Electrochimica Acta 51 (2006) 6025–6037

Review article

Electrochemical sensors based on conducting polymer—polypyrrole
A. Ramanaviˇ ius a,b,∗ , A. Ramanaviˇ ien˙ a,b , A. Malinauskas c c c e

Department of Analytical and Environmental Chemistry, Vilnius University, Naugarduko 24, 03225 Vilnius, Lithuania Laboratory of Immunoanalysis and Nanotechnology, Institute of Immunology of Vilnius University, Mol˙ t˛ pl. 29, 08409 Vilnius, Lithuania eu c Department of Organic Chemistry, Institute of Chemistry, Goˇ tauto 9, 01108 Vilnius, Lithuania s 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

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


1. Introduction Nanotechnology is rapidly evolving to open new materials useful in solving challenging bioanalytical problems, including specificity, stability and sensitivity. Here conducting poly-

Corresponding author. Tel.: +370 5 2330987; fax: +370 5 2330987. E-mail address: arunas@imi.lt (A. Ramanaviˇ ius). c

mers can be exploited as an excellent tool for the preparation of nanocomposites with entrapped nano-scaled biomolecules, mainly proteins and single stranded DNA oligomers. Some conducting polymers doped and/or covalently or not covalently modified by bionanomaterials mentioned exhibit unique catalytic [1] or affinity [2] properties that can be easily applied in the design of bioanalytical sensors (biosensors). Polypyrrole is one of the most extensively used conducting polymers in design of bioanalytical sensors [3] as well as

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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. Versatility of this polymer is determined by a number of properties: redox activity [4], ability to form nanowires with room temperature conductivity ranging from 10−4 to 10−2 S cm−1 [5], ion-exchange and ion discrimination capacities [6,7], electrochromic effect depending on electrochemical polymerization conditions and charge/discharge processes [8], strong absorptive 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 conducting 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 immobilized 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 easily monitored; capability to protect electrodes from interfering materials; easy ways for electrochemical deposition on the surface 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 applications of this polymer in design of catalytical biosensors, immunosensors, DNA sensors and molecularly imprinted polymer based sensors. 2. Discussion 2.1. Electrochemical polymerization of polypyrrole versus chemical polymerization Polypyrrole was firstly synthesized in 1912 [24]. Polypyrrole 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 chromatography 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 synthesized 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 increasing 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 precipitation [30]. However, the major obstacle for use of this deposition method for designing of Ppy based sensors is a poor adherence 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 electrochemical cell. That is the reason why electrochemical polymerization has found an application as a general deposition method if thin Ppy layers are requested. By using this method thickness and morphology of deposited layer might be controlled by application of well-defined potential and known current passing through the electrochemical cell [36]. Electrochemical deposition of Ppy might be performed from various solvents (e.g. acetonitrile, water, etc.). From the point of view of nanostructuring 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 buffering 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 properties 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 polymerization 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

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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 polymer. 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 positive potentials in water and/or oxygen-containing environment and in this case it is leading towards partial destruction of polymeric backbone and generation of oxygen-containing (carboxyl, carbonyl and hydroxyl) groups. Overoxidized Ppy has been used in many electroanalytical applications that utilize its permselectivity 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 conducting 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 polymer. Potential pulse techniques enable to increase concentration of entrapped biologically active material within nano-thin layers 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 polymerization 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 determined requirements. All factors mentioned enable to prepare large variation of nanostructured polymeric layers with different analytical characteristics even if the same bulk solution is used for polymerization. In general, electrochemical polymerization is more versatile if compared with chemical one in terms of possible variations and control of polymerization conditions. Moreover, combination of electrochemical techniques with some chemical surface modification techniques opens new opportunities for development of new nanobiostructural assemblies based on this polymer. More in detail, it was demonstrated that the surface of electrochemically deposited Ppy after some additional electrochemical/chemical functionalization 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 various substrates if different Ppy modification approaches are applied. However, there are some particular cases where chemical methods have some advantages if compared to electrochemical methods. Chemical methods are still mostly used if large extent of Ppy or appropriate Ppy structures e.g. nanoparticles

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 impedimetric techniques are mainly applied for analytical signal registration in design of electrochemical sensors and biosensors); (iii) establishment of efficient electron transfer if amperometric detection is applied. Consequently, immobilization of biologically active material is of pivotal importance in the creation of biosensors [42], since it allows application of the same biologically 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 biomaterial 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 measurements 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, polyaniline 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 various 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


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by regulation of passing current and/or potential applied. Here the application of pulsed potential techniques allows preconcentration of biologically active molecules (e.g. DNA, enzymes, etc.) by applying of proper potential between the pulses initiating 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 incorporated 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 electrochemically 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 substances 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 useful 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 conclusion, 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 characteristics 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 mediator 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 conversion of the analyte of interest by a bio-catalyst immobilized on the suitable signal transducer. The specific interaction of analyte with bio-recognition element results in a change of one or more physicochemical properties (e.g. electron transfer, capacity, optical properties) which can be detected and measured via signal transduction and registered by registration devices. Electrochemical catalytic biosensors are on special interest since they can be applied for detection of analytes in non-transparent samples 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. Cofactors 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 amperometric 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 dehydrogenases [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 embedment of enzymes within a conducting polymer film prevent the enzyme from being leached out, while at the same time maintaining accessibility of the catalytic sites due to the permeability of the film to analytes [75]. Pulse technique for the electrochemical 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 oxidation of enzymatically produced H2 O2 is only possible at high electrode potentials (Fig. 1). Some suitable redox mediators 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 oxidases 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

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Fig. 3. Generalized scheme of electrochemical biosensors based on PQQdependent glucose dehydrogenase. Mox , oxidized form of redox mediator; Mred , reduced form of redox mediator. 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 pyrrole 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 drawback 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 amperometric biosensors might be divided into two major subclasses: NAD+ -dependent dehydrogenases, and PQQ-dependent dehydrogenases. 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 advantage was achieved when polypyrrole based on osmium-complex modified redox polymer with entrapped PQQ-dependent glucose dehydrogenase was applied for glucose sensing independent on oxygen concentration fluctuations [64]. Some multicofactor enzymes, like PQQ and heme-c based alcohol dehydrogenase were found to be able to transfer electrons 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-

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 QHADH entrapped within polypyrrole exhibit direct electron transfer to this polymer [72]. It might be predicted that Ppy and hemec-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 limited 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-ferrocenyl4-nitrophenol [78]. Biosensors with different selectivity and activity characteristics 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 introduction 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 different Ppy functionalization ways might be applied one based

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


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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 biosensors, since this method allows to entrap significant amount of redox enzymes that are able to convert high amount of analyte 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 sensor 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 determined 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 electrons to the metal electrodes [17]. It seems that polypyrrole is an inherently biocompatible material. The sufficient water content ensures that the surface energy of this material is such that it causes minimum disturbance to the biologically active compound. 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. However, 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 performed; (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 alternative immunoassay technique in areas such as clinical diagnostics 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 turbid 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 without 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 performance liquid chromatography. This factor is important for many applications, especially in clinical diagnostics, where different 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 considerable 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 transfer energy as electrochemical transducer [20]. Affinity sensors mainly rely on immobilized biomolecules or artificially formed structures able to interact non-covalently with analyte as interaction partner and to form multimolecular complexes. Among such non-covalently interacting materials are DNA, antibodies, 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 reusability are among major topics and challenges in immunosensor development research. The conducting polymers and especially polypyrrole can be considered as effective material for immobilization of biomaterials and for transducing/amplification of analytical signal in design of immunosensing devices [72,93]. Electrochemical modification of electrodes by conducting polymers doped with biologically active compound included within polymeric backbone is a simple step that is often used for creation of different immunosensors. In the design of immunosensors antibodies [94,95], ligands/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 elements 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 nanoto pico-molar range in the presence of hundreds of other substances, even if they exceed the analyte concentration by 2–3 orders of magnitude [97]. At the time, antibodies can be generated against almost all analytes, even if the analyte is nonimmunogenic. 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].

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In many designs of electrochemical immunosensors secondary antibodies able to recognize analyte complex with immobilized 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 electrochemical signal can be applied as labels for indication. Labeled immunosensor format belongs to indirect analytical signal detection 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 fouling; chemical groups for coupling should be available [103]. Species such as nitrophenol, H2 O2 , and NH3 that can be determined 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 electrode, 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 sensitive 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 electrochemical labels; it makes this method more expensive, time consuming since additional procedures mainly based on incubation 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 selectivity 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 samples are under investigation or detection of analyte should be performed in the body of patient. The majority of label-free

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 exhibiting reagents after formation of immobilized receptor and analyte complex changes in capacitance/resistance are registered [106]. Here potentiometric [107], capacitive [102] and amperometric [99–101] transducers have been used for electrochemical immunoassays that indicate the binding of analyte directly. Amperometric techniques have been used to monitor binding of analyte in real-time without using a labeled compound. A polymer-modified antibody electrode has been used in combination 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; migration 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 electrical 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 working 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 electrolytic capacitors the capacitance depends on the thickness and dielectric behavior of a polymeric layer before and after interaction 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 difficult 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 electrochemical measurements have stimulated efforts towards the development 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 detection scheme, the enzyme label is registered via formed/degraded


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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 surface 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 2,6dichlorphenolindophenol [125] and 1,4-benziquinone [126], the oxidation of those is followed amperometrically. Several immunosensors were applied for continuous measurements [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 detection 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 electrodes were used for the repeat, intermittent on-line monitoring of tissue corticosteroids in experimental animals. Here, in a competitive assay, sample steroid competed with enzyme-labeled corticosteroid for the antibody immobilized on a platinum electrode 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]. However, 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 factors which are usually used for dissociation of Ag–Ab complexes 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], environmental analysis, food and beverages control. 2.5. Polypyrrole in the design of DNA sensors DNA is a unique biomolecule, which is served in known living 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 distinguished 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 diseases, 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 nanomaterial, 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 electrochemical 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 immobilization 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) entrapment 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 electrochemical 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

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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 electrochemical 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 detection 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 easily addressed towards appropriated electrode what is extremely useful for designing electrochemical-DNA arrays [156]. In some polypyrrole based DNA sensors, quartz crystal 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 electrochemical DNA sensor design [150,151,160]. 2.6. Application of polypyrrole in molecular imprinting technology Indispensable condition during the development of previously 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 immobilized, molecularly imprinted polymers and artificial receptor based sensors have been gaining importance as a possible alternative to other affinity sensors (e.g. immunosensors, DNA sensors, etc.) which are based on immobilized biomolecules [161].

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 molecularly 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 recognized 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 sensor 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 molecular 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 predetermined molecular recognition properties [170]. The preparation 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 constitute 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 tolerance towards acid, base, high temperature and organic phases [174]. It was found that the manufacture of composites consisting 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.


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Then within Ppy entrapped molecules are removed by solvent 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, chemical functionalities of the monomer residues become spatially positioned around the cavity in a pattern that is complementary 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. Sensors 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 synthesized 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 degradation of polypyrrole polymeric backbone and introduction of carboxylic, carbonilic and hydroxilic groups into polymeric backbone that determines semi-permeability as well as ability 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 electrochemical detection method [21,22]. 3. Conclusions and future developments Polypyrrole is the most extensively used among other conducting polymers for the construction of different types of bioanalytical sensors. The background presented illustrates that polypyrrole is a very attractive, versatile material, suitable for preparation of various catalytic and affinity sensors and biosensors. Both electrochemically and chemically induced pyrrole polymerization methods has potential application in the development 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 sensors like immunosensors, DNA sensors and sensors based on

molecularly imprinted polymers. The use of polypyrrole in conjunction with bioaffinity reagents has provident to be a powerful route that has expanded the range of applications of electrochemical detection and its future development is expected to continue. The interaction between the proteins, mainly negatively 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. Further 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 sensor 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 analyte. 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 addition, for the construction of more complex nanostructures, some degree of molecular self-assembly will be needed and conducting polymers become more complex, versatile and will find the new applications. New nanotechnological approaches to overcome 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. References
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