Direct Electrochemical Detection of Oligonucleotide Hybridization on Poly(thionine) Film

Chinese Journal of Chemistry, 2005, 23, 1665—1670 Full Paper

* E-mail: [email protected]; Tel.: 0086-021-62233508; Fax: 0086-021-62233508 Received September 20, 2004; revised July 8, 2005; accepted August 15, 2005. Project supported by the National Natural Science Foundation of China (No. 29875008).

© 2005 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Direct Electrochemical Detection of Oligonucleotide Hybridization on Poly(thionine) Film

XU, Ying(徐颖) JIANG, Ying(蒋莹) YANG, Lin(杨琳) HE, Pin-Gang(何品刚) FANG, Yu-Zhi*(方禹之)

Department of Chemistry, East China Normal University, Shanghai 200062, China

In this work, the application of a conducting polymer, poly(thionine), modified electrode as matrix to DNA immobilization as well as transducer to label-free DNA hybridization detection was introduced. The electropoly-merization of thionine onto electrode surface was carried out by a simple two-step method, which involved a pre-anodization of glassy carbon electrode at a constant positive potential in thionine solution following cyclic voltam-metry scans in the solution. Electrochemical detection was performed by differential pulse voltammetry in the elec-troactivity potential domain of poly(thionine). The resulting poly(thionine) modified electrode showed a good sta-bility and electroactivity in aqueous media during a near neutral pH range. Additionally, the pendant amino groups on the poly(thionine) chains enabled poly(thionine) modified electrode to immobilize phosphate group terminated DNA probe via covalent linkage. Hybridization process induced a clear decrease in poly(thionine) redox current, which was corresponding to the decrease in poly(thionine) electroactivity after double stranded DNA was formed on the polymer film. The detection limit of this electrochemical DNA hybridization sensor was 1.0×10－10 mol/L. Compared with complementary sequence, the hybridization signal values of 1-base mismatched and 3-base mis-matched samples were 63.9% and 9.2%, respectively.

Keywords electrochemical DNA hybridization sensor, conducting polymer, poly(thionine), label-free DNA hy-bridization detectione

Introduction

The knowledge of the structure, organization and sequence of nucleic acid molecules has many important applications to clinical diagnostics, new drug research, gene therapy, food technology, environmental science, forensic analysis, etc.1 As a result, the detection of spe-cific DNA is an emerging monitoring task. Conven-tional DNA hybridization-based methods require ex-pensive and time-consuming procedures as well as the use of carcinogentic or radioactive reagents. Recently, many research groups have focused their efforts on the development of electrochemical DNA hybridization biosensors.2 While many electrochemical DNA hy-bridization biosensors are based on the use of an elec-troactive hybridization indicator to label DNA probe or to react with DNA helix,3-7 the attention recently has been paid to the methods to directly detect DNA hy-bridization without using hybridization indicator. For example, hybridization altered the capacitance8,9 or in-creased the interfacial electronic transfer resistance of DNA probe modified electrodes.10,11 Hybridization of DNA on bilayer lipid membranes resulted in a decrease in the ion current through the lipid membranes.12 DNA helix can also be directly detected by electrochemically oxidating its bases13-15 and catalytically oxidating its

sugar moiety.16 Another successful method to directly detect DNA

hybridization is to apply conducting polymer modified electrode, which acts at the same time as immobilization matrix and transduction agent.17-21 To apply this method, two requirements must be satisfied: (1) the polymer is able to immobilize DNA either by adsorption or by co-valent grafting, and (2) the polymer is electro-active, and hybridization induces change in polymer electroac-tivity. For example, Cha et al.17 reported a DNA hy-bridization electrochemical sensor made of poly(thiophen-3-yl-acetic acid 1,3-dioxo-1,3-dihydroisoindol- 2-yl ester) modified electrode. The DNA hybridization induced a decrease in polymer conductivity, which was detected by cyclic voltammogram in acetonitrile/ tetrabutylammonium hexafluorophosphate between 0 and 1.2 V. Korri-Youssoufi et al.18 developed an elec-trode based on polypyrrole functionalized with both ferrocenyl group and DNA probe. The DNA hybridiza-tion induced a decrease in the polymer current density.

In this paper, the construction of a label-free DNA hybridization electrochemical sensor was based on poly(thionine) modified electrode. In the early studies, modification of electrodes with poly(thionine) was usu-ally achieved by one of the following two methods:22 (1)

1666 Chin. J. Chem., 2005, Vol. 23, No. 12 XU et al.


potential scans during a potential range in an acetonitrile solution containing thionine, and (2) constant potential oxidation at a positive voltage of 1.0—1.5 V (vs. SCE) in a strong acid solution containing thionine. However, poly(thionine) film was immediately desorbed from the electrode surface when the pH value of the solution was increased to be higher than 4. Therefore, such modified electrode could only be used in the solution with pH＜4, which was not suitable for DNA hybridization detection. The formation of poly(thionine) onto electrode surface in this paper was based on a two-step method23 accord-ing to the manner in Scheme 1,22 which enabled the re-sulting poly(thionine) to be electroactive in near neutral buffer solution during a moderate potential range be-tween －0.40 and 0.15 V. Additionally, the pendant

amino groups on the polymer chains enabled the poly(thionine) to react with phosphate group terminated DNA probe via phosphoramidate bond. These proper-ties made poly(thionie) modified electrode suitable to immobilize DNA probe and directly detect DNA hy-bridization by investigating the change in poly(thionine) electroactivity.

Experimental

Reagents

The various oligonucleotides were purchased from Shenggong Bioengineering Ltd. Company (Shanghai, China) with the following sequences: probe oligonu-cleotide: 5'-PO4-GAGCGGCGCAACATTTCAGGTCGA-

Scheme 1 The proposed thionine coupling scheme

Electrochemical DNA hybridization sensor Chin. J. Chem., 2005 Vol. 23 No. 12 1667


3' (hereafter referred to as PO4-ssDNA (single-stranded DNA) probe), complementary target oligonucleotides: 5'-TCGACCTGAAATGTTGCGCCGCTC-3', 1-base mismatched oligonucleotide: 5'-TCGACCTGAAACGTT- GCGCCGCTC-3', 3-base mismatched oligonucleotide: 5'-TCGTCCTGAAACGTTGCGCCTCTC-3', noncomplementary target oligonucleotide: 5'-GAGCGGCGC- AACATTTCAGGTCGA-3'.

0.1 mol/L PBS with pH 6.0 (0.1 mol NaCl＋0.0877 mol NaH2PO4＋0.0123 mol Na2HPO4 dissolved in 1000 mL of ultrapure water), 0.1 mol/L PBS with pH 7.0 (0.1 mol NaCl ＋ 0.0390 mol NaH2PO4 ＋ 0.0610 mol Na2HPO4 dissolved in 1000 mL of ultrapure water), 0.1 mol/L PBS with pH 8.0 (0.1 mol NaCl＋0.00530 mol NaH2PO4＋0.0947 mol Na2HPO4 dissolved in 1000 mL of ultrapure water), and 0.75 mol/L PBS with pH 6.0 (0.75 mol/L NaCl＋0.0877 mol NaH2PO4＋0.0123 mol Na2HPO4 dissolved in 1000 mL of ultrapure water) were used. Thionine was obtained from Fluka. 1-Ethyl-3-(3- dimethylaminopropyl) carbodimide (EDAC) was pur-chased from Sigma. All reagents were of analytical re-agent grade. All of the solutions were prepared with ultrapure water from an Aquapro system.

Apparatus

Differential pulse voltammetry (DPV) and cyclic voltammetry (CV) measurements were performed with a CH instrument Model 660 electrochemical analyzer (CH Instrument Inc., US). The three-electrode system consisted of a glassy carbon working electrode (2 mm of diameter), an Ag/AgCl reference electrode with KCl saturated, and a platinum wire counter electrode. All electrochemical measurements were carried out in a 10 mL cell.

Thionine electropolymerization

Working electrode was polished prior to use with a 0.3 µm alumina slurry and washed thoroughly with ul-trapure water. The electropolymerization of thionine was carried out by two steps: (1) holding the glassy carbon electrode at 1.5 V for 10 min in 0.1 mol/L PBS (pH 6.0) containing1.0×10－4 mol/L thionine and (2) potential scans for 15 cycles at 50 mV/s between －0.4 and 0.15 V (vs. Ag/AgCl) in the solution. The poly(thionine) modified electrode was washed thor-oughly in 0.1 mol/L PBS (pH 6.0) at 60 ℃ for 1 h to wash away the adsorbed thionine monomer.

DNA immobilization and hybridization

Poly(thionine) modified glassy carbon electrode was dipped into 500 µL of 0.75 mol/L PBS (pH 6.0) con-taining 4OD PO4-ssDNA probe in the presence of 1.0×10－2 mol/L EDAC at 60 ℃ with stirring. After 5 h, the reaction was stopped by removing the electrode from the reactive solution, and the PO4-ssDNA probe/ poly(thionine) modified electrode was washed in 0.75 mol/L PBS (pH 6.0) at 60 ℃ for 15 min with stirring to remove the non-covalently bound DNA.

The hybridization experiments were performed by

dipping the PO4-ssDNA probe/poly(thionine) modified electrode in 0.75 mol/L PBS (pH 6.0) containing target DNA for 1.5 h at 40 ℃ with stirring, and the electrode was rinsed with the same buffer solution before elec-trochemical detection.

Differential pulse voltammetry

The hybridization was detected by recording the de-crease in the reduction peak current of the PO4-ssDNA probe/poly(thionine) modified electrode. The following parameters were used: pulse amplitude 50 mV, pulse period 200 s, pulse width 50 ms, potential domain be-tween －0.40 and 0.15 V (vs. Ag/AgCl). The electro-lyte media were 0.1 mol/L PBS (pH 6.0).

Results and discussion

Polymer film characterization and electroactivity

In this paper, the poly(thionine) modified electrode was obtained by a simple two-step method. After a pre-anodization at 1.5 V in 0.1 mol/L PBS (pH 6.0) con-taining 1.0×10－4 mol/L thionine, the cyclic voltammo-gram scanning between －0.40 and 0.15 V was re-corded in Figure 1, which indicated the growth process of the poly(thionine) film on electrode surface with consecutive cycles. During the process of thionine elec-tropolymerization onto electrode surface, the currents of a pair of reversible redox peaks continued to build up with every successive sweep, indicating an increase in quantity of electroactive species on the electrode. On removal of the electrode from thionine-containing solu-tion, a golden with purple film was formed on the elec-trode surface. The pH value of the electropolymeriza-tion solution had a significant effect on the electro-polymerization process of poly(thionine). As shown in Figure 1, during the near neutral biocompatible pH

Figure 1 Repetitive cyclic voltammograms for the electro-polymerization of thionine onto glassy carbon electrode surface in 0.1 mol/L PBS containing 1.0×10－4 mol/L thionine at differ-ent pH values with 15 continuous cyclic scanning between

－0.40 and 0.15 V (vs. Ag/AgCl) at 50 mV/s, 20 ℃. Before the electropolymerization, the electrode went on a preanodization at 1.5 V in the thionine solution for 10 min.



range (pH 6.0—8.0), pH 6.0 resulted in the best reversi-ble and conductive poly(thionine) film onto electrode surface. Therefore, this work chose pH 6.0 as the pH value of the electropolymerization solution. Besides, the temperature also influenced the electropolymerization process in our experiment, and consequently, 20 ℃ was fixed as the electropolymerization temperature.

The preanodization operation was critical. During this period, a large amount of positive charges were accumulated on the electrode surface and used to create the thionine cation radicals. These active cation radicals were linked through the —NH— bridge to form poly(thionine) onto electrode surface with scanning between －0.40 and 0.15 V as the manner in Scheme 1. Therefore, if the cyclic voltammogram scans were di-rectly carried out without an oxidation pretreatment or with a preanodization potential lower than 1.1 V, there were no enough positive charges accumulated on elec-trode to create thionine cation radicals, which resulted in a failing electropolymerization of thionine as there was no current growth under the same polymerization conditions. However, if the preanodization was conducted below 1.5 V, the polymer film was not strongly attached to the electrode and destroyed by wash in PBS with stirring. Hence, present experiment chose 1.5 V as the pretreatment potential.

The poly(thionine) was electroactive because each monomer unit retained its electroactive heterocyclic nitrogen atom as shown in Scheme 1. Moreover, as po-lymerization occurred, more pendant amino groups were converted into nitrogen bridge between monomer units, and those bridges, like in polyaniline films, were electroactive.22 The important point was that the result-ing poly(thionine) formed by this method was instinc-tively electroactive in near neutral phosphate buffer so-lution during a moderate potential region (curve a in Figure 2A and 2B), and the pH was not necessary to be lower than 4. This result was accorded with Ref. 23. This property made poly(thionine) suitable to detect DNA hybridization under a biocompatible condition. Besides, the polymer was stable in phosphate buffer solution without change of the polymer redox peak cur-rent after 2 d.

Oligonucleotide probe immobilization

The pendant amino groups on poly(thionine) chains could react with phosphate group terminated DNA (PO4-ssDNA) probe via a phosphoramidate covalent bond in the presence of EDAC.24 The successful covalent grafting of DNA probe onto polymer led to a clear decrease in the polymer current intensity with a ratio of 71.37% (curve b in Figure 2A and 2B). It could be ex-plained that the grafted nucleic acid strands acted as an insulating layer on the polymer film19 and increased the stiffness of poly(thionine). The latter effect formed a barrier of poly(thionine) backbone rotating between structure I and II (Scheme 1) during redox process, and the similar result has been gained in Ref. 17 with

Figure 2 Differential pulse voltammograms for the poly(thionine) modified electrode (curve a), the PO4-ssDNA probe/poly(thionine) modified electrode (curve b) scanning from －0.40 to 0.15 V (A) and 0.15 to －0.40 V (B) in 0.1 mol/L PBS (pH 6.0). poly(thiophen-3-yl-acetic acid 1,3-dioxo-1,3-dihydroisoindol-2-yl ester) modified electrode to directly detect DNA hybridization.

However, DNA would be adsorbed to poly(thionine) for the electrostatic attraction between DNA negative phosphate backbone and the protonated amino groups on the poly(thionine) chains. In our experiment, it was found that an increased ionic strength in DNA solution weakened the nonspecific adsorption between DNA and poly(thionine) film. This electrostatic attraction experi-ment was conducted by holding the poly(thionine) modified electrode in 500 µL of phosphate buffer solu-tion (pH 6.0) containing 4OD noncomplementary DNA and different concentration of NaCl at 60 ℃ for 5 h. Results showed that when the concentration of NaCl was increased from 0 to 0.1, 0.3, 0.5 mol/L, the decrease in poly(thionine) reduction peak current via the electro-static attraction between poly(thionine) film and non-complementary DNA was decreased rapidly from 3.25 to 2.27, 1.07, 0.5 µA, then reached its minimum 0.25 µA when NaCl was higher than 0.75 mol/L. Therefore, in order to avoid this weak nonspecific adsorption, this investigation chose 0.75 mol/L PBS (pH 6.0) as the re-active media to link PO4-ssDNA probe onto polymer and as the hybridization media.

Electrochemical DNA hybridization sensor Chin. J. Chem., 2005 Vol. 23 No. 12 1669


Hybridization detection

The PO4-ssDNA probe/poly(thionine) modified electrode was incubated with complementary DNA or noncomplementary DNA in the same hybridization con-ditions. After the PO4-ssDNA probe/poly(thionine) modified electrode was incubated with noncomplemen-tary DNA for 1.5 h at 40 ℃, the different pulse voltammogram of poly(thionine) redox process was little changed (curve b in Figure 3) during reduction process. However, the complementary sequence led to a clear decrease in the poly(thionine) redox current density (curve c in Figure 3). It could be explained by an increase in the poly(thionine) stiffness when double stranded DNA was formed on the poly(thionine) film, which led to a higher barrier of the poly(thionine) rotat-ing between structures I and II during its redox process. The decrease of reduction peak current depended on the complementary DNA concentration from 10－8 to 10－6

mol/L and the regression equation was Y＝9.0901＋0.90025X [X: lg(the concentration of complementary DNA), Y: the decrease in poly(thionine) reduction peak current (µA)] with the correlation coefficient R 0.9862. The detection limit was 3.0×10－9 mol/L (S/N＝3, n＝11) of complementary DNA in solution. Additionally, the effect of hybridization time on hybridization signal was investigated when the hybridization experiment was conducted with 10－7 mol/L complementary DNA at 40

Figure 3 Differential pulse voltammograms in 0.1 mol/L PBS (pH 6.0) from 0.15 to －0.40 V (vs. Ag/AgCl) for the PO4- ssDNA/poly(thionine) modified electrode before hybridization (curve a, ―), after hybridization with 10－6 mol/L noncomple-mentary DNA (curve b, ∆) or 10－6 mol/L complementary DNA (curve c, ○) at 40 ℃ for 1.5 h under stirring.

℃ under stirring. Results showed that the decrease of poly(thionine) reduction peak current was increased rapidly from 5 to 30, 60 min with the value of 0.95, 1.75, 2.50 µA, then reached its maximum at 90 min with the value of 2.69 µA. Based on such results, the investiga-tion chose 1.5 h as the hybridization period.

To investigate the selectivity of this sensor, the de-crease in poly(thionine) reduction peak current was compared by incubating the PO4-ssDNA probe/poly- (thionine) modified electrode with perfectly matched, 1-base mismatched and 3-base mismatched DNA in the same hybridization conditions (Figure 4). The result showed that the poly(thionine)-based DNA hybridiza-tion sensor had the ability to distinguish DNA se-quences with a negligible signal for 3-base mismatched DNA sample and a low hybridization signal for 1-base mismatched DNA sample: when compared with com-plementary sequence, 1-base mismatched sample re-sulted in 63.9% hybridization amount, and 3-base mis-matched sample resulted in 9.2%.

It was demonstrated that the DNA hybridization signal was dependent on the thickness of the poly- (thionine) film and the thicker film resulted in the higher signal (Table 1). It could be explained that im-mobilization and hybridization occurred on polymer film surface as well as inside the polymer network, therefore, hybridization amount was increased with the polymer film thickness. Based on a thicker polymer film gained from electropolymerization of 45 cycles, the de-tection limit was improved to 1.0×10－10 mol/L.

Figure 4 The decrease in poly(thionine) reduction peak current vs. the target DNA sequence. The hybridization was conducted with 10－6 mol/L target DNA at 40 ℃ for 1.5 h under stirring.

Table 1 The reduction peak current of the poly(thionine) modified electrode (µA)

a The detection media were 0.1 mol/L PBS (pH 6.0). b The hybridization was conducted with complementary DNA of 10－8 mol/L at 40 ℃ for 1.5 h under stirring.

Electropolymerization cycles Poly(thionine) PO4-ssDNA probe/poly(thionine)a Double stranded DNA/poly(thionine)b (a-b)/a (%)

15 17.25 4.938 3.137 36.47

30 32.25 7.764 4.633 40.33

45 45.56 10.28 5.759 43.98



Conclusion

This study described the preparation of conducting poly(thionine) modified electrode to covalently immo-bilize nucleic acid probe and directly detect DNA hy-bridization. Differential pulse voltammetry, which was performed in a biocompatible condition, showed a clear current decrease in poly(thionine) redox process after hybridization occurred between the PO4-ssDNA probe/ poly(thionine) modified electrode and the complemen-tary DNA target in solution. While, no change of poly(thionine) redox current was observed when the PO4-ssDNA probe/poly(thionine) modified electrode was incubated with noncomplementary DNA in the same hybridization conditions. In addition, DNA se-quences between complementary and mismatched sam-ples could be differentiated distinctly by this method. This DNA biosensor was able to detect 1.0×10－10

mol/L complementary DNA target. Therefore, this poly(thionine)-based DNA hybridization sensor was biocompatible and suitable for gene diagnostics. A fur-ther improvement in sensitivity and selectivity was un-der way in our laboratory.

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Direct Electrochemical Detection of Oligonucleotide Hybridization on Poly(thionine) Film