Impedance DNA Biosensor Using Electropolymerized Polypyrrole/Multiwalled Carbon Nanotubes Modified Electrode

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Impedance DNA Biosensor Using ElectropolymerizedPolypyrrole/Multiwalled Carbon Nanotubes Modified ElectrodeYing Xu, Xiaoyan Ye, Lin Yang, Pingang He,* Yuzhi Fang*

Department of Chemistry, East China Normal University, Shanghai, 200062, P. R. China*e-mail: [email protected]; [email protected]

Received: March 20, 2006Accepted: May 3, 2006

AbstractIn this paper, we present an electrochemical impedance-based DNA biosensor by using a composite material ofpolypyrrole (PPy) and multiwalled carbon nanotubes (MWNTs) to modify glassy carbon electrode (GCE). Thepolymer film was electropolymerized onto GCE by cyclic voltammetry (CV) in the presence of carboxylic groupsended MWNTs (MWNTs-COOH). Such electrode modification method is new for DNA hybridization sensor. Aminogroup ended single-stranded DNA (NH2-ssDNA) probe was linked onto the PPy/MWNTs-COOH/GCE by usingEDAC, a widely used water-soluble carbodiimide for crosslinking amine and carboxylic acid group. The hybridizationreaction of this ssDNA/PPy/MWNTs-COOH/GCE resulted in a decreased impedance, which was attributed to thelower electronic transfer resistance of double-stranded DNA than single-stranded DNA. As the result of the PPy/MWNTs modification, the electrode obtained a good electronic transfer property and a large specific surface area.Consequently, the sensitivity and selectivity of this sensor for biosensing DNA hybridization were improved.Complementary DNA sequence as low as 5.0� 10�12 mol L�1 can be detected without using hybridization marker orintercalator. Additionally, it was found that the electropolymerization scan rate was an important factor for DNAbiosensor fabrication. It has been optimized at 20 mV s�1.

Keywords: Electrochemical DNA biosensor, DNA hybridization, Impedance, Polypyrrole (PPy), Multiwalled carbonnanotubes (MWNTs)

DOI: 10.1002/elan.200603544

1. Introduction

The discovery of carbon nanotube (CNT) in 1991 [1] openedup a new era in material science and nanotechnology. Theinteresting electronic and photonic properties make CNTattractive for many applications [2 – 7]. Due to its largespecific surface area, wide electrochemical window, flexiblesurface chemistry and unique property to accelerate elec-tronic transfer, CNT has been recognized as an idealnanomaterial to fabricate electrochemical DNA biosensorsincluding using CNT modified electrodes [8 – 18], alignedCNTelectrodes [19 – 24] and CNT-based hybridization label[25, 26]. Usually, CNT modified electrodes are fabricated bydispersing CNT solution onto electrode surface and waitingthe modification layer to be dry [3 – 18]. Thus preparedelectrodes are more powerful to transfer electrochemicalDNA hybridization signal than bare electrodes. However,the unordered CNTs are always random lying on theelectrode surface, and easily peel off from the substratesurface, badly depressing the sensors’ detection reproduci-bility. On the other hand, CNTarray electrode has not beenwidely used for biosensor application due to its complicatedand expensive synthesis.

In this work, PPy/CNT hybrid composite was synthesizedonto a glassy carbon electrode (GCE) surface by PPyelectropolymerization in the presence of carboxylic groups

ended multiwalled carbon nanotubes (MWNTs-COOH).During the polymer electropolymerization, MWNTs-COOH coated with PPy was firmly attached onto theGCE surface. Thus prepared material showed propertiescharacteristic of both constituent components, i.e. the goodelectronic transfer ability and the large surface area, as wellas maybe the potential synergetic effects. Results showedthe presented method for electrode modification is simpleand efficient, giving a stable and powerful modificationlayer for immobilizing DNA and transducing nucleic acidhybridization. As shown in Scheme 1, amino group endedsingle-stranded DNA (NH2-ssDNA) probe was covalentlylinked onto the PPy/MWNTs-COOH/GCE. The hybrid-ization of the ssDNA probe/PPy/MWNTs-COOH/GCEwith its target sequence was investigated by conventionalelectrochemical impedance measurements. As it is knownwhen electrochemical impedance is measured in a simpleredox reaction system, such as Fe(CN)3�=4�

6 , without form-ing a adsorbed layer onto electrode surface, the electro-chemical impedance value (Z) is a complex numbercomposed of electrode interfacial electronic transfer resist-ance (Ret), double layer capacitance (Cdl), the Warburgimpedance (Zw) of the mass transfer resistance, and solutionresistance (Rs) [27]. For double-stranded DNA (dsDNA)has a lower electronic transfer resistance than ssDNA [28],hybridization of the DNA electrode gave a decreased Ret

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Electroanalysis 18, 2006, No. 15, 1471 – 1478 © 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

and, consequently, a decreased Z. Results showed this is alabel-free DNA hybridization sensor with acceptable sensi-tivity and specificity.

2. Experimental

2.1. Reagents

All oligonucleotide sequences were customer-designed andsynthesized by Shanghai Shenergy Biocolor BiologicalScience & Technology Company (Shanghai, China).

The sequence of the amino group ended single-strandedDNA probe:24 mer-5’-NH2-GAGCGGCGCAACATTTCAGGTCGA-3’ (hereinafter, it was referred as NH2-ssDNA probe);its complementary oligonucleotide:5’-TCGACCTGAAATGTTGCGCCGCTC-3’;1-base mismatched oligonucleotide:5’-TCGACCTGAAACGTTGCGCCGCTC-3’;non-complementary oligonucleotide has the same basesequence of the DNA probe without amino group tag.

Multiwalled carbon nanotubes (MWNTs) with the endsopened were obtained from Shenzhen Nanotech Port Co.Ltd. (Shenzhen, China). The inner diameter was 10 – 20 nm,outer diameter 30 – 50 nm and the length 1 – 10 mm. Beforeelectropolymerization, MWNTs were refluxed in boiling65% nitric acid solution for 5 h to attach carboxyl groupsonto their tips [12], referred as MWNTs-COOH. After washand filtration for 10 times, the MWNTs-COOH was dried

under an infrared lamp. Ultimately, we got the recovery ofMWNTwas 91.25%. Pyrrole monomer was purchased fromSigma-Aldrich, Inc. (> 99%). Before electropolymeriza-tion, it was purified by distillation under vacuum. 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC) was pur-chased from Sigma-Aldrich, Inc. Daunomycin hydrochlor-ide was obtained from Shanghai Institute for Drug Controland used without further purification. The buffer solutionused in the experiments was 100 mmol L�1 phosphate buffersolution (1.0 L aqueous solution prepared from 6.084 gNaH2PO4 · 2H2O and 21.848 g Na2HPO4 · 12H2O, pH 7.0)containing 0.75 mol L�1 KCl. Hereinafter, it was referred asPBS. Other regents were all commercially available inanalytical reagent grade. All the solutions were preparedwith ultrapure water from an Aquapro system (Ever YoungEnterprises Development Co., LTD.).

2.2. Electropolymerization of Polypyrrole/MultiwalledCarbon Nanotubes onto Glassy Carbon Electrode

A glassy carbon electrode (GCE) was conventionallypolished by 0.3 mm alumina slurry. After washing theGCE, this fresh GCE, Ag/AgCl and Pt electrodes weredipped into 5 mL of electropolymerization aqueous solution(0.2 mol L�1 pyrrole, 0.2 mol L�1 KCl and 40 mg MWNTs-COOH) to carry out cyclic voltammetry between 0 V andþ0.70 V for 10 cycles, 20 mV s�1. A modest stir was going onthrough the electropolymerization.

Scheme 1. The whole detection scheme of this impedance-based DNA biosensor. Step 1: In the presence of MWNTs-COOH, pyrrolewas electropolymerized onto GCE. Step 2: In the presence of EDAC, NH2-ssDNA probe was linked onto PPy/MWNTs-COOH/GCE.Step 3: Hybridization was carried out at ssDNA/PPy/MWNTs-COOH/GCE.

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Electroanalysis 18, 2006, No. 15, 1471 – 1478 www.electroanalysis.wiley-vch.de © 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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2.3. DNA Probe Binding and Hybridization

After PBS washing the electrode thoroughly, the PPy/MWNTs-COOH/GCE was incubated at room temperaturefor 2 h in 500 mL of PBS containing 0.01 mol L�1 EDAC and1.0� 10�6 mol L�1 NH2-ssDNA probe. Then, the resultingssDNA probe/PPy/MWNTs-COOH/GCE was washed in5 mL of PBS containing 0.1% SDS for 20 min to remove thenon-bound ssDNA probe. After that, hybridization reactionwas carried out by incubating this ssDNA probe/PPy/MWNTs-COOH/GCE at room temperature for 20 min in500 mL of PBS containing target ssDNA. Ultimately, thedsDNA/PPy/MWNTs-COOH/GCE was washed in 5 mL of0.1% SDS PBS for 20 min to remove away the non-hybridized target DNA. SDS wash was important to removethe unspecific ssDNA probe adsorbing on electrode, as wellas the target DNA in this work.

2.4. Signal Detection

All the Cyclic Voltammetry (CV), Amperometric i – tCurve, and AC Impedance measurements were carriedout using a CHI660A electrochemical analyzer (CHIinstrument Inc., USA) in a 10 mL analytical cell. A three-electrode system consisted of a glassy carbon electrode asworking electrode for PPy/MWNTs modification, an Ag/AgCl electrode with KCl saturated as conference electrode,a platinum wire as counter electrode. The CV measurementswere carried out between �0.2 V and 0.6 V with scan rate20 mV s�1, the i – t measurements were with bias potential0.22 V, run time 20 s and sample interval 2� 10�6 s, and theimpedance measurements were from 105 Hz down to 1 Hzwith a sampling rate of 12 points per decade, AC amplitude5 mV, bias potential 0.22 V. The impedance detectionelectrolyte was aqueous solution containing 0.1 mol L�1

KCl and 0.001 mol L�1 K3[Fe(CN)6]/K4[Fe(CN)6] (1 :1) aselectroactive probe.

3. Results and Discussion

3.1. Polypyrrole Electropolymerization in the Presence ofMultiwalled Carbon Nanotubes

Electrode surface modification plays an important role infabricating DNA hybridization biosensor [29]. The largesurface area, coupled with good charge transport property,makes carbon nanotubes and conducting polymer idealmaterials to modify electrode for electrochemical DNAsensors [7]. We present an easy electrode modificationmethod in this brief work to offer a stable PPy/MWNTsmodification layer with desirable electrochemical features.We expected that it would be a functionalized systemexample based on PPy and CNT for DNA biosensingapplication. In the experiments, CNTwas electrochemicallydeposited a conducting PPy film. Simultaneously, thisconducting polymer-CNT composite regularly attached

onto the substrate surface firmly. The Scheme 1 insert givesSEM images of the PPy/MWNTs modified GCE withdifferent scales, showing a porous and dense modificationlayer on the electrode surface with a high specific surfacearea. In this case CNT served as the nanosized backbone forPPy polymerization, resulting that the porous PPy filmcovered around the CNT in a cylinder structure. Ultimately,the CNT covered up by PPy film can not show clearly in theSEM. It has been found that the scan rate and a modest stirduring the PPy electropolymerization significantly affectedthe growth of PPy/MWNTs onto electrode surface. Thecyclic voltammograms of PPy/MWNTs electropolymeriza-tion with (dotted line) and without (straight line) stirring theelectropolymerization solution were compared in Figure 1.As shown in the figure, the PPy electropolymerization withmodestly stirring the electropolymerization solution result-ed in a larger polymerization current and a lower polymer-ization beginning voltage when compared with the onewithout stirring. The configuration difference of these tworesulting electrodes can be easily found by our naked eyes.The former one was covered with much more PPy/CNTclusters than the latter one. This can be explained thatduring stirring, MWNTs were completely dispersed in thepolymerization solution, and had a higher probability to beattached onto the electrode surface during polymerizationthan those without stir. Consequently, the MWNTs on theelectrode surface further stimulated the PPy formation [17,18]. Figure 1 insert is the cyclic voltammogram of the PPy/MWNT electropolymerization with a stir at a slower scanrate, 10 mV s�1. The results showed that slower electro-polymerization scan rate resulted in a much larger polymer-ization current and a lower polymerization beginningvoltage, consequently giving a thicker modification layerwhen compared with the faster rate, for example 20 mV s�1.

Fig. 1. The cyclic voltammograms of PPy electropolymerizationin the presence of MWNTs-COOH between 0 and þ0.7 V for 10cycles with (dotted line) or without (straight line) stirring theelectropolymerization solution. Scan rate was 20 mV s�1. Inset:The cyclic voltammograms of PPy electropolymerization in thepresence of MWNTs-COOH with scan rate 10 mV s�1, stirring.

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The electrochemical performance of the PPy/MWNT-COOH/GCE was evaluated by carrying out cyclic voltam-metry to measure its detection area [30], as well asAmperometric i – tCurve. The cyclic voltammetric responseof the PPy/MWNTs/GCE in a K4Fe(CN)6 solution (Fig-ure 2A), its calculated detection area (Figure 2B), and itscapacitive current were all increased when the electro-polymerization scan rate decreased. This indicates that thusprepared PPy/MWNTs composite was highly electroactiveand offered increasing detection area when compared withbare GCE. Figure 2C shows the i – t curve of the differentelectrodes measured at 0.22 V in K4Fe(CN)6/K3Fe(CN)6

solution. The results showed that decreased polymerizationscan rate resulted in a modified electrode with an increasedcurrent response at the constant voltage, coupled with aconcomitant increased capacitive current. Therefore, thusprepared PPy-CNT composite is a promising material for

fabricating DNA biosensors, as well as other applications,such as powerful capacitors.

3.2. DNA Probe Immobilization onto Polypyrrole/MWNTs-COOH/GCE and Hybridization Detectionby AC Impedance

To demonstrate the potential DNA biosensing applicationof this PPy/MWNTs/GCE, ssDNA probe was linked ontothe electrode by amide bond. The ssDNA probe attachmentand the subsequent hybridization were investigated byrecording the Z vs. frequency (Figure 3A), Zre vs. frequency(Figure 3B), Zim vs. frequency (Figure 3C), and the Nyquistplots (Figure 3D) of the electrode. The results showed thatthe ssDNA probe attachment onto the PPy/MWNTs/GCEresulted in an increased Z from 105 Hz to 1.0 Hz (Figure 3A

Fig. 2. The electrochemical measurements of PPy/MWNTs-COOH/GCE. A) Cyclic voltammetry was carried out according Randles-Sevcik equation [30] in 20 mmol L�1 K4Fe(CN)6 aqueous solution at 20 mV s�1 between �0.2 V and 0.6 V to determine the detectionarea of bare GCE or PPy/MWNTs-COOH/GCEs from different electropolymerization scan rate. B) Electrode detection area of bareGCE or PPy/MWNTs-COOH/GCEs from different electropolymerization scan rate. C) The i – t curves detected at 0.22 V in aqueoussolution containing 0.1 mol L�1 KCl and 0.001 mol L�1 K3[Fe(CN)6]/K3[Fe(CN)6] (1 :1). The electrode was bare GCE or PPy/MWNTs-COOH/GCEs from different electropolymerization scan rate.

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curve a to b). Subsequently, hybridization gave a decreasedZ (curve b to c). This impedance response of hybridizationwas much higher than the one using a conventional CNTmodified electrode coated with ssDNA probe/PPy film [17],attributed to the better modified electrode used in this work.Comparing the changes of Z, Zre and Zim with each other(Figure 3A, B, C, E), it can be found theZ change was mostlyattributed to the Zre change. In the equations: whenw!1 ,Zre¼Rsþw2Ret/(w

2 þw02) and whenw! 0, Zre ¼Rs þRet þ

sw�1/2, Rs is the solution resistance, Ret the electrodeinterfacial electronic transport resistance, which equatesto the diameter of the Nyqusit plot’s semicircle [18, 27], Zre

the real component of impedance, Zim the imaginarycomponent of impedance, s a constant, w0 the angularfrequency where Zim in the semicircle of Nyquist plot ismaximum [18, 27]. Due to the negative charges of ssDNArepelling Fe(CN)3�=4�

6 and the insulator property of ssDNA[31], ssDNA electrode had an increased Ret than the PPy/MWNTs electrode. That resulted in an increased semicirclediameter in the Nyquist plots (Figure 3D, curve a to b).Additionally, the w0 little changed after ssDNA bondingonto the electrode and hybridization in the experiments.Consequently, based on the above two equations, Zre and Zwere increased as a result of the increase of Ret. After thehybridization, the Ret decreased because dsDNA has abetter electronic transfer ability than ssDNA [28], giving asmaller semicircle in the Nyquist plot (Figure 3D, curve b toc). Therefore, dsDNA electrode had lower Zre and Z thanssDNA electrode. In the experiments, we chose the decreaseof Z at 546.9 Hz as the hybridization signal.

The presence of dsDNA on the GCE could also beconfirmed by the electrochemical redox response of thedaunomycin-intercalated dsDNA (Figure 3A insert). Addi-tionally, the heat denaturation was workable to renew thissensor electrode. After the dsDNA electrode denaturationin 90 8C water for 3 min and wash in 0.1% SDS for 20 min,the Z curve recovered to that of the ssDNA electrode in thefirst two cases. However, after the third hybridization andsubsequent heat denaturation using this electrode, the Zcurve could only go back half to the original one of thessDNA electrode.

3.3. The Sensitivity and Selectivity of This Biosensor

This ssDNA probe/PPy/MWNTs/GCE was used to monitorthe target DNA’s concentration for its sensitivity assay. Itwas carried out by varying the concentration of comple-mentary sequence to react with the ssDNA probe electrodeunder the same hybridization condition. As shown inFigure 4A, a linear response of the impedance decrease vs.the concentration of complementary sequence was obtainedfrom 1.0� 10�11 mol L�1 to 1.0� 10�7 mol L�1. The linearregression is Y¼ 90.966þ7.374 lg X (Y: the Z decrease at546.9 Hz from ssDNA/PPy/MWNTs-COOH/GCE todsDNA/PPy/MWNTs-COOH/GCE,W; X: the complemen-tary DNA concentration, mol L�1), regression coefficient(R) 0.9952, detection limit 5.0� 10�12 mol L�1. This result

was more sensitive than the one based on casting MWNTs-COOH onto GCE for PPy/ssDNA electropolymerization(detection limit 1.0� 10�8 mol L�1) [17]. This can beattributed to the stable CNT-supported conducting polymerlayer on GCE providing large surface area, as well asmechanical stability and efficient thermal/electrical con-duction.

The ssDNA probe/PPy/MWNTs/GCE was also demon-strated to be selective for target DNA sequence when wecompared the hybridization signals of non-complementarysequence and one-base mismatched sequence with that ofthe complementary DNA. The specificity assay was carriedout by hybridizing ssDNA probe electrode with 1.0�10�8 mol L�1 different target DNA under the same con-ditions. The results showed that only the complementarysequence gave an obvious decreased Z (Figure 4B). Thehybridization signal of one-base mismatched sequence was35.5% when compared with that of complementary one.Non-complementary sequence gave little change of Z afterthe hybridization reaction. In our another impedance-basedDNA biosensor based on casting MWNTs-COOH ontoGCE for PPy/ssDNA electropolymerization [17], the datawas 51% for one-base mismatched DNA. Therefore, it canbe concluded that thus prepared ssDNA probe/PPy/MWNTs/GCE is useful for biosensing DNA hybridizationwith acceptable sensitivity and selectivity.

3.4. The Optimized Scan Rate of PolypyrroleElectropolymerization

In the experiments, it has been found that the decreasedelectropolymerization scan rate resulted in an increasedamount of PPy/MWNTs onto GCE, which has beendiscussed in Section 3.1. Therefore, it was expected thatthe electropolymerization scan rate would significantlyaffect the surface-bound DNA amount, as well as thehybridization signal as a result. To investigate this, PPyelectropolymerization was carried out with varied scan ratefrom 10 mV s�1 to 200 mV s�1 in the same polymerizationsolution (MWNTs-COOH, pyrrole monomer, KCl) from0 V toþ0.7 V, 10 cycles, stirring. The resulting different PPy/MWNT-COOH/GCEs were used for ssDNA probe immo-bilization and hybridization under the same conditions. Asshown in Figure 5, we obtained two impedance changetrends after hybridization. When the electropolymerizationscan rate was slower than 50 mV s�1, the dsDNA formationresulted in a decrease of impedance. However, when therate was faster than 50 mV s�1, the impedance increasedafter hybridization. We attributed the different impedancechanges to the different DNA amounts on the electrodesurfaces. When the scan rate was slower than 50 mV s�1, theGCE surface was coated with lots of PPy/MWNTs-COOH,subsequently linked many ssDNA probe chains. As a result,the electron transfer from Fe(CN)3�=4�

6 to the electrode wasmostly carried out through the DNA molecules. Therefore,dsDNA formation resulted in a decreasedZdue to the lowerelectronic transfer resistance of dsDNA than ssDNA. Lee’s




group has fabricated a dense DNA modified Au electrodeby 5-day self-assembly to investigate the electronic transferfrom the Fe(CN)3�=4�

6 in the solution to the electrode [32].

Their results showed that if the DNA covered up theelectrode surface, electrons transferred through the DNA,and the electronic transfer ability of DNA had a significant

Fig. 3. The impedance plots for PPy/MWNTs-COOH/GCE (a), ssDNA probe/PPy/MWNTs-COOH/GCE (b) and dsDNA/PPy/MWNTs-COOH/GCE (c). The complementary DNA was 1.0� 10�8 mol L�1. A) Impedance value (Z) vs. the frequency. Insert: thedifferential pulse voltammery of dsDNA/PPy/MWNTs-COOH/GCE after it reacting with 1.0� 10�6 mol L�1 daunomincy PBS solutionfor 2 min. B) Impedance real part (Zre) vs. the frequency. C) Impedance image part (Zim) vs. the frequency. D) Nyquist plots (Zim vs. Zre).E) the changes of Z, Zre and Zim vs. the frequency from electrode (b) to (c).

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influence on the impedance, especially the Ret of the Auelectrode. Contrarily, the faster scan rate than 50 mV s�1

resulted in a modification layer containing less PPy/CNTs,as well as linking less ssDNA probe subsequently. As aconsequence, electrons were mainly transferred through theremaining DNA-unlinking sites of PPy/CNT film. There-fore, dsDNA formation resulted in a increased Z due to themore negative charges of dsDNA repelling the Fe(CN)3�=4�

6

molecules than ssDNA. In the experiments of the PPyelectropolymerization scan rates slower than 50 mV s�1,20 mV s�1 gave a maximum Z change for investigating DNAhybridization when compared with 10 mV s�1 and 50 mVs�1, which was chosen as the optimized electropolymeriza-tion scan rate in fabricating DNA biosensor. It can beexplained that 20 mV s�1 resulted in a modest ssDNAsurface coverage on the electrode for DNA hybridization.However, 10 mV s�1 gave a too packed ssDNA probesurface coverage, discouraging the complementary se-

quence approaching electrode and decreasing the hybrid-ization efficiency [33]; 50 mV s�1 gave a too sparse one, alsoresulting in a low signal. Similarly, when the scan rate wasfaster than 50 mV s�1, 200 mV s�1 gave a smaller hybrid-ization signal than 100 mV s�1, for such a fast scan rateresulted in few probe DNA chains on the electrode surface,consequently giving a very low hybridization signal.

4. Conclusions

A composite material of carbon nanotube and conductingpolymer was synthesized by electropolymerization method.The new material showed properties characteristic of bothconstituent components, as well as the potential synergeticeffects. Such prepared PPy/CNT modified electrode pro-vided a stable modification layer for linking DNA chains,and subsequently transducing the hybridization. Due to thespecific properties of CNT and conducting polymer, thesensitivity and selectivity of this biosensor were all bothimproved. The hybridization was investigated by label-freeimpedance detection approach.

5. Acknowledgement

We thank the National Nature Science Foundation of China(NSFC), which financially supported this work (No.29875008).

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Fig. 4. A) The change of Z at 546.9 Hz from ssDNA probe/PPy/MWNTs-COOH/GCE to dsDNA/PPy/MWNTs-COOH/GCEwith complementary DNA sequence from 1.0� 10�11 mol L�1 to1.0� 10�6 mol L�1. B) The change of Z of ssDNA/PPy/MWNTs-COOH/GCE after it reacted with different 1.0� 10�8 mol L�1

DNA target sequences.

Fig. 5. The change of Z from ssDNA probe/PPy/MWNTs-COOH/GCE to dsDNA/PPy/MWNTs-COOH/GCE based onthe different PPy electropolymerization scan rate. The comple-mentary DNA sequence was 1.0� 10�8 mol L�1.




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Impedance DNA Biosensor Using Electropolymerized Polypyrrole/Multiwalled Carbon Nanotubes Modified Electrode