems likeiotine-avidine clips [148]. The most distinct electrochemicalignals are generated after DNA hybridization. Two differentays of ssDNA entrapment might be distinguished: (i) entrap-ent in the presence of other counter ions [21,144,147,149];

ii) doping of Ppy film by ssDNA if ssDNA is present asingle ion in electrochemical-polymerization solution and iserved as counter ion [20,153]. In this way polypyrrole bearing21,144,147] and doped [20,153] with single-stranded DNAas applied for the development of DNA sensors. On the otherand, polypyrrole is capable to transfer energy as an electro-
hemical transducer and direct label-free, electrical detection ofNA hybridization has also been accomplished by monitoring
hanges in the impedance and capacity/resistance of conductingolymer molecular interfaces [21]. Electrochemical impedance

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echnique seems most informative in label-free DNA detectionince this method brings the highest number of information onarget DNA interaction with immobilized ssDNA [150,151].ignificant differences in impedance between electrochem-

cal systems containing single-stranded DNA immobilizedithin polypyrrole and double-stranded DNA (dsDNA) cane converted into electrical signals that are easily monitored152]. However, this method can be successfully replaced byther less sophisticated electrochemical techniques like cyclicotammetry [144], pulsed amperometric detection [21] or basicmperometry [153]. Garnier et al. have reported a study in whichucleic acid probes were linked to the polypyrrole surface andyclic voltammetry investigations were performed. This studyemonstrated a potential shift and wave broadening in the cyclicoltammograms registered after interaction with target DNA144]. Cyclic voltammetry was applied for biosensing of DNAybridization by polypyrrole functionalized with ferrocenylroups [154]. Our group has showed that pulsed amperometricetection might be applied for detection of target DNA if ssDNAntrapped within Ppy is deposited over working electrode [21].nvestigations of Wang’s group illustrated that short termurrent peaks provoked by the hybridization event at constantotential might be registered if short complementary poly-A,oly-G, poly-T and poly-C oligonucleotides are interacting withomplementary ones. However, if not-complementary DNA isresent in the sample the distinctly opposite current peaks werebserved [153]. High stability of Ppy/DNA films allows detec-ion of target DNA in flow-trough systems what is a significantdvantage for continuous routine measurements [155]. On thether hand, it was demonstrated that pyrrole–DNA might be eas-ly addressed towards appropriated electrode what is extremelyseful for designing electrochemical-DNA arrays [156].

In some polypyrrole based DNA sensors, quartz crys-al microbalances or fluorescence methods were successfullypplied and such sensors were used for multiple determinationf target DNA [148]. It might be predicted that new DNA sensorsill be developed where intrinsic electron transfer properties ofNA [157] and conducting polymers will be combined together.ext promising direction which is offered by nanotechnology

s application of DNA in combination with nanoparticles [158].ere polypyrrole based nanoparticles [30] might be applied,

ince previously described DNA sorption on polypyrrole-silicaanocomposites was very efficient [159]. Carbon nanotubesodified with Ppy/ssDNA seem very promising for electro-

hemical DNA sensor design [150,151,160].

.6. Application of polypyrrole in molecular imprintingechnology

Indispensable condition during the development of previ-usly described affinity sensors is that the bind-able reagentshould be immobilized. However, given to poor chemical andhysical stability of biomolecules even if they are well immo-
ilized, molecularly imprinted polymers and artificial receptorased sensors have been gaining importance as a possible alter-ative to other affinity sensors (e.g. immunosensors, DNA sen-ors, etc.) which are based on immobilized biomolecules [161].
wPap

ica Acta 51 (2006) 6025–6037 6033

he preparation of molecularly imprinted polymers requiresolymerization around a print species using monomers thatre selected for their capacity to form specific and definablenteractions with the print species. Within polymer entrapped

olecules can be removed by solvent extraction and the molec-larly imprinted polymer is ready for use. Cavities are formedn the polymer matrix with ‘images’ of the size and shape of themprinted molecules. Furthermore, chemical functionalities ofhe monomer residues become spatially positioned around theavity in accord with complementarity to the chemical structuref the imprint molecule. Molecularly imprinted polymers areery promising during the development of synthetic recognitionystems and are of great interest to workers in the field of sensorechnology. Molecular imprinting is increasingly becoming rec-gnized as a versatile technique for the preparation of artificialeceptors based on molecularly imprinted conducting polymersMIPs) containing tailor-made recognition sites. MIP is anotherlass of substances of great interest in the field of chemical sen-or technology [162]. Moreover, these sensors are able to detectow molecular mass organic molecules [31–33,163,164]. It is theeason why the development of synthetic recognition systems isf great interest to workers in the field of sensor technology165]. These highly stable synthetic polymers possess molecu-ar recognition properties due to cavities formed in the polymeratrix that are complementary to the analyte (ligand) both in

hape and in positioning of functional groups [166]. Some ofhese polymers have shown very high selectivity and affinityonstants fully comparable to natural recognition systems suchs antibodies [167–169]. In general, molecular imprinting is aechnology for the manufacture of synthetic polymers with pre-etermined molecular recognition properties [170]. The prepara-ion of molecularly imprinted polymers requires polymerizationround the print species using monomers that are selected forheir capacity to form specific and definable interactions with themprinted species [171]. Furthermore, chemical functionalitiesf the monomer residues become spatially positioned aroundhe cavity in a pattern which is complementary to the chemicaltructure of the print molecule [172]. These imprints consti-ute a permanent memory for the print species and enable themprinted polymer to rebind the print molecule from a mixturef closely related compounds selectively [173]. Finally, the printolecules are removed by solvent extraction and the molecularly

mprinted polymer is ready for use. Some of these polymers haveeen shown to be useful in sensor applications, exhibiting tol-rance towards acid, base, high temperature and organic phases174]. It was found that the manufacture of composites con-isting of molecularly imprinted conducting polymers resultsn obtaining materials that exhibit both predetermined selective

olecular recognition and electrical conductivity [175]. Thisype of materials is of special interest for use in the field ofensor technology [176]. Here overoxidized polypyrrole is mostrequently applied.

Overoxidized polypyrrole exhibits an improved selectivity,
hich is attributed to the removal of positive charges frompy films due to introduction of oxygen functionality, suchs carbonyl groups. The preparation of molecularly imprintedolypyrrole requires polymerization around printed species.

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hen within Ppy entrapped molecules are removed by sol-ent and the molecularly imprinted polymer is ready for use29]. Such polymer possesses nano-pores and nano-cavities thatre complementary to removed dopant. Furthermore, chemi-al functionalities of the monomer residues become spatiallyositioned around the cavity in a pattern that is complemen-ary to the chemical structure of the print molecule. Thesemprints constitute a permanent memory for the print speciesnd enable the imprinted polymer to selectively rebind the printolecule from a mixture of closely related compounds. Sen-

ors based on molecularly imprinted Ppy for serotonin and-naphthalensulfonate [177], amino acids [31,175], caffeine32,163], atropine [178], sacharide [179], glycoproteins [22]ere reported. Both chemically and electrochemically synthe-

ized Ppy can be applied in the development of molecularlymprinted polymers. The electrochemical properties of Ppytrongly depend on their redox state. At positive potentialsveroxidation of polypyrrole occurs. It leads to the partial degra-ation of polypyrrole polymeric backbone and introduction ofarboxylic, carbonilic and hydroxilic groups into polymericackbone that determines semi-permeability as well as abil-ty to recognize imprinted molecules [38]. The best results arechieved if during electrochemical deposition overoxidized Ppys imprinted by small molecular weight molecules [31–33,176].

oreover, attempts to imprint Ppy by large molecular weightigid structure possessing proteins were reported as well as, inhis case viral envelope proteins possessing rigid structure weremprinted within overoxidized polypyrrole [22].

The conversion of the binding event into a measurable signaln particular at a low concentration of the analyte, the preventionr elimination of non-specific interactions, the regenerability,nd reusability among other topics are the major challengesn molecularly imprinted polymer based biosensors. In suchensors differences in capacitance and/or resistance arising inlectrochemical system during interaction of affinity agents cane converted into signals that are easily monitored [180]. Hereulsed amperometric detection can be applied as basic electro-hemical detection method [21,22].

. Conclusions and future developments

Polypyrrole is the most extensively used among other con-ucting polymers for the construction of different types of bio-nalytical sensors. The background presented illustrates thatolypyrrole is a very attractive, versatile material, suitable forreparation of various catalytic and affinity sensors and biosen-ors. Both electrochemically and chemically induced pyrroleolymerization methods has potential application in the devel-pment of analyte-recognizing/converting layers.

In several studies it was shown that Ppy is able to transducenalytical signal generated by some redox enzymes directly ifedox center of enzyme is deeply buried in the protein globule.olypyrrole modified by covalently attached redox groups might
e applied to facilitate electron transfer. From the other side, theresented overview shows that polypyrrole might be applieds immobilization matrix in the design of various affinity sen-ors like immunosensors, DNA sensors and sensors based on
ica Acta 51 (2006) 6025–6037

olecularly imprinted polymers. The use of polypyrrole in con-unction with bioaffinity reagents has provident to be a powerfuloute that has expanded the range of applications of electro-hemical detection and its future development is expected toontinue. The interaction between the proteins, mainly nega-ively charged at neutral pH, and the delocalized positive chargeslong the polypyrrole chains induces changes in capacitance ofhis nanostructured material. Consequently, such interactions,videnced from electrochemical measurement are the basis ofioaffinity signal. The use of a wide range of counterions willrovide significant change in affinity at the Ppy ion-exchangeites. The application of nanoelectrode (e.g. carbon nanotubes)echnologies already established in “electronic-nose” devicesill be beneficial to polypyrrole based immunosensors. Fur-

her exploitation of this technology to immobilize bioaffinityeagents with the polymer matrix may enable the design ofmaller, more compact and portable biosensing systems.

Current achievements show that electrochemical affinity sen-or based on molecularly imprinted polypyrrole could have areat potential for direct electrochemical sensing. It is straightorward that in the future molecularly imprinted polymer basedensors will require deliberate control of the molecular structuret the surface of the electrode to exhibit higher affinity to ana-yte. As the surface microstructure becomes more complicated,

ore chemical methods of molecular structure construction wille required. These methods will use “molecular technology”nstead of bulk technology mainly used at present time. In addi-ion, for the construction of more complex nanostructures, someegree of molecular self-assembly will be needed and conduct-ng polymers become more complex, versatile and will find theew applications. New nanotechnological approaches to over-ome these challenges are still in their infancy and application ofonducting polymers, in particular cases polypyrrole, will findroper place in future molecular technology.

cknowledgement

This work was partially financially supported by Lithuaniantate Science and Studies Foundation project number C 03047nd COST program D33.

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