Molecularly Imprinted Polypyrrole for DNA Determination

Molecularly Imprinted Polypyrrole for DNA Determination

Vilma Ratautaite,a Seda Nur Topkaya,b Lina Mikoliunaite,a Mehmet Ozsoz,c Yasemin Oztekin,d, e

Almira Ramanaviciene,d Arunas Ramanavicius*a, f

a Department of Physical Chemistry, Faculty of Chemistry, Vilnius University, Vilnius, Lithuaniatel: +370(5) 2336310, +370(5) 2193115, +370(5) 2193185, fax: +370 (5) 2330987

b Department of Analytical Chemistry, Faculty of Pharmacy, Ege University, Bornova, Izmir, Turkeyc Department of Biomedical Engineering, Izmir Katip Celebi University, Izmir, Turkeyd NanoTechnas-Center of Nanotechnology and Material Science, Faculty of Chemistry, Vilnius University, Vilnius, Lithuaniae Department of Chemistry, Faculty of Science, Selcuk University, Konya, Turkeyf Laboratory of NanoBioTechnology, Department of Materials Science and Electronics, Institute of Semiconductor Physics, State

Scientific Research Institute Centre for Physical Sciences and Technology, Vilnius, Lithuania*e-mail: [email protected]

Received: February 2, 2013Accepted: February 6, 2013Published online: March 19, 2013

AbstractIn this study graphite electrodes modified by a thin DNA-imprinted polypyrrole layer, which was able to bind spe-cific target-DNA, are reported. For this aim, electrochemical synthesis of polypyrrole was performed on a pencilgraphite electrode by cyclic voltammetry (CV) or by potential pulse sequences (PPS). The modified electrode sur-face was used for electrochemical determination of target-DNA by differential pulse voltammetry. According to ourbest knowledge this is a first report on the application of DNA-imprinted polymer for the determination of target-DNA. The results showed that the molecularly imprinted polypyrrole (MIPPy) layer that formed on the carbonelectrode surface was sensitive for target-DNA, while the nonimprinted polypyrrole layer was not sensitive to thesame target-DNA. Comparison of electrodes modified using PPS and CV techniques is presented.

Keywords: Molecularly imprinted polymers (MIPs), Polypyrrole, DNA, Electrochemical DNA biosensor, DNA-imprinted polymer

DOI: 10.1002/elan.201300063

Supporting Information for this article is available on the WWW under http://dx.doi.org/10.1002/elan.201300063.

1 Introduction

Nanotechnology is rapidly evolving to open new combi-nation of methods, resolve challenging bioanalytical prob-lems, including specificity, stability and sensitivity. In-creasing attention has been paid to the preparation ofnew sensing surfaces. Carbon based electrodes are oftenused as substrates for such modifications due to their ad-vantages such as chemical inertness, broad potentialwindow, low residual current, low cost, easy modificationand great versatility. Various forms of carbon includingglassy carbon [1], pencil lead [2], pyrolytic graphite [3],carbon ceramics [4], impregnated graphite, carbon fibers[5], filaments, graphene [6], carbon nanotubes [7], carbonfilms and carbon paste have been used as electrode mate-rials suitable for various analytical purposes. To enhanceand/or extend application of any kind of carbon forms inelectrochemical detection systems, the modification ofcarbon surfaces have been applied. Among number ofmodification methods, electrochemical formation of con-ducting/nonconducting polymers is the subject of great in-terest.

The interest in molecularly imprinted polymers (MIPs)arises from their potential to recognize/bind selected mol-ecules. The principle of MIP-based technology was in-spired by mimicking of some native processes, e.g.: anti-gen-antibody interaction, which is based on selective rec-ognition. In the typical synthesis of the MIPs two maincompounds are involved: monomers and template mole-cules. The template molecules that are used for MIPspreparation usually are similar to the target molecules orin many cases they are the same type of molecules.Hence after polymerization three-dimensional polymernetworks with entrapped template molecules are formed.For different MIP applications, polymerization of one orcopolymerization of few different types of monomers hasbeen performed [8–11]. After polymerization, the tem-plate molecules are removed, leaving cavities, which arecomplementary to the target molecules, therefore theycan be involved in a selective recognition of target mole-cules.

For development of sensors such conducting polymersas polypyrrole (PPy), polyaniline and others have beenused. Among other conducting polymers PPy is prefera-ble for sensor development because of some advantages

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including rapid electrochemical response, low cost, widedynamic range, low detection limits, and operation atphysiological pH [12]. The application of MIP modifiedpolypyrrole for the determination of low molecularweight molecules including caffeine [9,13], ascorbic acid[8, 14], paracetamol [15], tripeptide glutathione (GSH)[16] was performed. Moreover high molecular weightmolecules including glycolproteins [17] and enzymes [18]were also imprinted in polymeric structures.

Electrochemical sensors for target-DNA detection areapplied in various practical applications including envi-ronmental monitoring [19], evaluation of polymorphism[20], detection of specific pathogens [21] or toxic agentslike herbicide – atrazine [22,23]. Chronopotentiometricand voltammetric methods are applied in electrochemicalDNA sensors [21,24, 25]. Early works on electrochemicalDNA sensors have been mainly based on the applicationof electrochemical labels suitable for the indication of im-mobilized single stranded DNA (ssDNA) interaction withtarget-DNA, which is present in the sample. Other appli-cations of PPy for DNA determination involved thedirect electrochemical detection, which is carried out bymonitoring of target-DNA hybridization induced conduc-tivity changes of DNA/PPy composite [26]. A label-freeelectrochemical detection of DNA hybridization based onelectrostatic modulation of the electrochemically drivenchloride ion-exchange of modified polypyrrole-bilayerfilms deposited at microelectrodes has been also reported[27].

The above described methods for the target-DNA anal-ysis are based on the immobilization of biomolecules onthe conducting polymer surface. However such sensorshave limited applicability due to poor chemical and physi-cal stability of immobilized ssDNA even if it is efficientlyimmobilized. Therefore molecularly imprinted polymersand artificial receptor based sensors gained the impor-tance as a possible alternative to other affinity sensors(e.g. immunosensors, DNA sensors, etc.), which are basedon immobilized biomolecules.

It has been demonstrated that the influence of the nu-cleic acid, which is applied as a dopant, upon the redoxactivity of the resulting PPy-modified electrode impartsa high degree of discrimination between synthetic oligo-nucleotides and chromosomal DNA [28]. The behavior ofPPy films, prepared in the oligonucleotide- and DNA-based mixtures has been mostly influenced by smaller oli-gonucleotides. Hence the recognition of oligonucleotidesin such cases has been a result of discrimination of oligo-nucleotides and DNA molecules by size. Application ofpolypyrrole as a linker between a substrate and oligonu-cleotide probes has been demonstrated by Szunerits et al.[29]. The modification step has been based on the elec-trochemical copolymerization of pyrrole and oligonucleo-tides bearing a pyrrole group on its 5’ end. This strategyhas been employed for the immobilization of oligonucleo-tides on millimeter-sized electrodes, microelectrodearrays, as well as for the local structuring of homogeneousgold surfaces [29]. It has been found, that localized im-

mobilization could be achieved by an electrospottingtechnique, where a micropipette has been served as anelectrochemical cell. In this case formation of PPy wasperformed by potential cycling. This electrochemical pro-cedure allowed the formation of a copolymer, which wasa mixture of polypyrrole and polypyrrole bearing cova-lently linked oligonucleotides [29]. The study, which de-scribes an electrochemical DNA sensor based on polypyr-role and 3-pyrrolylacrylic acid (PAA) copolymer followedby electrochemical impedance spectroscopy based evalua-tion redox couple, has been reported [30]. Other studydescribes the application of poly[pyrrole-co-(N-pyrrolyl)-caproic acid] copolymer for detection of target-DNA bycyclic voltammetry [31]. However, no reports on applica-tion of polymers with DNA molecular imprints were pub-lished up to now.

The aim of this study was to perform electrochemicalpolymerization of molecularly imprinted polypyrrole(MIPPy) with template-DNA. The comparison of twoelectrochemical polymerization methods (electrochemicalsynthesis by sequence of potential pulses and by potentialcycling) was performed. Nonimprinted polymer (NIP) orMIP modified pencil graphite electrodes were investigat-ed using differential pulse voltammetry (DPV). Differen-ces of guanine oxidation peaks were evaluated as analyti-cal signals.

2 Experimental

2.1 Apparatus and Chemicals

Detection of target-DNA was performed by differentialpulse voltammetry using an AUTOLAB PGSTAT 30electrochemical analysis system Eco Chemie (Utrecht,The Netherlands). The three electrode system applied forelectrochemical measurements consisted from the dispos-able pencil graphite electrode as working electrode, Ag/AgCl/KClsat. as a reference electrode and a platinum wireas an auxiliary electrode. Electrical contact for graphiteleads holder with the lead was obtained by solderinga metallic wire to the metallic part [20].

All chemicals used for experiments were purchasedfrom Sigma Ltd and were of analytical grade. �Reagentgrade quality� pyrrole purchased from Aldrich (Germany)was used for polymer formation. Fish sperm doublestranded DNA purchased from Sigma-Aldrich (Stein-heim, Germany) was used as template-DNA duringMIPPy preparation and as target-DNA during the evalua-tion of specific or nonspecific interaction. Stock solutionsof DNA (1000 ppm) were prepared with ultra pure waterand they were kept frozen at �4 8C. More diluted solu-tions of DNA were prepared using 0.5 M acetate buffer(AcB), pH 4.8, containing 20 mM of NaCl. Pyrrole solu-tions were prepared in 50 mM phosphate buffer (PBS),pH 7.4, with 20 mM of NaCl. All experiments were con-ducted at room temperature (25 8C).

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2.2 Preparation of Electrodes

Pencil graphite electrodes (PGEs) of 0.5 mm diameterwere purchased from Tombo Ltd (Japan) and were usedas disposable electrodes. PGEs of 3.0 cm length were pre-pared as previously described in literature [32]. Theworking area of the electrodes during measurements was20.6 mm2. PGEs were pretreated applying constant poten-tial (+1.40 V) for 30 s in AcB, pH 4.8, while the solutionin electrochemical cell was mixed.

2.3 Modification of Electrodes by Nonimprinted Polymerand Molecularly Imprinted Polymer

Electrochemical modification of pencil graphite electro-des with polypyrrole (PPy) layer molecularly imprintedwith template-DNA was performed by potential pulse se-quences (PPS) and cyclic voltammetry (CV). Similarlywere prepared two types of nonimprinted PPy basedpencil graphite electrodes.

Electrochemical synthesis by means of potential pulsesequence (PPS) was carried out as previously describedby Ramanaviciene et al. [13]. During this procedure thePPy film was formed applying 25 potential pulses. Thepulse potential profile was: +1000 mV vs. Ag/AgCl/KClsat

for 1 s (synthesis of PPy was performed at this stage) and0 mV vs. Ag/AgCl/KClsat for 10 s (the pyrrole monomerequilibration in the neighborhood of the electrode oc-curred at this stage). Both nonimprinted polypyrrole(NIPPy-PPS) and molecularly imprinted polypyrrole(MIPPy-PPS) were deposited on the electrode using thesame potential pulse profile. NIPPy-PPS was depositedfrom 50 mM solution of pyrrole in PBS, while MIPPy-PPS was deposited from 50 mM of pyrrole and 5 ppm oftemplate-DNA dissolved in PBS. After the deposition ofNIPPy-PPS and MIPPy-PPS the electrodes were washedunder stirring for 5 min in PBS in order to remove notpolymerized compounds. The template-DNA from MIP-modified electrode was removed by washing out with in-tensively stirred PBS for 30 min.

Electrochemical synthesis by means of cyclic voltam-metry was carried out as described by Ozcan and Sahin[15]. Thus electrochemical formation of polymer was per-formed by 10 potential cycles in the range from �0.6 V to+0.8 V vs. Ag/AgCl/KClsat, at the sweep rate of 100 mV/s.Nonimprinted polypyrrole (NIPPy-CV) was depositedfrom 50 mM solution of pyrrole and molecularly imprint-ed polypyrrole (MIPPy-CV) was deposited from 50 mMof pyrrole solution containing 5 ppm of template-DNA inAcB, pH 4.8. Differently from synthesis conditions, whichhas been described by Ozcan and Sahin [15], the uppervertex potential was reduced down to +800 mV becauseof possible guanine oxidation at +1000 mV.

After the polymerization in order to remove the tem-plate-DNA from polymer, MIPPy-modified electrodeswere washed by intensively stirred PBS for 30 min. Toreduce pretreatment/washing effects on analytical signals,

NIPPy-based electrode was pretreated/washed in thesame manner as MIPPy-based one.

2.4 Evaluation of Target-DNA Interaction with NIPPyand MIPPy

In order to evaluate the specific interaction of target-DNA with MIPPy and nonspecific interaction withNIPPy, all these layers-based electrodes were divided intwo groups containing 10 electrodes modified by MIPPyand 10 electrodes modified by NIPPy each. The first fiveelectrodes modified by NIPPy after washing were left forfurther investigation without any additional incubationand it was estimated as a �blank� polymer, which is re-quired in order to obtain baseline for differential pulsevoltammetry. Second group of five electrodes modified byNIPPy were used for the investigation of target-DNAcontaining solution. The same was done using electrodeswith MIPPy, for this five electrodes with MIPPy were notincubated in target-DNA containing sample and analyzedby DPV, the last five electrodes were analyzed by DPVafter the incubation in the sample containing target-DNA.

In order to evaluate specific and nonspecific interactionof target-DNA with NIPPy or MIPPy, the electrodesmodified by NIPPy and MIPPy were incubated for30 min in the sample containing 5 ppm of target-DNAdissolved in AcB, pH 4.8. Specific and nonspecific interac-tions with target-DNA occurred after incubation in ana-lyte containing aliquot. After the incubation the electro-des were washed by dipping for 3 times into the AcB,pH 4.8, for 30 s. DI/DE for MIPPy (or NIPPy) represent-ed in figure 4 was calculated using the Equation 1:

DI=DE ¼ DI 0=DE0�DI 00=DE00 ð1Þ

where:

DI – peak current evaluated by DPV of MIPPy (orNIPPy);

DE – peak voltage in DPV of MIPPy (or NIPPy);DI’ – peak current registered by DPV of MIPPy (or

NIPPy) incubated in target-DNA;DE’ – peak voltage in DPV of MIPPy (or NIPPy) incu-

bated in target-DNA containing sample;DI’’ – peak current registered by DPV of MIPPy (or

NIPPy) not incubated in target-DNA containing sample;DE’’ – peak voltage in DPV of MIPPy (or NIPPy) not

incubated in target-DNA containing sample.

2.5 Electrochemical Measurements and Assessment ofthe Results

The oxidation signal of guanine was registered by DPV inthe blank AcB, pH 4.8, at 50 mV modulation amplitudewith an initial potential of 0 V and end potential of+1400 mV applying 8 mV step [32].

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2.6 Evaluation of Surface Dominant Features withAtomic Force Microscopy (AFM)

Surface dominant features were measured using atomicforce microscope �Catalyst� from Bruker Ltd, (Santa Bar-bara, USA). The topography images were obtained usingcontact mode with soft silicon nitride tip covered with re-flective gold coating, in air with a 18 mm scanned area.The bending spring constant of the tip was 0.06 N/m, thescanning rate was 2 lines/s. All images were processedusing “flatten” procedure of the software NanoScope 8.0provided by Bruker Ltd, (Santa Barbara, USA). Theheight of dominant features of the MIPPy and NIPPycovered electrode surface was evaluated using �Depth�function, which represent the height difference of twodominant features that occur at distinct heights.

3 Results and Discussion

Electrochemical polymerization of PPy was performed inorder to modify electrodes by NIPPy and MIPPy. Duringthe formation of the MIPPy layer the polymerization wascarried out in 50 mM of pyrrole with 5 ppm template-DNA. Figure 1 represents the procedures applied in thisstudy.

The polymerization mixture was produced by thoroughmixing of the monomer with the template-DNA at a cer-tain ratio. During the electrochemical polymerization ofPPy the template-DNA molecules were captured in thematrix of PPy. After electrochemical polymerization thewashing procedure was performed in order to ensure thecomplete extraction of template-DNA from PPy matrixwhile forming cavities complementary to target-DNA. It

Fig. 1. Schematic presentation of procedures and manipulations applied for MIP formation and target-DNA detection. A) Activationof PGE, at a potential of +1.4 V. B) Electrochemical polymerization of MIPPy and NIPPy. C) Extraction of template-DNA fromMIPPy and incubation in sample. D) Detection of target-DNA by oxidation of guanine by DPV.




should be noted that the template-DNA used during elec-trochemical polymerization process is identical to thetarget-DNA.

In Figure 2 the electrochemical formation of the poly-mer on the working electrode is represented. During elec-trochemical formation of NIPPy-PPS and MIPPy-PPS itwas detected that the current registered during polymerformation has slowly decreased (Figure 2A). One of themost plausible explanations of this effect is related to theconductivity difference of blank PGE and PGE modifiedby NIPPy-PPS and MIPPy-PPS. Electrochemical forma-tion of NIPPy-CV- and NIPPy-CV-based layers was per-formed by potential cycling (Figure 2B). During electro-chemical formation of MIPPy-CV and NIPPy-CV theCVs obtained were very similar (data not shown) in theseCVs two specific oxidation and reduction peaks (at 0.2 Vand �0.15 V, respectively) were observed. This effect of

polymer formation is in agreement with cyclic voltammo-grams presented in other studies [15,33].

DPV was selected as one of the most suitable methodsfor the detection of target-DNA. Guanine and adeninebases are the most important for DPV-based determina-tion of target-DNA, because oxidation of guanine inAcB, pH 4.8, occurs at +1000 mV potential and adenineat +1250 mV vs. Ag/AgCl/KClsat. (Figure 1) [31]. More-over it was determined that oxidation of guanine can beaffected by the pH of the selected buffer [34]. Thereforethe evaluation of guanine oxidation was performed onlyin AcB, pH 4.8. The oxidation of guanine is clearly de-tectable in voltammograms obtained by anodic stripping,therefore the evaluation of the concentration of target-DNA specifically bounded to MIPPy-CV and/or nonspe-cifically adsorbed on the electrode surface could be per-formed by estimation of these peaks. If MIPPy-CV elec-trode after the extraction of template-DNA was incubat-ed in target-DNA containing solution and then evaluatedby DPV the typical DPV-peak, which is attributed to gua-nine oxidation, was observed. In this study during the de-termination of target-DNA only guanine oxidation peakcurrent was estimated.

The results of the previous studies showed that theelectrochemical synthesis method is important for theproperties of the formed PPy. In order to evaluate the in-fluence of the PPy formation method on the performanceof MIPPy-based electrodes different types of electrodeswere designed. Potential pulse sequences were appliedfor the deposition the NIPPy-PPS and MIPPy-PPS layerson the electrode surface. The selection of the PPSmethod for MIP preparation was based on several of ourprevious studies [13, 17]. In one of these studies it hasbeen shown that the PPS method is more suitable for thedesign of molecularly imprinted PPy because suchMIPPy-PPS film possess better electrochemical activity,which is determined by their high specific surface area[12]. It should be noted that the comparison of potentio-static and potentiodynamic electrochemical PPy synthesesperformed at the disposable graphite electrode haveshowed that depending on the preparation method theguanine oxidation potential can be shifted [35]. The diffu-sion of monomers in the polymerization solution is anoth-er important issue; the PPS in this respect is the mostsuitable polymerization method because it allows veryeasy adaptation of polymerization periods and durationof monomer equilibration by adjustment of the appliedpulse profile [13]. Therefore in this study electrochemicalpolymerization by means of PPS polymerization methodwas performed by consecutive pulses of: (i) higher volt-age (of 1000 mV), which is suitable for pyrrole polymeri-zation, for 1 s and (ii) lower voltage (of 0 mV) for 10 s,expecting that monomers and molecules of the templatewill equilibrate at the electrode surface during this frameof time. Hence due to the applied PPS-based Ppy forma-tion method the template-DNA molecules are equallydistributed within the polymer matrix.

Fig. 2. Amperometric signals registered during: A) electro-chemical formation of NIPPy-PPS and MIPPy-PPS by means ofpotential pulse sequence (PPS); B) electrochemical formation ofNIPPy-CV by means of cyclic voltammetry (CV).




Four groups of disposable pencil graphite electrodes(based on NIPPy-PPS, MIPPy-PPS, NIPPy-CV andMIPPy-CV layers) were prepared. All of them after poly-merization were pretreated using the same standardizedwashing procedure. In order to demonstrate that the re-moval of the target-DNA from the polymer by the ap-plied MIPPy regeneration procedure is effective, twogroups of NIPPy and MIPPy based electrodes were evalu-ated. One group of electrodes was incubated in target-DNA containing the sample and analyzed by DPV. Thesecond group of electrodes was analyzed by DPV omit-ting the incubation step. In this way it was confirmed thattemplate-DNA molecules were effectively washed outfrom the polymer surface during MIP preparation step.Figure 4 demonstrates the differences between DPV sig-nals of incubated and not-incubated in target-DNA con-taining sample obtained by NIPPy and MIPPy. In thisway possible interfering effect of not removed template-DNA on DPV signal was eliminated. Figure 4A illustratesthat the target-DNA was not adsorbed on NIPPy-PPS-based electrode and in opposite – it was adsorbed onMIPPy-PPS-based electrode, because oxidation peak ofguanine was registered. As follows from studies evaluat-ing copolymers of polypyrrole, 3-pyrrolylacrylic acid [30]and poly[pyrrole-co-(N-pyrrolyl)-caproic acid] [31] somenonspecific adsorption of target-DNA can occur directlyon the polymer surface. Therefore in our study method ofsubsequent comparison of not incubated and incubatedNIPPy was applied and in the same way the comparisonof not incubated and incubated MIPPy eliminates anypossible distortion of results by nonspecifically adsorbedtarget-DNA.

The MIPPy formation and the principle of functioningare explained by three dimensional polymer matrix for-mation, which is complementary to the imprinted tem-plate molecule [15]. The complementary cavities areformed within the polymer during chain branching andcross-linking processes in the presence of the template-DNA molecule. As the >N�H group of the pyrrole unitcan form the hydrogen bond with the >C=O groups ofDNA nucleobases, the cavities formed are geometricallyand functionally complementary to the structure of thetarget-DNA [13–17]. In contrast to some other studies, inpulsed amperometric polymerization we applied theupper potential equal to 1000 mV, which is similar to theguanine oxidation potential [12]. It means that during thepolymerization process guanine oxidation could occur. Asshown in Figure 4A the guanine oxidation peak registeredby the MIPPy-PPS-based electrode was higher than thatobserved by the NIPPy-PPS-based electrode. Thereforewe conclude that guanine oxidation during the polymeri-zation process had no negative effect on the three dimen-sional polymer matrix formation and cavities, which arecomplementary to the shape of the template-DNA mole-cule and to the location of functional groups, wereformed in the PPy film. For the formation of NIPPy-PPStemplate-DNA was not used therefore no complementarycavities were formed. For this reason the target-DNA is

adsorbing on NIPPy-PPS only nonspecifically and just atrelatively low quantities. Therefore no peak of guanineoxidation on the NIPPy-PPS was observed.

During the formation of NIPPy-CV- and MIPPy-CV-based layers the electrode potential was cycled in therange from �600 mV to +800 mV. The main reason forthe selection of relatively low oxidation vertex potential(+800 mV) was based on the hypothesis that during the

Fig. 3. Comparison of 10th cycles of CV�s obtained during elec-trochemical formation of NIPPy-CV and MIPPy-CV.

Fig. 4. Differences in differential pulse voltammograms ofNIPPy and MIPPy modified electrodes (presented differenceswere calculated between analytical signals before and after incu-bation). A: NIPPy-PPS and MIPPy-PPS; B: NIPPy-CV andMIPPy-CV. 1) at NIPPy surface, 2) at MIPPy surface, G: guanineoxidation peak at 1000 mV potential. 5 ppm of target-DNA wasdissolved in 50 mM phosphate buffer, pH 7.4, with 20 mM ofNaCl. DPV was carried out in 0.5 M acetate buffer solution,pH 4.8, with 20 mM of NaCl.




preparation of MIP the target-DNA can be oxidized ifa higher potential would be applied. Comparison of 10th

cycles of cyclic voltammograms obtained during electro-chemical formation of NIPPy-CV and MIPPy-CV weresimilar (Figure 3). Therefore the similarity of both cyclicvoltammograms can be exploited as evidence illustrating

that guanine was not oxidized during the electrochemicalformation of MIPP-CV. Evaluation of NIPPy-CV- andMIPPy-CV-based electrodes demonstrated that no non-specific target-DNA adsorption on NIPPy-CV-based elec-trode was detected and significant specific interaction

Fig. 5. The AFM images of: A) MIPPy-PPS and B) MIPPy-CV. MIPPy-PPS was formed applying potential pulse sequence andMIPPy-CV, using cyclic voltammetry.




with target-DNA was registered by the MIPPy-CV-basedelectrode (Figure 4B).

Some researchers reported that potential cycling isa very efficient method for the formation of molecularlyimprinted PPy [14,15]. However results presented in ourstudy (Figure 4A,B) showed that pulsed amperometry ismore efficient for the formation of MIPPy-PPS films.

In the case of PPS-based synthesis, the polymerizationprocess consists of two major steps: (i) the polymerizationof the monomer occurs during the higher potential phase(at 1000 mV) and (ii) during low potential phase (at0 mV) monomer and target molecule concentrations be-comes equilibrated close to the surface of the electrode.In the case of electrochemical synthesis by means ofcyclic voltammetry the potential is linearly changedversus time and at higher potentials the polymerization ofthe monomer is more intensive. In this case due to thecontinuous polymerization process equilibration of tem-plate and monomer concentrations was not possible.Hence during the preparation of MIPPy template-DNAmolecules are entrapped within the three-dimensionalpolymeric matrix. But the linear decrease of the potentialis influenced by some uncertainty of the slow diffusion oftemplate-DNA molecules in polymerization bulk solu-tion. Consequently in the case of potential cycling, whichwas applied as an electrochemical polymerizationmethod, some difficulties related to the formation of thepolymer matrix can arise. Next factor influencing thetemplate molecule distribution in the final polymer struc-ture is the ratio of monomer and template molecules. Inthis study the concentration of the monomer was 50 mMwhile the concentration of template-DNA was 5 ppm.Other studies have reported that the ratio of monomerand template molecule can vary from 2.5 :1 to 25 :1 [13–15]. Hence the ratio of template and monomer used forthe preparation of MIPPy layer was significantly higher.Taking into account the high molecular weights of tem-plate-DNA molecules and slow macromolecular diffusionrates, the electrochemical formation of MIPPy layer bymeans of potential pulse sequence is more efficient in thecomparison to cyclic voltammetry; because during thelow potential phase, which is 10 times longer than thehigh potential polymerization phase, the equilibration oftemplate-DNA concentration close to electrode surface ismore efficient. The presented results of this study confirmthat if electrodes are modified using the PPS method(Figure 4) the specific interaction of target-DNA is stron-ger on MIPPy-PPS- than on MIPPy-CV-based films.

AFM was used to evaluate the surface structure of theobtained PPy-modified electrodes (Figure 5). The AFMimaging provides useful information about the polymersurface topography and the structure features. The AFMimage illustrated the spheres-based structure of the PPy.It was found that approximately 95 % of the formedMIPPy-PPS structures were from 0.2 to 0.9 mm height andthe most of dominant surface features were of 0.55 mmheight (Figure 6). The evaluation of features in MIPPy-CV showed that height describing values were shifted and

this shift was in the range from 0.4 to 1.1 mm. Also it wasfound that the most of dominant surface features were of0.74 mm height. In some studies nucleation mechanismand elongation of polymer during electrochemical poly-merization was evaluated, which is in agreement withhere presented AFM features [36,37].

4 Conclusions and Future Developments

In this study electrochemical synthesis of MIPPy withtemplate-DNA was performed. Comparison of MIPPy,which was formed using two different electrochemicalmethods ((i) by potential pulse sequence and (ii) by po-tential cycling electrochemical synthesis) showed thatMIPPy formed by both methods suitable for target-DNAdetermination in the sample. The obtained results illus-trated that potential pulse sequence based electrochemi-cal polymerization was more suitable for MIPPy forma-tion in comparison with polymerization performed by thepotential cycling based method. Adsorption of target-DNA on the NIPPy was lower or unnoticeable if com-pared to interaction with MIPPy. The presented resultsindicate that the oxidation of guanine during MIP forma-tion is not a crucial factor and MIP is successfully formedeven if the upper vertex potential of potential cycling isvarying within a range of 800–1000 mV vs. Ag/AgCl/KClsat. It should be noted that the sequence specificity ofthe formed MIPPy was not evaluated and this issue needssome further investigations.

Acknowledgement

Financial support from postdoctoral fellowship funded bythe European Union Structural Funds Project “Postdoc-toral Fellowship Implementation in Lithuania” within theframework of the Measure for Enhancing Mobility ofScholars and Other Researchers and the Promotion ofStudent Research (VP1-3.1-SMM-01) of the Program of

Fig. 6. MIPPy-PPS and MIPPy-CV surface roughness distribu-tion measured by AFM.




Human Resources Development Action Plan is acknowl-edged.

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