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Analytical Letters
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Nano-ZnO/Chitosan Composite Film Modified Electrode for Voltammetric Detection of DNA Hybridization
Zhi-Min Liu , Yan-Li Liu , Guo-Li Shen & Ru-Qin Yu
a c a b c

College of Chemistry and Chemical Engineering, Henan University of Technology, Zhengzhou, P.R. China
b

College of Materials Science and Engineering, Hunan University, Changsha, P.R. China
c

State Key Laboratory for Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha, P.R. China Version of record first published: 05 Jun 2008.

To cite this article: Zhi-Min Liu , Yan-Li Liu , Guo-Li Shen & Ru-Qin Yu (2008): NanoZnO/Chitosan Composite Film Modified Electrode for Voltammetric Detection of DNA Hybridization, Analytical Letters, 41:6, 1083-1095 To link to this article: http://dx.doi.org/10.1080/00032710801978608

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Analytical Letters, 41: 10831095, 2008 Copyright Q Taylor & Francis Group, LLC ISSN: 0003-2719 print/1532-236X online DOI: 10.1080/00032710801978608

BIOSENSORS

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Nano-ZnO/Chitosan Composite Film Modified Electrode for Voltammetric Detection of DNA Hybridization
Zhi-Min Liu,1 Yan-Li Liu,2 Guo-Li Shen,3 and Ru-Qin Yu3
College of Chemistry and Chemical Engineering, Henan University of Technology, Zhengzhou, P.R. China 2 College of Materials Science and Engineering, Hunan University, Changsha, P.R. China 3 State Key Laboratory for Chemo=Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha, P.R. China
1

Abstract: A sensitive electrochemical DNA biosensor based on nano-ZnO= chitosan composite matrix for DNA hybridization detection was developed. The Nano-ZnO was synthesized by the hydrothermal method and dispersed in chitosan, which was used to fabricate the modification of the glassy carbon electrode (GCE) surface. The ZnO=chitosan-modified electrode exhibited good biocompatibility and excellent electrochemical conductivity. The hybridization detection was monitored with differential pulse voltammetry (DPV) measurement using methylene blue (MB) as an indicator. The established biosensor can effectively discriminate complementary target sequence and two-base-mismatched sequence, with a detection limit of 1.09 10 11 mol L1 of complementary target.
Keywords: Chitosan; DNA biosensor; Methylene blue; ZnO

Received 25 November 2007; accepted 15 December 2007. The authors are grateful for the financial support provided by the National Natural Science Foundation of China (No. 20435010) and Henan University of Technology (No. 150196). Address correspondence to Zhi-Min Liu, College of Chemistry and Chemical Engineering, Henan University of Technology, Zhengzhou 450052, P.R. China. E-mail: zhimin@haut.edu.cn 1083

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INTRODUCTION In recent years, electrochemical DNA biosensors based on nucleic acid hybridization have received considerable attention due to their increasing importance in the diagnosis of disease (Palecek and Fojta 2001; Kerman et al. 2002; Wang, 2002; Erdema et al. 2005). DNA biosensors offer great benefits for their high sensitivity, small dimensions, low cost, and compatibility with microfabrication technology (Pang and Abruna 1998). The principle of a DNA electrochemical sensor basically is to convert a nucleic acid hybridization event engendered at an electrode surface into a useful electrical signal. A variety of approaches have been developed in the construction of electrochemical nucleic acid biosensors, such as those based on metal nanoparticles tags (Authier et al. 2001; Liu et al. 2005), catalytic oxidation of guanine (Gore et al. 2003), redox active hybrid indicators (Millan and Mikkelsen 1993), and enzyme-amplified nucleic acid assays (Dequaire and Heller 2002; Bakker and Telting-Diaz 2002). With the development of electrochemical DNA biosensors, the detection of DNA hybridization at surfaces becomes more important. The detection of DNA hybridization using electrochemical readout is particularly attractive for the development of clinical diagnostics (Zhu et al. 2006). The use of nanomaterials in electrical detection is relatively new and offers unique opportunities for electrochemical transduction of DNA sensing events. Zhu and coworkers (Zhu et al. 2005) developed a method of immobilizing DNA based on platinum nanoparticles combined with multiwalled carbon nanotubes (MWCNTs). In another biosensor, based on nanoparticles ZrO2 for monitoring DNA hybridization, sensing was carried out with an MWCNTs=nano ZrO2= chitosan-modified glassy carbon electrode (Yang et al. 2007). It has been established that ZnO is a very attractive semiconductor material. Bulk ZnO is widely used in rubber products, ceramics, paints, pharmaceuticals, and sensors (Sberveglieri et al. 1995). Topoglidis and coworkers reported the immobilization and bioelectrochemistry of proteins on nanoporous TiO2 and ZnO films (Topoglidis et al. 2001). Therefore, ZnO nanoparticles (nano-ZnO) deserve further investigation as an important and promising candidate for support material in the fabrication of the biosensors. Chitosan is a kind of b-1, 4-linked glucosamine oligomer. It is a natural cationic polymer and can form a stable complex with the polyanionic phosphodiester backbones of DNA, either native or denatured. DNA electrochemical biosensors based on chitosan to immobilize singlestranded DNA (ssDNA) have been investigated (Li et al. 1997), although the conductivity of the films has been found to be less than ideal, leading

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to the conclusion that the electron transfer may therefore be retarded to some extent. Recently, the use of composite materials based on integration of chitosan with some other materials to combine properties of the individual components with a synergistic effect, have gained growing interest (Yang et al. 2007; Li et al. 2005). For example, chitosan doped with carbon nanotube resulted in the remarkable improvement of redox activity of methylene blue due to the excellent electron-transfer ability of carbon nanotubes (Li et al. 2005). Chitosan films mixed with ZnO have been used to develop an IgG immunosensor (Wang et al. 2006). However, whether ZnO=chitosan composite film can be used to enlarge the electrochemical signal of the DNA indicator and increase sensitivity for DNA detection has not been investigated previously. The aim of this work is to develop a simple and sensitive DNA immobilization strategy that will provide a well-defined recognition surface for hybridization. In the current study, nano-ZnO was prepared by a hydrothermal method and dispersed in the chitosan solution to form a ZnO=chitosan composite film. Then the ssDNA probe sequence was immobilized on the surface of the glassy carbon electrode (GCE) modified with ZnO=chitosan films. The hybridization reaction was conducted by immersing a probe-modified electrode into its complementary DNA sequence in solution and detected by differential pulse voltammetry (DPV) with the electroactive methylene blue (MB) as an indicator. Results showed that the probe ssDNA immobilized on the ZnO=chitosan film modified electrode could be successfully used for a complementary target sequence and two-base-mismatched strand. The performance of the DNA biosensor with respect to the sensitivity and linear range was discussed.

EXPERIMENTAL Reagents and Materials Initially, 17-base DNA oligonucleotides were purchased from Dalian Biotechnology Co., Ltd. (Liaoning, China). Their base sequences are as follows: target DNA (17-mer base sequence A) 50 -TAA GCA ACC TGA TTT GA-30 probe DNA (17-mer base sequence B) 50 -TCA AAT CAG GTT GCT TA-30 mismatch DNA (17-mer base sequence A0 ) 50 -TAA GCA AGG TGA TTT GA-30

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The 17-mer base sequence A is complementary to 17-mer base sequence B; the 17-mer base sequence A0 is a mutant of the 17-mer base sequence A with two bases changed, as indicated by the underline. Chitosan (MW $ 1 106, 85% deacetylation) was supplied by Sigma (St. Louis, MO, USA). The MB was obtained from Shanghai Chemical Reagent Company and used without further purification. Stock solutions of the DNA target with various concentrations were prepared in a 10 mmol L1 phosphate buffer solution and stored in a freezer until use. Other reagents were commercially available and were all of analytical reagent grade.

Apparatus Powder X-ray diffraction (XRD) experiments were conducted on a Rigaku D=max 2550 X-ray diffractometer (Rigaku, Tokyo, Japan) with Cu Ka radiation (k 1.5418 A), and the data were collected in steps of  1 0.020 (2-theta) min from 10 to 90(2-theta). The database of the Joint Committee on Powder Diffraction Standards (JCPDS) was used for phase identification. The scanning electron microscope (SEM) image was taken with JSM-5600LV (JEOL, Japan), using an accelerating voltage of 20 kV. Cyclic voltammetry (CV) and differential pulse voltammetry (DPV) experiments were carried out using a CHI 650 C electrochemical analyzer (Chen Hua Instruments, Inc., Shanghai, China). The three-electrode system consisted of glassy carbon electrode (3 mm diameter) as the working electrode, a saturated calomel electrode (SCE) as the reference electrode, and a platinum foil as the counter electrode.

Preparation of Nano-ZnO Zn(Ac)2 2H2O and NaOH were dissolved in water to prepare 0.50 and 5.0 mol L1 solutions, respectively. The two solutions were mixed (molar ratio 1:10) under constant stirring and to form a transparent Zn(OH)42 solution. A 2.0 mL volume of the above solution was added to 22 mL of H2O under ultrasonic agitation. Then the mixture was transferred into a 30-ml Teflon-lined autoclave. The autoclave was sealed and maintained at 180C for approximately 13 h and then naturally cooled to room temperature. The precipitate was collected by centrifugation, washed with absolute ethanol and distilled water several times, and then dried under reduced pressure at 100C for 4 h.

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Preparation of ZnO/Chitosan Solution An appropriate amount of nano-ZnO was dispersed in 0.1% chitosan solution (1.0% acetic acid), and the mass ratio was 1:100 (ZnO: chitosan). The mixture was stirred for 8 h at room temperature. Finally, a highly dispersed viscous solution was formed.

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Electrode Modification Before each experiment, the glassy carbon working electrode (GCE) was polished with 0.050 mm a-alumina powder and rinsed thoroughly with absolute alcohol and water in an ultrasonic bath and dried in air. A ZnO= chitosan composite solution (10 mL) was pipetted onto the surface of an inverted GCE and air dried at room temperature overnight. After the electrode was rinsed with phosphate buffer (pH 7.0), the ZnO=chitosanmodified GCE was prepared. The immobilization of ssDNA probe on the ZnO=chitosan-modified GCE was carried out as follows: The ZnO=chitosan-modified GCE was inverted, and 10 mL of ssDNA probe (3.57 105 mol L1) was pipetted onto the electrode surface. The probe droplet was air dried at room temperature for 1 h to obtain a probe-modified GCE. After that, the electrode was rinsed three times with phosphate buffer (pH 7.0) before hybridization. Hybridization and Indicator Binding to the Hybrid Hybridization was conducted at 45C by incubating the modified electrode in 10 mmol L1 phosphate buffer containing 0.1 mol L1 NaCl and 1.0 103 mol L1 KCl in the presence of target DNA. The electrode was washed with phosphate buffer to remove the unhybridized DNA. After that, the hybridized electrode was placed in the stirred phosphate buffer (pH 7.0) containing 2.0 105 mol L1 MB with 20 mmol L1 NaCl for 5 min without applying any potential. After accumulation of MB, the electrode was rinsed three times with phosphate buffer (pH 7.0). Electrochemical Detection The response signal of MB was measured by using DPV in a phosphate buffer (pH 7.0) with the hybridized working electrode, with accumulated MB, the SCE reference electrode, and the platinum wire counter electrode.

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RESULTS AND DISCUSSION Structural Characterizations The structure of the obtained ZnO was first characterized by XRD (Fig. 1). All peaks are consistent with the peaks of ZnO (JCPDS Card No. 361451) with high crystallinity. Figure 2 shows the scanning electron micrograph morphology of ZnO=chitosan film. It can be seen that ZnO nanoparticles were regularly dispersed in chitosan solution, exhibiting uniform porous structure. This structure provides a significant increase of effective electrode surface for the ssDNA probe loading.

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Electrochemical Characteristics of Modified-GCE The cyclic voltammograms of a bare glassy carbon electrode (GCE), a GCE treated with ZnO=chitosan (ZnO=chitosan=GCE), and a ZnO= chitosan=GCE-modified with ssDNA probe (ssDNA=ZnO=chitosan= GCE) in 5.0 mmol L1 K3Fe(CN)6 solution are shown in Figure 3. As can be seen, the peak current increased in the order of ssDNA= ZnO=chitosan=GCE, GCE and ZnO=chitosan=GCE. The peak current of GCE (Fig. 3a) is less than that of ZnO=chitosan=GCE (Fig. 3b), which contributed to the excellent electronic conductivity of ZnO and the accumulation ability of chitosan with amino groups for the negatively charged ferricyanide ions. After the ssDNA probe immobilized on the ZnO=chitosan=GCE, the peak current decreased (Fig. 3c). The reason seems to be related to the electrostatic repulsion between the polyanionic

Figure 1. XRD image of ZnO nanoparticles.

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Figure 2. SEM image of ZnO=chitosan composite film.

DNA immobilized on the ZnO=chitosan=GCE and the anionic redox couple ions (Zhu et al. 2004). The differential pulse voltammograms for the cathodic signals of MB at different electrodes immobilized with ssDNA probe were also recorded (shown in Fig. 4). As can be seen, the electrode modified only with

Figure 3. Cyclic voltammograms of 5 mmol L1 ferricyanide=ferrocyanide in 10 mmol L1 KCl at 100 mV s1 using (a) the bare GCE, (b) the ZnO=chitosanmodified GCE (ZnO=chitosan=GCE), and (c) the ZnO=chitosan-modified GCE treated with ssDNA probe (ssDNA=ZnO=chitosan=GCE).

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Figure 4. The DPV response of MB as indicator in phosphate buffer (pH 7.0) recordedfor the chitosan film modified GCE (a) and the ZnO=chitosan composite film modified GCE (b) after immobilization with the same concentration (3.57 105 mol L1) ssDNA probe. Stirred in phosphate buffer (pH 7.0) containing 2.0 1 05 mol L1 MB with 20 mmol L1 NaCl for 5 min. Scan rate was 100 mV s1.

chitosan film (ssDNA=chitosan=GCE) could also provide the enhanced MB signal (Fig. 4a), owing to the ability of chitosan, a polycationic polymer to bind with DNA and other polyanions. Immobilization of ssDNA probe onto the ZnO=chitosan composite film resulted in an increase in the peak current of MB (Fig. 4b), which was attributed to the high surface area of nano-ZnO dispersed in chitosan film. The electroactive surface area of the modified electrode was estimated to be 0.38 cm2, according to the RandlesSevcik equation (Hrapovic et al. 2004). The experimental results showed that the ZnO=chitosan composite film is effective in increasing the loading of ssDNA probe and improving the sensitivity of the analysis.

Effect of MB Concentration The concentration of MB employed in the experiments directly affected the amount of MB accumulated with ssDNA probe. Therefore, the sensitivity of a biosensor can be influenced by MB concentration. The results showed that the signal of MB increased rapidly with its concentration, from 2.0 106 to 1.5 105 mol L1, then slowly, and a signal plateau

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appeared at 2.0 105 mol L1 or above. So, 2.0 105 mol L1 MB was used as the optimum concentration.

Selectivity of the DNA Biosensor

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MB as an electroactive redox indicator has been used for electrochemical sensing of DNA hybridization at the electrode surface (Kerman et al. 2002; Erdema et al. 2000). We wanted to investigate whether the ssDNA=ZnO=chitosan=GCE can be used to efficiently distinguish the complementary sequence from the noncomplementary sequence, using MB as an indicator. Figure 5 shows the DPV curves of MB recorded for the ssDNA=ZnO=chitosan=GCE without hybridization (a), after being hybridized with complementary sequences (b), and two-basemismatched DNA sequences (c). All the peaks obtained were well-defined. One notices that ssDNA=ZnO=chitosan=GCE (Fig. 5a) presented the highest signal due to the strong affinity of MB for the free guanine bases (Ozkana et al. 2002). A significant decrease in the signal of MB was observed when hybridizing with the complementary target sequence (Fig. 5b), which was because the interaction of MB and guanine residues of the probe was prevented by duplex formation on the electrode

Figure 5. Differential pulse voltammograms using MB as indicator in phosphate buffer (pH 7.0) for (a) ssDNA probe-modified ZnO=chitosan=GCE, (b) after hybridization with the complementary target DNA sequence (3.57 108 mol L1), and (c) after exposure to 3.57 108 mol L1 two-base-mismatched DNA sequence.

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surface (Ozkana et al. 2002). While incubation with two-base-mismatched sequence (Fig. 5c), the reduction signal of MB changed very little, indicating that complete hybridization was not accomplished due to the base mismatch. The results demonstrated that the probemodified electrode has high selectivity for hybridization detection, and the response of MB on the probe electrode can be considered an efficient intercalator to distinguish the complementary sequence from the mismatch sequence. Analytical Performance The analytical performance of the DNA sensor was investigated by using ssDNA probe to hybridize with the target DNA sequences of different concentrations, according to the procedure described. The peak current values of MB were measured by DPV under optimum conditions after ssDNA probe hybridized. As can be seen from Figure 6, the reduction peak current values were linear with the logarithmic value of the complementary target DNA sequence concentration ranging from 3.57 10 11 mol L1 to 3.57 107 mol L1, with a detection limit of 1.09 10 11 mol L1. The regression equation was y 0.7103 log x 3.8892, where y(107 A) was the DPV current value of MB, and x(pmol L1) was the concentration value of target DNA. The correlation coefficient of the linear curve was 0.9925. Reproducibility of the DNA Sensor To characterize the reproducibility of the sensor, repetitive experiments, including electrode modification, the immobilization of ssDNA, and the detection of DNA hybridization were carried out. Five DNA sensors, made independently, showed an acceptable variation coefficient of 4.58% (n 5). This study indicated that a satisfactory reproducibility could be obtained by this system. CONCLUSION In this study, the fabrication of a DNA biosensor based on the capture probe on ZnO=chitosan-modified GCE was reported. The ZnO=chitosan composite matrix incorporated the advantages of nano-ZnO and chitosan, which could form a thin film on the surface of the GCE for the immobilization of ssDNA probe. The results showed that the electrode modified with ZnO=chitosan composite film could offer a more enhanced

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Figure 6. (A) Differential pulse voltammograms for different target concentrations: (a) 0.00 pmol L1, (b) 3.57 101 pmol L1, (c) 3.57 102 pmol L1, (d) 3.57 103 pmol L1, (e) 3.57 104 pmol L1. (f) 3.57 105 pmol L1. (B) The resulting logarithmic standard plot.

signal than the electrode modified only with chitosan could provide. Preliminary experiments have shown that ssDNA=ZnO=chitosan=GCE could be used to efficiently identify the complementary sequence from the two-base-mismatched sequence. Compared with other DNA detection techniques, this study provides a simple and practical method that

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displays a satisfactory detection limit and a relatively wide linear range, indicating that the ZnO=chitosan system can be used as a stable and sensitive platform for DNA detection. The detection signals were enlarged by the introduction of nano-ZnO, indicating that nano-ZnO has great potential to be used to improve sensor performance.

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