dna polymer

Review Conducting polymers for electrochemical DNA sensing Hui Peng a , * , Lijuan Zhang a , Christian Soeller a , Jadranka Travas-Sejdic a , b , ** a Polymer Electronic Research Centre, The University of Auckland, Private Bag 92019, Auckland, New Zealand b MacDiarmid Institute for Advanced Materials and Nanotechnology, New Zealand article info Article history: Received 30 September 2008 Accepted 24 December 2008 Available online xxx Keywords: Conducting polymers Electrochemical DNA sensor Electropolymerization abstract Conducting polymers (CPs) are a class of polymeric materials that have attracted considerable interest because of their unique electronic, chemical and biochemical properties, making them suitable for numerous applications such as energy storage, memory devices, chemical sensors, and in electro- catalysis. Conducting polymer-based electrochemical DNA sensors have shown applicability in a number of areas related to human health such as diagnosis of infectious diseases, genetic mutations, drug discovery, forensics and food technology due to their simplicity and high sensitivity. This review paper summarizes the advances in electrochemical DNA sensing based on conducting polymers as active substrates. The various conducting polymers used for DNA detection, along with different DNA immo- bilization and detection methodologies are presented. Current trends in this field and newly developed applications due to advances in nanotechnology are also discussed. Ó 2008 Elsevier Ltd. All rights reserved. 1. Introduction DNA analysis plays an ever-increasing role in a number of areas related to human health such as diagnosis of infectious diseases, genetic mutations, drug discovery, forensics and food technology. Conventional methods for the analysis of specific gene sequences are based on either direct sequencing or DNA hybridization. The sequencing technology was invented by Maxam and Gilbert [1] and Sanger et al. [2] in the 1970s. In the same period, solid-supported hybridization became a widespread method for DNA analysis using membrane-based blots [3,4]. However, these approaches have some disadvantages, such as the inability to use a large number of DNA samples, low selectivity between closely related sequences and they are often time consuming. In the early 90s, gene array technologies which relied on the anchoring of multiple specific probe DNA fragments or oligonucleotides (ODNs) onto solid surfaces and detection of fluorescently or radioactively tagged analyte oligonucleotides appeared as promising tools for the simultaneous analysis of multiple DNA sequences [5–7]. These array technologies have had a huge impact on genomics and pro- teomics applications, although they have shortcomings arising from, for example, limited tagging efficiency, hazardous waste disposal and complex multi-step analysis. In order to seek faster, sensitive and label-free DNA detection, a number of approaches have been suggested based on optical [8–11], acoustic [12] and electrochemical [13–15] techniques. Electrochemical DNA sensors are regarded as particularly suit- able for direct and fast biosensing since they can convert the hybridization event into a direct electrical signal [16–18]. This means that there is no need for complex signal transduction equipment and the detection can be accomplished with an inex- pensive electrochemical analyzer. Electrochemical DNA sensing approaches include the intrinsic electroactivity of DNA [19–22], electrochemistry of DNA-specific redox reporters [23,24], electro- chemistry of nanoparticles [25–27] and conducting polymers (CPs) [18,28]. Conducting polymers (CPs) are polyconjugated polymers with electronic properties resembling those of metals, while retaining properties of conventional organic polymers. Since the observation of the remarkably high electrical conductivity of a halogen-treated polyacetylene [29], a number of other conjugated polymers have been transformed from an insulating into a highly conductive state. The most widely investigated conducting polymers include poly- aniline, poly(phenylenevinylene), polypyrrole and polythiophene (Fig. 1 ). The award of the Nobel Prize in Chemistry in 2000 to H. Shirakawa, A. MacDiarmid and A. Heeger for their pioneering work on conducting polymers widely recognized the importance of these materials and has prompted even more vigorous research in the field. Compared to saturated polymers, CPs have a unique elec- tronic structure which is responsible for their electrical conduc- tivity, low ionization potentials and high electron affinity. For CPs in * Corresponding author. ** Corresponding author. MacDiarmid Institute for Advanced Materials and Nanotechnology, New Zealand. E-mail addresses: h.peng@auckland.ac.nz (Hui Peng), j.travs-sejdic@auckland. ac.nz (J. Travas-Sejdic). Contents lists available at ScienceDirect Biomaterials journal homepage: www.elsevier.com/locate/biomaterials ARTICLE IN PRESS 0142-9612/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2008.12.065 Biomaterials xxx (2009) 1–17 Please cite this article in press as: Hui Peng et al., Conducting polymers for electrochemical DNA sensing, Biomaterials (2009), doi:10.1016/ j.biomaterials.2008.12.065 the ground state (insulating or semiconducting state), p -bonds ( p – p *) are partially localized due to a phenomenon called the Peierls distortion [30]. During the doping process, the excitation across the p – p * band gap creates self-localized excitations of conjugated polymers with localized electronic states in the gap region [30]. These self-localized excitations are called polarons, bipolarons and solitons and underlay electrical conduction in CPs. The unique properties of CPs have led to a variety of applications for these materials, such as light emitting diodes (LEDs) [31], electrochromic materials [32], anti-static coatings [33], solar cells [34], batteries [35], anti-corrosion coatings [36], chemical sensors and biosensors [37] and drug release systems [38–40]. Conducting polymers can be synthesized chemically [41,42] and electrochemically [43,44]. In terms of biological applications, electrochemical polymerization is widely used because of several advantages: (i) it is performed at ambient temperatures and microelectrodes or electrodes with a large surface area can be used; (ii) the polymer film formed is confined to the electrode and its shape can thus be controlled by electrode design, while the thick- ness can be controlled in the nanometer to micrometer range; (iii) the properties of the CP film can be widely modulated by varying electrochemical polymerization conditions. Electrochemical poly- merization can be carried out potentiostatically, amperometrically or with potential scanning and the whole process may only take a few seconds [45]. During polymerization, the monomers are oxidized to form radical cations, followed by coupling reactions to form oligomers that eventually lead to deposition of the polymers on the electrode surface. More detailed descriptions of electro- chemical polymerization can be found elsewhere [41,46]. The electronic structure of CPs is highly sensitive to changes in the polymeric chain environment and other perturbations in the chain conformation caused by, for example, a biological recognition event such as DNA hybridization. The changes in the delocalized electronic structure or in other CP properties are manifested in altered optical and electrical properties, and, when measured, can provide a signal for the presence of a target analyte molecule [47]. These advantages of CPs make them suitable materials for chemical sensors and biosensors. An excellent review on chemical sensors based on CPs by Swager and collaborators [47] outlines numerous synthetic approaches towards the specific recognition probes attached to a conjugated polymer backbone. More recently, Bai et al. reviewed the application of CPs as gas sensors [48]. This review paper focuses on the applications of conducting polymers specifically in DNA sensing, with a special attention paid to current trends and applications developed recently in the field due to advances in nanotechnology. 2. Immobilization of DNA probes A typical configuration for DNA sensors based on CPs is shown in Fig. 2. Single-stranded DNA probes are immobilized on or within a conducting polymer layer. The target DNA is captured by base- pairing to generate a recognition signal, which is recorded through an electrode (gold, platinum, glassy carbon, etc.). Because the recognition event takes place at the CP/electrolyte interface and the recognition signal generated reaches the transducer through the CP layer, the properties of the CP and the orientation of the immobi- lized DNA probes on the CP are crucial to the sensor performance. The procedure of DNA probe immobilization should retain the probe’s affinity for complementary target DNA. Ideally, the orien- tation of probes should be predictable and readily accessible to the analyte DNA [49]. Generally, immobilization methods fall into the classes of electrochemical entrapment, covalent immobilization or affinity interactions. The electrochemical entrapment method originates from the pioneering work on enzyme sensors by Umana and Waller [50].It involves the electrochemical oxidation of a suitable monomer to the corresponding conducting polymer from a solution that contains oligonucleotide (ODN) probes. Wang et al. first illustrated that ODNs can act as the sole dopant during the growth of n N H n S n N N N H N H n n polyacetylene poly(aniline) poly(phenylene vinylene)polypyrrole polythiophene Fig. 1. Structures of some of the most common conducting polymers. target DNA immobilized DNA probe conducting polymer laye r transducer signal Fig. 2. General DNA sensor design based on CPs. H. Peng et al. / Biomaterials xxx (2009) 1–172 ARTICLE IN PRESS Please cite this article in press as: Hui Peng et al., Conducting polymers for electrochemical DNA sensing, Biomaterials (2009), doi:10.1016/ j.biomaterials.2008.12.065 polypyrrole films while maintaining their hybridization activity [51,52]. The significant advantage of this immobilization method is its simplicity. Potential drawbacks include the possibility of damaging ODN probes due to the high potentials employed during polymerization, and poor target accessibility to the incorporated probe in the bulk of the resulting film [49]. Covalent attachment can overcome the disadvantages of the electrochemical entrapment method and improve probe accessi- bility by target DNA [53]. Generally, ODN probes are functionalized with –NH 2 , –COOH, etc., and are then covalently attached to either a functionalized monomer or a functionalized polymer. The reac- tions used for the covalent immobilization are shown in Fig. 3. Livache et al. developed a process that utilizes pyrrole monomer bearing an ODN to copolymerize with pyrrole (Fig. 3a) [54,55] allowing the immobilization of multiple probes on electrode arrays. Later the same group used a similar process to prepare an ODN array consisting of a matrix of 48 addressable 50- m m microelectrodes that was applied to detect hepatitis C virus in blood samples [56]. In another approach for covalent ODN attachment a film is first electropolymerized from a solution containing func- tionalized monomers (either exclusively or in a mixture with non- functionalized monomers), followed by covalent attachment of a5 0 -end modified ODN onto CP functional groups. In this method, the conducting polymer films can be prepared under conditions that are potentially incompatible with the maintenance of ODNs, such as organic solvents and high polymerization potentials, while the subsequent attachment of the ODN probes is performed under mild conditions that ensure ODN integrity. Garnier et al. prepared a functionalized polypyrrole, poly(3-acetic acid pyrrole-co-3-N- hydroxyphthalimide pyrrole), which bears an easy leaving group, N-hydroxyphthalimide (Fig. 3b) [28,57]. In a further step, an amino- substituted ODN was then grafted onto this precursor copolymer by direct chemical substitution of N-hydroxyphthalimide. The authors concluded that 3-substitution is more favorable than N H 3 C O NH(CH 2 ) 6 NHOC(CH 2 ) 6 CONH(CH) 2 N O HO P OO O ODN + N H NH(CH 2 ) 6 NHOC(CH 2 ) 6 CONH(CH) 2 N N H 3 C O O HO P O O O ODN N H N H Electropolymerizaiton a N H N H + H 2 N ODN Electropolymerization b OH O O N O O O N H N H OH O O N O O O N H N H OH O O H N O N H O OH N H N H O OH N H + Electropolymerization ODN probe H 2 N EDC N H O HN N H c S S + O S N O O S S O S N O O S S O S Cl O O S S O S NH O O H 2 N ODN Electropolymerization 1. Cathodic cleavage 2. Chlorination d Fig. 3. Reactions for covalent immobilization of ODN probes. a, b, c and d are from Refs. [18,28,54] and [61], respectively. H. Peng et al. / Biomaterials xxx (2009) 1–17 3 ARTICLE IN PRESS Please cite this article in press as: Hui Peng et al., Conducting polymers for electrochemical DNA sensing, Biomaterials (2009), doi:10.1016/ j.biomaterials.2008.12.065 N-substitution with regard to maintaining the high intrinsic conductivity of the polymer. Peng et al. reported an acid func- tionalized polypyrrole, poly[pyrrole-co-4-(3-pyrrolyl) butanoic acid], where the amino-functionalized ODN was covalently attached to the polymer film using 1-ethyl-3-(3-dimethylamino- propyl) carbodiimide (EDC) as a catalyst (Fig. 3c) [18]. Thompson et al. proposed a different approach, in which a single-stranded ODN probe was linked to the conducting polymer by forming a bidentate complex between Mg 2þ and an alkyl phosphonic acid group on the polymer and the phosphate group of the ODN [58]. Recently Gautier et al. developed a process that involves electro- chemical copolymerization of 3-methylthiophene and 3-(oxy- alkyl)-thiophene bearing an arylsulfonamide group. The sulfonamide terminal functional group of the copolymer film was electrochemically cleaved and chemically modified with N-chlorosuccinimide (NCS) which led to a sulfonyl chloride func- tionality as a prerequisite for amino-ended single-stranded DNA probe immobilization (Fig. 3d) [59–61]. An advantage of this approach is the ability to electrochemically cleave immobilized ODN probes, which leads to the potential application for the immobilization of addressable multiple ODN probes on an elec- trode array. Due to this advantage, the research group developed an electrochemically controlled DNA delivery system by using similar procedure [62]. The immobilization of ODN probes on the CP film can also be achieved via an affinity interaction. The most common approach involves the avidin–biotin interaction which is extremely specific and the strongest known non-covalent biological bond (association constant K a ¼ 10 15 M ). This approach can be highly versatile due to the ability to anchor different biomolecules on the same support and the sensor can be regenerated by treatment with a detergent solution that breaks the avidin-biotin bridge but does not affect the support matrix. Cosnier and Lepellec first prepared poly(pyrrole- biotin) films for grafting glucose oxidase [63]. Later Dupont-Filliard et al. exploited this principle to reversibly immobilize ODN probes [64]. Briefly a biotinylated polypyrrole film was synthesized, fol- lowed by immobilization of avidin units by biotin-avidin interac- tion, and the film was exposed to biotinylated ODN probes, as shown in Fig. 4. The reversibility was achieved by a treatment in aqueous solution of sodium dodecylsulfate to cleave the avidin– biotin connection [64,65]. 3. Transduction mechanisms The transduction process converts the recognition event into a measurable signal. According to the quantity measured, the transducing mechanisms can be classified as electrochemical, optical, mass or thermal. Among these, electrochemical trans- duction is well suited to DNA sensing where a biorecognition event results in a direct electrical signal and the whole sensing system can be very effectively miniaturized. Indeed, portable systems for diagnosis and on-site environmental monitoring are now being developed [66]. For electrochemical DNA sensors based on CPs, the polymer is not only used as an immobilization matrix but also plays an active role in transduction. CPs can be reversibly doped and dedoped using electrochemical techniques, with doping and dedoping resulting in significant changes in electrical and spec- troscopic properties. These changes can be modulated by probe– analyte interactions [67,68], and then become an analytical signal for analyte detection that can be quantified as a change in current at a fixed applied potential (amperometry), a change in conductivity (conductometry), impedance (impedimetry) or potential (potentiometry). 3.1. Amperometric detection and cyclic voltammetry Amperometry is the most common approach used generally with biosensors based on CPs due to the simplicity of the method and fast response. In this approach, the performance of biosensors is governed by the efficacy of the electron transfer between the biomolecules, such as oxidoreductase, and the underlying electrode surface, involving the CP layer. In the case of DNA sensors, the efficacy of modulating the electrical conductivity and the redox properties of the CP by hybridization plays a key role. PPy/probe- modified electrodes, made by Wang et al. [51,69] using oligonu- cleotide probes as sole counter anions, displayed transient anodic peaks upon adding a complementary target, and opposite (cathodic) signals upon spiking of the solution with non- Fig. 4. Schematic of a sensor design based on electrocopolymerization of biotinylated pyrrole and the use of biotin–avidin interactions. Reprinted with permission from Ref. [64] with permission. Copyright Elsevier 2008. H. Peng et al. / Biomaterials xxx (2009) 1–174 ARTICLE IN PRESS Please cite this article in press as: Hui Peng et al., Conducting polymers for electrochemical DNA sensing, Biomaterials (2009), doi:10.1016/ j.biomaterials.2008.12.065 complementary strands. The direction of signals (relative to the base line) was dependent on the sequence of probe dopants and of the added target oligomers. The authors suggested that such a response reflected the change in the conductivity of the host PPy network caused by an increase in charge density in the case of a complementary target, or induced by electrostatic repulsion in the case of non-complementary sequences. Cyclic voltammetry is widely used with CP-based DNA sensors as the readout method, because the doping and dedoping processes monitored during potential scanning can be efficiently modulated by thehybridizationevent. Garnieret al.[28,70,71]illustrated thisby using polypyrrole grafted with single-stranded ODNs that was electropolymerized onto a Pt electrode. The cyclic voltammograms showed a significant decrease in the oxidation current and a positive shift in oxidation potential of the polymer film after hybridization with complementary ODN samples (Fig. 5). Such a change has been ascribed to the formation of bulky and rigid double-stranded DNA upon hybridization, thus increasing the energy required to planarize the polymer upon oxidation. The detection sensitivity increased with increasing length of the recognition base sequence, while the oxidation potential in cyclic voltammograms was shifted to even higher potentials. In similar studies, it was found that better sensi- tivities are achieved by using thinner sensor films due to the larger surface to volume ratio [18,71]. Furthermore, modulation of elec- trical properties of the conjugated backbone of CPs by single bases has been confirmed by Emge and Bauerle [72,73]. First, they func- tionalized bithiophene monomers with pyrimidine and triazine bases. After polymerization, the addition of small quantities of complementary bases strongly affected the electrochemical prop- erties of the obtained polymer, also resulting in the decrease of the oxidation currents and positive potential shift that described above. 3.2. Conductometric detection Conductometry measures the conductivity change of CPs arising between a pair of electrodes. Krishnamoorthy et al. electrochemically synthesized poly(3,4-ethylenedioxythiphene) microtubules in the presence of ssDNA using a polycarbonate membrane as a template, and developed a label-free conducto- metric DNA sensor based on these microtubules [74]. The conductivity measurements were done at a gate potential of þ0.8 V (vs. SCE) where the response was at its highest. The authors investigated the effect of ssDNA length on the sensor response. The results showed that the sensor response, as well as the linear range, increased with an increase in the ssDNA length. For the sensor based on ssDNA with 20 bases, the linear range was 8.0 Â 10 À8 to 1.0 Â 10 À5 gmL À1 and the detection limit was 8.0 Â 10 À8 gmL À1 . Ramanathan et al. prepared polypyrrole nanowires in the presence of avidin-conjugated ZnSe/CdSe quantum dots by electrodeposition within a channel between two electrodes on the surface of a silicon wafer [75]. After addition of biotin end-modified DNA, the resis- tance of the nanowires increased significantly. 3.3. Impedimetric detection Electrochemical impedance spectroscopy (EIS) has become a powerful tool in the study of corrosion, semiconductors, batteries and electro-organic synthesis because it provides kinetic and mechanistic information. In EIS experiments, a sinusoidally varying and interrogating voltage (typically in the range of 5–10 mV peak- to-peak) relative to a suitable reference electrode is applied to the working electrode and the resulting current response is measured. The real (Z re ) and imaginary (Z im ) components (or magnitude çZj and phase q ) of the impedance are calculated as the ratio between the system voltage phasor and the current phasor, which are generated by a frequency response analyzer during the experiment [16]. Compared to dc techniques, EIS offers the following advan- tages: (i) a very small excitation amplitude is used which causes only minimal perturbation of the electrochemical test system, reducing errors introduced by the measurement technique; (ii) EIS provides valuable mechanistic information, because electro- chemical impedance experiments obtain data on both electrode capacitance and charge-transfer kinetics. Furthermore, a purely electronic model consisting of a specific combination of resistors and capacitors can be used to represent the electrochemical system, and then the electrochemical system can be characterized by using established AC circuit theory in terms of equivalent circuits. The immobilization of DNA probes and the hybridization event not only induce changes in the intrinsic properties of the CP film but also bring about changes in the various interfacial film prop- erties, such as the capacitance and interfacial electron transfer resistance [17]. EIS can detect these changes, which can be exploited in the development of truly label-less biosensing. Indeed, EIS has been successfully employed in the label-free detection of DNA hybridization based on conducting polymers, although a full mechanistic understanding of the above-mentioned contributions to the overall EIS spectra change for a CP films has still to be developed. Peng et al. showed that there is a considerable differ- ence in the AC impedance spectra of a functionalized polypyrrole containing covalently grafted ODN probes before and after hybridization with different concentrations of complementary ODNs obtained in the presence of Fe(CNrad) 6 4À / Fe(CNrad) 6 3À at open circuit potential [18]. The spectra were modeled using a modified Randles equivalent circuit, but with a constant phase element (CPE) used instead of a double layer capacitance. The fitting results showed that the charge-transfer resistance increased with an increase in the concentration of complementary ODNs. The values of the heterogeneous standard charge-transfer rate constant (k a 0 ), which represents the kinetic facility of a redox couple, decreased with an increase in complementary ODN concentration, suggesting that the ODN duplexes form a barrier to ion movement Fig. 5. Cyclic voltammograms of poly(3-acetic acid pyrrole-co-3-ODN-acetamido pyrrole) deposited on a platinum electrode, after incubation in a buffered aqueous solution of (a) non-complementary ODN (b), increased concentration of complemen- tary ODN (c–e, 66, 165 and 500 nmol, respectively). The incubation solution was 5 mL. Reprinted with permission from Ref. [28]. Copyright Elsevier 20 08. H. Peng et al. / Biomaterials xxx (2009) 1–17 5 ARTICLE IN PRESS Please cite this article in press as: Hui Peng et al., Conducting polymers for electrochemical DNA sensing, Biomaterials (2009), doi:10.1016/ j.biomaterials.2008.12.065 and the redox couple (Fe(CNrad) 6 4À / Fe(CNrad) 6 3À ) was less acces- sible to the electrode due to electrostatic repulsion. Tlili et al. reported detection of DNA hybridization using non-Faradic elec- trochemical impedance spectroscopy [76]. The DNA probes were covalently attached to the precursor copolymer, poly(3-acetic acid pyrrole, 3-N-hydroxyphthalimide pyrrole) and the impedance measurements were performed without any redox species at a DC potential of À1.4 V. The authors chose this potential in order to minimize the Warburg impedance and to emphasize the contri- bution of the impedance at the PPy-DNA/electrolyte interface. At this potential, polypyrrole was in non-doped and semiconducting state; therefore, no parasitic electrochemical reaction occurred during the measurement. The resulting impedance spectra were fitted using a modified Randles equivalent circuit with a CPE in order to reflect the non-homogeneity of the layer. The results showed that the charge-transfer resistance decreased upon graft- ing of DNA probes and increased upon hybridization with complementary DNA. The authors explained these observations as being due to the effect of the negative charge of ss-DNA and ds-DNA and to their conformational structures. In the case of grafting of ss- DNA onto functionalized polypyrrole, which could be considered as a p-type semiconductor under the experimental conditions, the grafting of negatively charged ss-DNA led to an increase in the majority carrier density and a decrease in the resistance of space charge region. On the other hand, the ss-DNA is in a flexible random structure and can penetrate into the polymer pores to increase the ionic concentration in the polymer film. After hybridization, the ds- DNA is in a helical formation, resulting in significant stiffness of the functionalized polypyrrole that leads to a decrease in intrinsic conjugation of the polymer backbone and causes an increase in the charge-transfer resistance. Peng et al. suggested a DNA detection system in which DNA sample fragments were entrapped in the PPy film and CdS nano- particle labeled ODN probes were used to amplify the impedance change [77]. The equivalent circuit model consisted of a solution resistance (R s ), an element of resistance (R e ) in parallel with a capacitance (C e ), representing the Au electrode, and an element of interfacial charge-transfer resistance (R ct ) in series with a Warburg impedance (W). A constant phase element (CPE) (in parallel with W and R ct ) acts as a non-ideal capacitor. The experimental data was well fitted by this model and the fitting results showed an increase in charge transfer resistance upon binding of complementary CdS– ODN nanoparticle probes. Besides taking the change of charge- transfer resistance as the sensor response, the authors also suggested that the change in impedance at a fixed frequency could be used as the sensor response. In another study a terthiophene polymer bearing a carboxyl group was polymerized onto a glassy carbon electrode and used for the preparation of a DNA sensor [78]. The impedance spectra were recorded before and after hybridization at open circuit potential without any redox species. A decrease in total impedance was obtained after hybridization, and the highest differences in admittance (¼1/jimpedancej) were observed at w1 kHz. The mechanism for the decrease in the impedance was not investigated in detail, which the authors attributed to the higher conductivity of double stranded ODN compared with ss-ODN. Peng et al. reported the synthesis of a terthiophene bearing an unsaturated side chain and used the resulting polymer for DNA hybridization detection [79]. The impedance spectra were measured without a redox probe at 800 mV, a potential at which the polymer is in an oxidized state. The hybridization caused a decrease in the impedance, similar to Ref. [78]. Peng et al. further studied the mechanism using electro- chemical quartz crystal microbalance (EQCM) and the authors suggested that the dopants play an important role in the imped- ance change [80]. When CF 3 SO 3 À was used as the dopant (which provided the best sensor response), EQCM results illustrated that the dominant ion movement during the polymer redox processes is cation movement. Thus, the hybridization, which caused an increase in the negative charge due to the formation of duplex DNA, facilitated cation movement during the doping process, resulting in an increase in the conductance of the polymer film. Gautier et al. prepared a functionalized polythiophene matrix for label-free DNA hybridization detection by copolymerizing an arylsulfonamide modified thiophene with 3-methylthiophene [61,81]. The impedance spectra were recorded without any redox species at þ1.1 V. The hybridization caused a decrease in the impedance (Fig. 6A). An equivalent circuit was suggested to describe this system, as shown in Fig. 6B. The resistive component R e was attributed to the sum of the electrolyte resistance and the resistance of the electrode material. The first parallel element circuit (C 1 , R 1 ) and the second one (Q 2 (R 2 Q 1f )) are responsible for the semicircles observed in experimental data in the high frequency and low frequency domains, respectively. The fitting results showed that the resistance R 1 decreases while the capaci- tance C 1 did not significantly change upon hybridization. For the second circuit element (Q 2 (R 2 Q 1f )), R 2 also decreased while the pseudo-capacitance C 02 increased slightly. The authors suggested that the decreases of the resistive components under hybridization were caused by the formation of the double helix structure which liberates the surface from the random coil conformation of the ss- DNA and restores a partial anionic exchange at the interface between film and electrolyte. The increase in the density of nega- tive phosphate groups at the surface was responsible for the increase of the pseudo-capacitance C 02 . Fig. 6. A: Nyquist diagrams recorded at þ1.1 V vs. SCE from 100 kHz to 0.1 Hz with perturbation amplitude of 10 mV, onto a DNA probe modified-copolymer-coated Pt quartz crystal. The working electrode was dipped in a deaerated TE/0.6 M NaCl buffer solution (triangles), next in the same solution after exposure to the non-comple- mentary DNA sequence (squares) and finally after exposure to the fully complementary DNA target (circles). The filled symbols correspond to the experimental data and the empty to the calculated data. B: Equivalent circuit. Reprinted with permission from Ref. [61]. Copyright Elsevier 2008. H. Peng et al. / Biomaterials xxx (2009) 1–176 ARTICLE IN PRESS Please cite this article in press as: Hui Peng et al., Conducting polymers for electrochemical DNA sensing, Biomaterials (2009), doi:10.1016/ j.biomaterials.2008.12.065 More recently, the same group compared non-Faradaic imped- ance spectra with Faradaic impedance spectra for the same system to reveal different changes in the impedance modulus [82]. Generally, the Faradaic impedance measurement described the kinetics of electron transfer processes while the non-Faradaic impedance was related to alterations in the capacitance and molecular layer organization [16]. The authors found that the impedance features obtained by the Faradaic approach were dramatically different from those obtained using the non-Faradaic approach. For the non-Faradaic impedance, the hybridization caused a decrease in the semicircle diameter in Nyquist plots, while an increase in the semicircle diameter was observed in Faradaic measurements due to electrostatic repulsion between the negative charges of the redox probes and negatively charged DNA. Further- more, the effect of the length of target DNA has been investigated. Target ss-DNA with 675 bases that was much longer than the probe (42 bases) caused an increase in the impedance modulus, which was diametrically opposite to the change in non-Faradaic imped- ance observed upon hybridization with a 37-base DNA target. The authors ascribed to differences in the organization of the DNA modified layer after hybridization. When the probe and the target have the same length, the hybridization resulted in an opening of the interface to mobile ions in solution. When the target was much longer than the probe, the double helix extended into the solution by way of a flexible single-stranded DNA sequence, which pre- vented access of the anions and caused an increase in the impedance. 3.4. Photocurrent spectroscopic detection Photoelectrochemical techniques have been employed increas- ingly for investigating thin photoconducting films and corrosion layers on metals and alloys [83,84], because of the simplicity of the experimental setup, the possibility of monitoring in situ surface changes with time, and the ability to investigate very thin films. Recently this technique has been used for the detection of DNA hybridization [85–87] and damage [88,89]. Conducting polymers can exhibit a good photocurrent response, so this technique has great potential for label-free DNA hybridiza- tion detection. However, DNA sensing based on the photocurrent properties of conducting polymers has not been fully exploited. The initial work on photocurrent spectroscopy as a transduction tool for direct detection of DNA hybridization based on CPs was undertaken by Lassalle et al. [90–92]. The photocurrent response under white light illumination was analyzed for a polypyrrole copolymer with one monomer unit modified by grafted ODN, and the copolymer exposed to a blank buffer solution, and buffer solutions containing either complementary or non-complementary ODNs (Fig. 7). When exposed to the blank buffer, and af ter interaction with non- complementary ODN, the pyrrole-ODN showed a similar photo- current response, while the photocurrent was much lower following hybridization. The reason for the decrease of photocur- rent was not clear. It was suggested that it may originate from a different physiochemical behavior for the polymer film caused by formation of a DNA double helix. The band gap energies for direct electronic transition in these three cases were estimated, with values of 2.9, 2.85, and 3.1 eV for the polymer, the polymer after interaction with non-complementary ODN, and for the hybridized film, respectively. These values were higher than those obtained for polypyrrole (e.g. 2.2–2.6 eV), indicating a less photosensitive film due to the presence of oligonucleotides. The photocurrent evolu- tion during hybridization revealed that the kinetics of the process could be followed. However, these sensing films were not completely characterized in terms of sensitivity, reproducibility and the linearity of a calibration curve. 4. DNA sensors based on different classes of CPs 4.1. DNA sensors based on polypyrrole and its derivatives Polypyrrole is one of the most extensively used conducting polymers in biosensor designs due to its good biocompatibility and polymerization at neutral pH [93]. Wang et al. illustrated that short ss-ODN probes could be entrapped in a polypyrrole film as the dopant during film growth and still maintain an affinity for target ODNs [51,52]. Alocilja et al. reported a DNA sensor for rapid detection of Escherichia coli prepared using same methodology [94]. The recognition element was a 25-base pair oligonucleotide specific for E. coli derived from the uidA gene that codes for the enzyme b - D glucuronidase. A DNA concentration of 1 m g m L À1 was detected by cyclic voltammetry and the analysis was complete in 15 min. A pulsed amperometric detection of target DNA in PCR- amplified amplicons with platinum electrodes modified by single- stranded DNA (20 bases) entrapped within polypyrrole has also been reported [95]. The detection time was 30–35 min and the sensor response to a complementary target was higher than for a non-complementary target by a factor of at least 6–8. Komarova et al. prepared ODN-doped PPy sensor films to detect the pathogen Variola major by means of chronoamperometry [96]. It was estab- lished that thinner films with smaller or more highly concentrated dopant ions produced stronger amperometric signals. Blocking of the film surface with fragmented half thymus DNA resulted in complete disappearance of the non-specific signal when ultra-thin (Langmuir–Blodgett) films were tested, while the specific signal from complementary ODN remained unaffected. Additionally, lowering the potential during the hybridization reduced the non- specific signal. Under optimal conditions a detection limit of 1.6 fmol of target ODN in 0.1 mL (16 p M ) was achieved. For this type of DNA sensors, the obvious advantages are simplicity and fast detection, however, steric hindrance and poor accessibility of the probe entrapped within the film to the analyte result in poor hybridization efficiency that greatly limits sensitivity and selectivity. In order to overcome these disadvantages, a variety of func- tionalized pyrrole monomers have been developed and used for DNA detection, as shown in Fig. 8. Livache et al. developed a pyrrole monomer bearing an ODN (Fig. 8, monomer 1) and copolymerized this with pyrrole to realize addressable multiple probe immobili- zation [54,55]. The hybridization was detected by photocurrent Fig. 7. Normalized photocurrent spectra of copolymer film (:), of copolymer film in presence of a non-complementary oligonucleotide (A) and copolymer film in pres- ence of a complementary oligonucleotide (-). Reprinted with permission from Ref. [92]. Copyright Elsevier 2008. H. Peng et al. / Biomaterials xxx (2009) 1–17 7 ARTICLE IN PRESS Please cite this article in press as: Hui Peng et al., Conducting polymers for electrochemical DNA sensing, Biomaterials (2009), doi:10.1016/ j.biomaterials.2008.12.065 spectroscopy [92]. The same group also prepared a biotinylated pyrrole monomer (Fig. 8, monomer 2) [64]. The resulting bio- tinylated polypyrrole film could be used for reversible biotinylated ODN probe immobilization using the strong biotin/avidin interac- tion. Thompson et al. prepared a pyrrole monomer modified with a phosphonic acid group (Fig. 8, monomer 3) [58]. The DNA probe was immobilized onto the resulting polymer film with the help of magnesium cations that served as a bridge between the phos- phonic acid group of the grafted polymer and the phosphate group of the oligonucleotide probe. This type of linkage makes the oligonucleotide offset from the surface of the polymer, giving it some freedom of movement and easing the effect of steric hindrance on the hybridization event. The hybridization was detected using cyclic voltammetry, while the sensitivity was not investigated. The same group developed this system further at a microelectrode [97] and applied the sensor to hepatitis C virus detection [98]. Ionescu et al. used poly(pyrrole-NHS) (Fig. 8, monomer 4) to covalently anchor an amino-21-mer oligonucleotide probe for detecting a short cDNA sequence from the West Nile Virus (WNV) by an amperometric method [99]. After incubation with a target model of the WNV cDNA, the modified electrode was further incubated in a complementary biotinylated 15-mer WNV cDNA solution followed by specific attachment of a biotinylated glucose oxidase via an avidin bridge. The hybridization event was then monitored at 0.6 V vs. Ag/AgCl by amperometric detection of H 2 O 2 , generated by the enzyme marker in the presence of glucose. Since the product of the enzyme-catalyzed reaction was detected, the sensitivity of the sensor was related to the permeability of the (pyrrole-NHS) film. With the aim of increasing permeability, the polymer film was overoxidized until its conductivity significantly decreased. A relatively short hybridization period (2 h) allowed the convenient quantification of the WNV DNA target in the range of 10 À10 –10 À15 gmL À1 and a detection limit of 1 fg mL À1 . Because 3-substitution of pyrrole is more favorable than N- substitution, with regard to maintaining the high intrinsic conductivity of the polymer, Garnier et al. prepared a functional- ized pyrrole monomer bearing an easy leaving group, N-hydroxy- phthalimide (Fig. 8, monomer 5) [28,57]. An amino-substituted ODN was grafted onto the precursor copolymer, poly(3-acetic acid N NH N N O O O HO P OO O ODN N P O OH O Mg 2+ O P O HO O ODN N H O O N O O N H O OH N H HO O N H O OH 12 35 67 N (CH 2 ) 13 8 NH C (CH 2 ) 4 NH HN O O N H 9 HN O Fe O N O O O N 4 O O N O O S S Fig. 8. Structures of functionalized pyrrole monomers for DNA sensors. H. Peng et al. / Biomaterials xxx (2009) 1–178 ARTICLE IN PRESS Please cite this article in press as: Hui Peng et al., Conducting polymers for electrochemical DNA sensing, Biomaterials (2009), doi:10.1016/ j.biomaterials.2008.12.065 pyrrole-co-3-N-hydroxyphthalimide pyrrole), by a direct chemical substitution of the easy leaving group, N-hydroxyphthalimide (the sensor response to the target ODN is shown in Fig. 5). The detection limit was found to be 2 n M . A pyrrole monomer bearing a relatively long butanoic acid side chain (Fig. 8, monomer 7) reported by Peng et al., was expected to position the ODN probe away from the copolymer backbone and allows easy hybridization with comple- mentary ODN sequences [18]. In both of the above cases, the hybridization was detected by cyclic voltammetry and the sensi- tivities were related to the redox properties of polymers. In order to increase the sensitivity of DNA detection, a ferrocenyl-functional- ized pyrrole (Fig. 8, monomer 6) was prepared [100] and ODN probes were subsequently grafted onto the obtained ferrocenyl- functionalized polymer film. In this arrangement the ferrocenyl group on the polymer was used as a probe for DNA detection due to its high sensitivity to changes in the electronic and steric envi- ronments and narrow and reversible redox signature. Upon hybridization, a shift in the oxidation wave of the ferrocenyl groups to more positive potentials and a decrease in the oxidation current were observed. The results were explained by a decrease in the permeability of the polymer film to dopant ions and changes in the polymer backbone conformation. The estimated detection limit was 2 p M of the target ODN molecule. In that study, the electro- polymerization was carried out first in an organic solvent, followed by grafting of DNA probes in an aqueous medium. This strategy would be unsuitable for multiprobe addressing on a chip. Bouchet et al. developed a technique allowing one-step electro-addressing of probes on a microarray and sensitive and label-less multi- detection of DNA targets in solution [101]. Firstly, a pyrrole–ferro- cene derivative (Fig. 9, monomer 1) that could be electropolymerized in an aqueous medium was synthesized. The electrochemical copolymerization of this monomer with monomer 2, bearing different sequences of ODN, and monomer 3 was then carried out in a buffered solution using a miniaturized graphite electrode network, as shown in Fig. 9. The hybridization was detected by changes in the ferrocene oxidation current. Good selectivity between Human Immunodeficiency Virus and Hepatitis B Virus targets was achieved and the detection limit reached 100 p M . More recently, Peng et al. investigated the effect of the ‘linker’ group (a functionalized side chain that links the polymer backbone and the bioprobe) on the resulting sensor properties [102,103]. Pyrrole monomers with unsaturated side chains were synthesized (Fig. 8 , monomers 8 and 9). The motivation for using a conducting polymer with an unsaturated carbon side chain was based on the idea that extension of the main chain conjugation into the side chain may improve polymer susceptibility to changes caused by DNA hybridization. The results demonstrated that a longer side chain improves the sensor response. It was also shown that copolymers with unsaturated side-chain functionalization have superior properties for use in biosensor applications compared to those with a saturated side chain. The resulting sensor had good selectivity and a detection limit of 0.5 n M . Recent advances in nanotechnology have opened up new possibilities for DNA sensor design. The sensitivity and other attributes of sensor can be improved by using nanomaterials which have superior physical and chemical properties over their bulk counterparts, because of effects such as quantum confinement, a mini-size effect, surface effects and macro-quantum tunnelling effects. Until now, conducting polymer nanomaterials with different morphologies such as nanoparticles, nanowires and nanotubes have been prepared by chemical or electrochemical methods (see a recent review paper [104]). Ramanathan et al. [75] reported the preparation of biologically functionalized polypyrrole nanowires by electropolymerization from an aqueous solution of pyrrole monomer in the presence of a model biomolecule (avidin- or streptavidin-conjugated ZnSe/CdSe quantum dots) and within a 100 or 200 nm wide and 3 m m long channels between gold electrodes (Fig. 10). After addition of biotin–DNA, the avidin– and streptavidin–polypyrrole nanowires generated a rapid change in resistance to 1 n M of biotin–DNA. The authors suggested the possibility of single-molecule detection by adjusting the nanowire’s conductivity to a value closer to the lower end of the semi- conductor. This work illustrated the concept of biological modifi- cation of PPy nanowires, and the detection of complementary DNA targets based on this principle is currently underway in author’s laboratory. Cai et al. prepared polypyrrole and multi-walled carbon nanotubes functionalized with carboxylic group (MWNTs–COOH) nanocomposites for indicator free DNA hybridization detection [105]. Firstly, MWNTs–COOH were attached to the glassy carbon electrode and ODN probes were doped within PPy films electro- polymerized on top of this, and the ODN probes served as the sole counter anion during the growth of the film. After hybridization, a decrease in impedance was observed, attributed to a decrease in impedance owing to the higher conductivity of double stranded ODNs compared with the ssODN. The process of optimization of hybridization conditions revealed that the sensitivity of the sensor increased dramatically with an increase in the amount of multi- walled carbon nanotubes (MWNT) used. The response (difference in logarithmic impedance values) obtained was five times larger when the optimum amount of MWNTs was used (as compared to the sensor without MWNTs). The estimated detection limit for this simple method was 10 n M , which was further improved to 0.05 n M by the formation of metallized double-stranded DNA [106]. Fu et al. used Au–Ag nanocomposites that were adsorbed onto the PPy film by electrostatic interactions and mercapto ODN probes were self- assembled onto the surface of the modified electrode [107]. This sensor was applied to the detection of human immunodeficiency Fig. 9. Monomers involved in the co-electropolymerization onto microelectrodes. Reprinted with permission from Ref. [101]. Copyright Elsevier 2008. H. Peng et al. / Biomaterials xxx (2009) 1–17 9 ARTICLE IN PRESS Please cite this article in press as: Hui Peng et al., Conducting polymers for electrochemical DNA sensing, Biomaterials (2009), doi:10.1016/ j.biomaterials.2008.12.065 virus (HIV) sequences by electrochemical impedance spectroscopy and the detection limit reached 0.5 n M of target ODN. Table 1 summarizes characteristics of the electrochemical DNA sensors based on polypyrrole and its derivatives. 4.2. DNA sensors based on polyaniline and its derivatives Compared to polypyrrole, polyaniline is less widely employed in biosensor designs, due to the polymerization commonly requiring an acidic environment not suitable for biomolecules. However, polyaniline undergoes two redox processes, mechanically resilient and environmentally stable, and can be functionalized, making it an attractive material for DNA sensing applications. Gu et al. reported the immobilization of DNA onto a thin layer of polyaniline/poly (acrylic acid) (PANI/PAA) composite polymer film that was elec- trodeposited on boron-doped diamond (BDD) thin films [108]. The carboxylic acid residues in the polymer film acted as binding sites for DNA attachment, whilst the conductive polymer matrix enhanced electron-transfer between DNA and the diamond surface. The direct oxidation of guanine and adenine in the double helix DNA was used to detect the hybridization event. The advantage of such a design was a minimal non-specific DNA adsorption, which was confirmed by fluorescence microscopic and cyclic voltam- metric measurements. This PANI/PAA modified BOD electrode was further characterized by electrochemical impedance spectroscopy [109]. The impedance measurements showed changes in the impedance modulus as well as electron-transfer resistance upon probe DNA immobilization and after hybridization with target DNA. Good selectivity between the complementary DNA targets and the one-base mismatch targets was demonstrated, as well as sensor reusability. The detection limit was 20 n M measured at 1000 Hz. Davis et al. investigated the differences between polyethylenimine, polyaniline and polydiaminobenzene modified screen-printed carbon electrodes containing single-stranded DNA by an AC impedance approach [110]. Complementary DNA hybridization gave rise to a lowering of the capacitance of the electrode/polymer film in solution. More recently, Arora et al. reported an ultrasensitive DNA hybridization biosensor based on polyaniline electrochemically deposited onto a Pt disc electrode, as shown in Fig. 11 [111]. Acti- vated avidin was covalently attached to the PANI film, followed by the immobilization of a 5 0 -biotin end-labeled oligonucleotide probe via the biotin/avidin interaction. The hybridization was detected using both direct electrochemical oxidation of guanine and a redox electroactive indicator methylene blue. Compared to a direct elec- trochemical oxidation of guanine, hybridization detection using methylene blue resulted in an enhanced detection limit by about 100 times and reached 2 f M . This sensor has been utilized for direct detection of E. coli by immobilizing a 5 0 -biotin-labeled E. coli probe, and using differential pulse voltammetry in the presence of methylene blue as a DNA hybridization indicator [112]. The detec- tion limits for complementary target probe, E. coli genomic DNA and E. coli were 0.009 ng m L À1 , 0.01 ng m L À1 and 11 E. coli cells mL À1 without PCR amplification and it can be used 5–7 times at temperatures of 30–45  C. PANI nanocomposites have also been used in DNA sensing. Wu et al. synthesized a polyaniline intercalated graphite oxide nano- composite (PAI/GO) [113]. Square wave voltammetric measure- ments showed that single-strand DNA and double-strand DNA alter the redox characteristics of PAI/GO at the PAI/GO modified carbon paste electrode. The PAI/GO-modified electrode immobilized with ssDNA by physical adsorption can be utilized to monitor hybrid- ization of complementary ssDNA which resulted in a new peak at À0.27 V. A nanocomposite of polyaniline and mercaptosuccinic- acid-capped gold nanoparticles (MSAG NP ) was also prepared by the layer-by-layer methodology [114]. The MSAG NP inside multilayer films can effectively shift the electroactivity of polyaniline to a neutral pH which greatly facilitates its biological applications. After immobilization of DNA probes onto the gold nanoparticles, DNA hybridization was detected either by electrochemical methods (cyclic voltammetry and AC impedance) or by surface plasmon Fig. 10. (A) SEM image of an Aqd-embedded polypyrrole nanowire (200 nm wide). The EDX analysis of polypyrrole nanowire with embedded Aqd (B) and without embedded Aqd (C). Reprinted with permission from Ref. [75]. Copyright American Chemical Society 2008. H. Peng et al. / Biomaterials xxx (2009) 1–1710 ARTICLE IN PRESS Please cite this article in press as: Hui Peng et al., Conducting polymers for electrochemical DNA sensing, Biomaterials (2009), doi:10.1016/ j.biomaterials.2008.12.065 [...]... easily electropolymerized on a glassy carbon electrode [78] A DNA sensor based on this polymer has shown good specificity The sensor responses for centre one-base and end two-base mismatched DNA targets were only 14.3% that for the complementary target This polymer was further used as a matrix for hydrazine-catalyzed ultrasensitive detection of DNA and proteins [132] The detection limit for the DNA was estimated... electrochemical DNA biosensor with conducting polymer film and nanocomposite as matrices for the detection of the HIV DNA sequences Anal Lett 2006;39(3):467–82 [108] Gu H, Su X, Loh KP Conductive polymer- modified boron-doped diamond for DNA hybridization analysis Chem Phys Lett 2004;388(4–6):483–7 [109] Gu H, Su XD, Loh KP Electrochemical impedance sensing of DNA hybridization on conducting polymer film-modified... multidetection of DNA Biosens Bioelectron 2007;23(5):735–40 [102] Peng H, Soeller C, Vigar NA, Caprio V, Travas-Sejdic J Label-free detection of DNA hybridization based on a novel functionalized conducting polymer Biosens Bioelectron 2007;22(9–10):1868–73 [103] Peng H, Soeller C, Travas-Sejdic J Novel conducting polymers for DNA sensing Macromolecules 2007;40(4):909–14 [104] Jang J Conducting polymer nanomaterials... dye-labeled singlestranded DNA J Am Chem Soc 2007;129(11):3048–9 [11] Peng H, Soeller C, Travas-Sejdic J A novel cationic conjugated polymer for homogeneous fluorescence-based DNA detection Chem Commun 2006;(35):3735–7 [12] Okahata Y, Niikura K DNA sensor with quartz crystal microbalance (QCM) Denki Kagaku oyobi Kogyo Butsuri Kagaku 1998;66(1):7–13 [13] Wang J Electrochemical DNA biosensors Electroanalytical... after interaction with ss -DNA and ds -DNA [124–130] Generally, most thiophenes with functional groups such as –NH2 and –COOH, suitable for the immobilization of biomolecules, are difficult to electropolymerize, because such functional groups exhibit substantial nucleophilicity and attack the radical cation intermediates formed during electropolymerization, hence inhibiting the polymerization process [131]... Cai H, Xu Y, He P-G, Fang Y-Z Indicator free DNA hybridization detection by impedance measurement based on the DNA- doped conducting polymer film formed on the carbon nanotube modified electrode Electroanalysis 2003;15(23–24):1864–70 [106] Xu Y, Jiang Y, Cai H, He P-G, Fang Y-Z Electrochemical impedance detection of DNA hybridization based on the formation of M -DNA on polypyrrole/carbon nanotube modified... monomers for DNA sensors Please cite this article in press as: Hui Peng et al., Conducting polymers for electrochemical DNA sensing, Biomaterials (2009), doi:10.1016/ j.biomaterials.2008.12.065 ARTICLE IN PRESS 14 H Peng et al / Biomaterials xxx (2009) 1–17 Fig 14 Schematic description of the electrochemical detection of unlabelled DNA targets, (a) without and (b) with non-complementary ss -DNA Reprinted... detection of DNA hybridization based on polypyrrole/ss -DNA/ multi-wall carbon nanotubes paste electrode Talanta 2007;72(3):1030–5 [142] Xu Y, Ye X, Yang L, He P, Fang Y Impedance DNA biosensor using electropolymerized polypyrrole/multiwalled carbon nanotubes modified electrode Electroanalysis 2006;18(15):1471–8 [143] Livache T, Maillart E, Lassalle N, Mailley P, Corso B, Guedon P, et al Polypyrrole based DNA. .. materials for DNA sensing to date Table 3 summarizes the characteristics of the electrochemical DNA sensors based on polythiophene derivatives discussed in this section 5 Conclusions and future developments This review outlines advances in application of CPs to electrochemical DNA sensing The unique electronic structures of CPs make them highly suitable materials for electrochemical label-free DNA detection... DNA sensors based on polyaniline discussed in this section 4.3 DNA sensors based on polythiophene and its derivatives Numerous functionalized polythiophenes have been synthesized due to their favorable properties [122,123] Water-soluble cationic polythiophene derivatives have, in particular, been widely used in optical DNA detection schemes based on the principle of a conformational change in the polymer . 2008 Available online xxx Keywords: Conducting polymers Electrochemical DNA sensor Electropolymerization abstract Conducting polymers (CPs) are a class of polymeric materials that have attracted considerable. advances in electrochemical DNA sensing based on conducting polymers as active substrates. The various conducting polymers used for DNA detection, along with different DNA immo- bilization and detection. Immobilization of DNA probes A typical configuration for DNA sensors based on CPs is shown in Fig. 2. Single-stranded DNA probes are immobilized on or within a conducting polymer layer. The target DNA is