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Electrochemical detection of avian influenza virus H5N1 gene sequence using a DNA aptamer immobilized onto a hybrid nanomaterial-modified electrode
Electrochimica Acta 56 (2011) 6266–6270
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Electrochimica Acta journal homepage: www.elsevier.com/locate/electacta
Electrochemical detection of avian influenza virus H5N1 gene sequence using a DNA aptamer immobilized onto a hybrid nanomaterial-modified electrode Xianggang Liu a , Ziqiang Cheng b,∗ , Hai Fan a , Shiyun Ai a,∗ , Ruixia Han a a b
College of Chemistry and Material Science, Shandong Agricultural University, Taian 271018, Shandong, PR China College of Animal Science and Technology, Shandong Agricultural University, Taian 271018, Shandong, PR China
a r t i c l e
i n f o
Article history: Received 23 February 2011 Received in revised form 9 May 2011 Accepted 17 May 2011 Available online 23 May 2011 Keywords: DNA aptamer Avian influenza virus H5N1 gene sequence Electrochemical detection DNA recognition Hybrid nanomaterial
a b s t r a c t A sensitive electrochemical method for the detection of avian influenza virus (AIV) H5N1 gene sequence using a DNA aptamer immobilized onto a hybrid nanomaterial-modified electrode was developed. To enhance the selectivity and sensitivity, the modified electrode was assembled with multi-wall carbon nanotubes (MWNT), polypyrrole nanowires (PPNWs) and gold nanoparticles (GNPs). This electrode offered a porous structure with a large effective surface area, highly electrocatalytic activities and electronic conductivity. Therefore, the amount of DNA aptamer immobilized onto the electrode was increased while the accessibility of the detection target was maintained. The biosensor is based on the hybridization and preferred orientation of a DNA aptamer immobilized onto a modified electrode surface with its target (H5N1 specific sequence) present in solution. It is selective for the H5N1 specific sequence, and the signal of the indicator was approximately linear to log(concentration) of the H5N1 specific sequence from 5.0 × 10−12 to 1.0 × 10−9 M (R = 0.9863) with a detection limit of 4.3 × 10−13 M. These studies showed that the new hybrid nanomaterial (MWNT/PPNWs/GNPs) and the DNA aptamer could be used to fabricate an electrochemical biosensor for gene sequence detection. Furthermore, this design strategy is expected to have extensive applications in other biosensors. © 2011 Elsevier Ltd. All rights reserved.
1. Introduction AIV has become an increasingly severe threat to human health. Until September 2009, 442 cases of the AIV H5N1 directly crossing barriers and infecting humans occurred and lead to 262 deaths [1]. Obviously, an effective method for monitoring highly pathogenic AIV is urgently needed. Previously, several previously developed methods have been used for the detection and identification of AIV. Such as enzyme-linked immunosorbent assays (ELISA) [2] and the polymerase chain reaction (PCR) [3,4]. However, these methods are labor-intensive and time-consuming for large numbers of clinical samples. Recently, increasing interest has been focused on the development of electrochemical DNA biosensors for the simple diagnosis of infectious agents [5,6]. Most of these biosensors are based on the hybridization of a DNA-specific sequence (probe) previously immobilized onto an electrode surface with its complementary sequence (target) present in solution. The DNA-specific sequence is based on the target sequence and is synthesized using chemical methods. It has dimensional structural differences with spatial
∗ Corresponding author. Tel.: +86 538 8247660; fax: +86 538 8242251. E-mail addresses: [email protected] (Z. Cheng), [email protected], [email protected] (S. Ai). 0013-4686/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.electacta.2011.05.055
recognition sites of the target sequence. Therefore, the preferred orientation is not well defined. In this case, hybridization is the main mode of binding to the target DNA sequence. Aptamers are a new class of single-stranded DNA or RNA oligonucleotides obtained using the “systematic evolution of ligands by exponential enrichment (SELEX)” from random RNA or DNA libraries [7]. This process is different from classical chemical synthesis method in that DNA aptamers have a uniform dimensional structure with spatial recognition sites of target sequence. The aptamers can discriminate targets on the basis of subtle structural differences, such as the presence or absence of chemical groups [8,9] and the dimensional structure [10,11]. Therefore, they have two different modes of binding to target DNA sequences: (i) hybridization and (ii) dimensional preferred orientation. They have been used in a wide range of applications that involve proteins [12], peptides, amino acids, nucleotides, drugs, carbohydrates and other small organic and inorganic compounds [13,14]. Many aptamer-based protocols have been proposed for electrochemical monitoring of DNA [15,16]. The key issues with any electrochemical DNA biosensor include the enhancement of aptamer immobilization amount, maintenance of target accessibility [17,18], improvement of the catalytic capabilities and conductivity of the modified electrode, and the demonstration of useful detection signals [19]. To solve these problems, various types of strategies have been developed [20,21] and nanomaterials have
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Fig. 1. A schematic diagram depicting the experimental procedures.
attracted significant attention in research and practical applications. MWNT has attracted substantial interest due to its unique properties such as fast electron transfer ability, mechanical strength, chemical stability and catalysis effect. They have had a significant impact on the development of new methods and instrumentation for chemical analysis [22]. Conducting polymeric nanomaterials are very effective substrates for biomaterial immobilization. Some of these nanomaterials are doped and/or covalently or physically modified by bio-nanomaterials, especially proteins and nucleic acids, which exhibit unique catalytic [23] or affinity properties that can be easily employed in the design of biosensors. Nanomaterials, such as PPNWs are easily deposited [24] and are electroactive in neutral environments. Moreover, they provide a suitable interface on electrode surfaces due to their numerous attractive properties, including high surface free energy, good ion-exchange capacity, small cross-sectional dimension and high electronic conductivity [25]. Noble metal (gold) nanoparticles (GNPs) have also been extensively utilized in recent years because of their good conductivity, useful electrocatalytic ability and biocompatibility [26]. Several research groups have fabricated electrochemical sensors and biosensors [27–30]. However, for many applications, greater flexibility and control of the properties and functionalities of nanomaterials is desired. One approach is to use hybrid nanomaterials that have properties that differ from those of single component nanomaterials. The use of multiple components offers a higher degree of flexibility for altering and controlling the properties and functionalities of nanomaterials. Multiple components are often used in electrode surface modification for DNA biosensor fabrication, but the vast majority of approaches are based on mixed coatings and do not provide a large effective surface area on electrode surfaces. To the best of our knowledge, there are no prior reports on the electrochemical detection of target sequence using this type of porous hybrid nanomaterial (MWNT/PPNWs/GNPs) modified electrode. In this paper, a sensitive electrochemical method for the detection of AIV H5N1 gene sequences using a DNA aptamer immobilized hybrid nanomaterial-modified electrode was developed, as shown in Fig. 1. The hybrid nanomaterial (MWNT/PPNWs/GNPs) provides a porous structure with a large effective surface area, highly electrocatalytic activities and electronic conductivity. In order to enhance the recognition and selectivity of the biosensor, DNA aptamers were used as capture probes. The results indicate that this strategy may provide a sensitive, selective, simple, and inexpensive detection method for the AIV genotype.
2. Experimental 2.1. Apparatus and chemicals Cyclic voltammetry (CV) and DPV measurements were conducted with a BAS Epsilon Electrochemical Analyzer (Bioanalytical Systems, Inc. USA). Electrochemical impedance spectroscopy (EIS) was obtained with a CHI 660 C electrochemical analyzer (Shanghai Chenhua Co., China). A three-electrode set-up was used and consisted of a platinum auxiliary electrode, a saturated calomel (SCE) reference electrode and an Au working electrode (1.6 mm diameter). All potentials were referenced to the SCE reference electrode, and all measurements were performed at room temperature. The target sequence of H5N1 was selected based on the past research [6]. All oligonucleotides were prepared and purified by Takara Biotechnology (Dalian) CO., Ltd. (China). Their base sequences are as follows: 5 -HS-(CH2 )6 -TTG-CCC-GTT-TAC-TTTGGG-TCT-3 (thiol-terminated DNA aptamer (HS-DNA)), 5 -AGACCC-AAA-GTA-AAC-GGG-CAA-3 (target DNA), 5 -AGA-CCC-AAAGTA-AAC-GCG-CAA-3 (one-base mismatch DNA), 5 -AGA-CACAAA-GAA-AAC-GCG-CAA-3 (three-base mismatch DNA), 5 -CCTAGG-GCT-TGC-GCG-ATA-GAT-3 (non-complementary DNA). All synthetic oligonucleotide stock solutions were prepared using TE (10 mM Tris–HCl, 1 mM EDTA, pH 8.00) and were kept frozen. The tris(1,10-phenanthroline) cobalt (III) perchlorate indicator was synthesized as previously described [31]. All other chemicals were of analytical grade. Doubly distilled water (DDW) was used throughout the experiments. 2.2. Preparation of hybrid nanomaterials modified electrodes Before each use, the Au electrode was polished with a wet slurry of 0.05 m Al2 O3 on a felt pad. Then the electrode was successively ultrasonic rinsed with Piranha (a mixture with 3:7 (v/v) of 30% H2 O2 and 98% H2 SO4 ), ethanol, and DDW for 5 min each. The electrode was then dried under a stream of high purity nitrogen. Finally, 2 L of 1.5 mg/mL MWNT was placed on a previously conditioned Au electrode that was held upside-down and was exposed to the air for 1.0 h at room temperature. The PPNWs were fabricated according to previous work [26]. They were electrochemically deposited by repeatedly cycling the potential from 0.0 to 0.80 V in 0.1 M PBS (pH 7.4) solution containing 0.1 M LiClO4 and 0.015 M pyrrole at a sweep rate of 50 mV/s for 20 cycles. The GNPs were electrochemically deposited on the prepared electrode by CV scanning from 0.2 V to −1.0 V in 0.1 M
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Fig. 2. (A) CV responses of various modified electrodes in a 0.1 M KCl solution containing 2.0 mM K3 [Fe(CN)6 ]. Scan rate: 50 mV/s. (B) Nyquist relationships of EIS data performed in a 0.1 M KCl solution containing 5.0 mM K3 [Fe(CN)6 ]/K4 [Fe(CN)6 ] (1:1) for various modified electrodes. The amplitude and potential were 0.005 and 0.165 V, respectively. (a) Bare Au, (b) Au/MWNT/PPNWs, (c) Au/MWNT/PPNWs/GNPs, (d) Au/MWNT/PPNWs/GNPs/DNA-aptamer.
KCl solution containing 0.5 mM HAuCl4 at a sweep rate of 50 mV/s for 20 cycles. The modified electrode was obtained and denoted as Au/MWNT/PPNWs/GNPs. 2.3. Preparation of the DNA aptamer monolayer For this process, 4 L of the DNA aptamer (9.6 M HS-DNA) was placed on a previously conditioned Au electrode that was held upside down, and the end of the electrode was fitted with a plastic cup to protect the solution from evaporation. The assembly was kept standing for 3.0 h at room temperature. After rinsing, the modified electrode was treated with 1.0 mM 6-mercapto-1-hexanol (MCH) solution for 1 h to mask the unmodified Au sites. 2.4. Recognition and transduction After modification with the DNA aptamer monolayer, the modified electrode was used to recognize various concentrations of different signal-stranded DNA diluted with 1× SSC solution by incubation at room temperature for 1 h. After incubation, the modified electrode was then rinsed with PBS and DDW to remove the free DNA oligonucleotides. The electrode was then immersed in a 10 M solution of indicator for 20 min to perform electrochemical detection. 2.5. Electrochemical measurements CV measurements were performed in 2 mM K3 [Fe(CN)6 ] solution containing 0.1 M KCl with a scan rate of 50 mV/s. DPV measurements were performed in 0.1 M PBS by scanning from 0 to +0.40 V with an pulse amplitude of 10 mV, pulse width of 0.05 s, and pulse period of 0.2 s. EIS was performed in a 0.1 M KCl solution within the frequency range of 10−2 to 105 Hz using a 5.0 mM K3 [Fe(CN)6 ]/K4 [Fe(CN)6 ] (1:1) mixture as an electroactive probe. The amplitude and potential were 0.005 and 0.165 V, respectively.
peak current at the Au/MWNT/PPNWs and a 3.9-fold increase at the Au/MWNT/PPNWs/GNPs were observed compared to that of bare Au. A significant acceleration of the probe redox reaction occurred due to the presence of the assembly (MWNT/PPNWs and MWNT/PPNWs/GNPs). As shown in Fig. 2A-d, it was apparent that the oxidation peak currents (ip ) decreased dramatically to approximately 27 A. This was due to the DNA aptamer attached to the surface of the modified electrode, which led to an increase in the negative charge on the surface of the electrode and prevented access of negatively charged probe ions (e.g., the redox couple Fe(CN)6 ]3−/4− ) to the surface due to electrostatic repulsion. EIS is also a highly effective method for probing the features of a surface-modified electrode [32]. In EIS, the semicircle diameter of EIS reflects the electron transfer resistance, which controls the electron transfer kinetics of the redox-probe at the electrode interface. In this study, EIS was conducted to investigate the changes of electron transfer resistance that originated from each surface modification step as shown in Fig. 2B. These results were consistent with those obtained in CVs. The superior characteristics of the MWNT/PPNWs/GNPmodified electrode are illustrated by the CV and EIS results. This electrode had highly electrocatalytic activities and electronic conductivity and increased the electron transfer rate between electrode surface and redox probe. 3.2. Response studies of various modified electrodes The influences of MWNT, PPNWs and GNPs on the electrochemical detection of DNA were investigated using DPV measurements. As illustrated in Fig. 3, the bare Au (Fig. 3a) showed a very small DPV
3. Results and discussion 3.1. Electrochemical characterization of the modified electrodes CV is an effective method for probing the features of a surface-modified electrode using the redox probe Fe(CN)6 ]3−/4− . The CV curves of various modified electrodes were shown in Fig. 2A. It is clear from Fig. 2A-a that Fe(CN)6 ]3−/4− has a few well-defined redox peaks at the bare Au. However, the peak obviously increased after the electrode was modified with MWNT/PPNWs and MWNT/PPNWs/GNPs, respectively (curves Fig. 2A-b and c). An approximately 3.5-fold increase in the
Fig. 3. (A) DPV responses of indicator oxidation signals at different DNA aptamer modified electrodes after recognition with 1 M complementary target DNA sequence. (a) Bare Au, (b) Au/GNPs, (c) Au/PPNWs/GNPs, (d) Au/MWNT/ PPNWs/GNPs.
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Fig. 4. (A) The corresponding DPV responses of indicator at Au/MWNT/ PPNWs/GNPs/DNA-aptamer before recognition (a) and after recognition with the non-complementary DNA sequence (b), three-base mismatch DNA sequence (c), one-base mismatch DNA sequence (d) and complementary target DNA sequence (e). (B) The corresponding histograms. Error bars are ±RSD. Experimental conditions as described in the text.
peak current of the indicator whereas a large DPV peak current was observed after immobilizing GNPs onto the bare Au electrode (Fig. 3b). An approximately 4-fold increased peak current was obtained in comparison with that of bare Au. The current increase can be attributed mainly to the increased DNA aptamer immobilization on the GNPs modified layer. The Au/PPNWs/GNPs (Fig. 3c) resulted in excellent amplification of the indicator oxidation response with a large peak current value. An approximately 35-fold increased peak current was obtained in comparison with that of bare Au. This increase was because the PPNWs provided a porous structure with a large effective surface area. The indicator’s largest oxidation peak was observed at Au/MWNT/PPNWs/GNPs (Fig. 3d). The large current increase was mainly ascribed to the catalytic and conductive effects of MWNT. The response was also associated with the MWNT’s ability to improve the effective surface area of the modified electrode. This phenomenon clearly showed that the novel modified electrode provided a good environment for DNA aptamer immobilization and promoted the hybridization and preferred orientation of the DNA aptamer with the target sequence. 3.3. Sequence-specific recognition studies The DPV technique can provide better peak resolution and current sensitivity [33]. In addition, the charging current contribution to the background current, which is a limiting factor in the analytical determination, is negligible in DPV mode [34,35]. To test whether the method was sufficiently selective to distinguish complementary target DNA, the prepared electrode was reacted with the target in the buffer. The recognition of different ss-DNA using the modified electrodes was conducted with DPV using 10 M tris(1,10-phenanthroline) cobalt (III) perchlorate PBS solution (pH 7.4) as shown in Fig. 4A. It was apparent that similar responses were acquired at Au/MWNT/PPNWs/GNPs/HS-DNA before and after treatment with the non-complementary sequence (Fig. 4A-a and b). Almost no recognition occurred with the noncomplementary sequence. The signal increased slightly when the DNA aptamer modified GNPs were treated with the three-base mismatch sequence (Fig. 4A-c) suggesting that the recognition efficiency was not high. However, the signal increased substantially after the DNA aptamer modified GNPs were treated with a one-base mismatch (Fig. 4A-d) and complementary target sequence (Fig. 4Ae), respectively. This was due to the fact that the indicator cannot bind to the single-stranded DNA aptamer before recognition. After recognition, the response was associated with the redox process involving intercalation of the indicator into the double-stranded DNA duplex. The resulting histograms of the relevant experiments are displayed in Fig. 4B. Each sample was tested five times to verify the reproducibility. The relative standard deviation (RSD) of the
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Fig. 5. Semi-log calibration curve for complementary DNA with concentrations varying from 5.0 × 10−12 to 1.0 × 10−9 M. Error bars are ±RSD and n = 3. Upper inset shows DPV responses of the indicator at Au/MWNT/PPNWs/GNPs/DNA-aptamer after recognition with the following increasing concentration of the complementary target DNA: (a) 0, (b) 0.5, (c) 1, (d) 2.5, (e) 5, (f) 10, (g) 25, (h) 50, (i) 100, (j) 250, (k) 500, and (l) 1000 pM. Lower inset indicates the general curvilinear coordinate system for target DNA with concentrations varying from 5.0 × 10−12 to 1.0 × 10−9 M.
DNA aptamer modified electrode treated with complementary target DNA sequence was 5.0% (Fig. 4B-e), and the other recognition situation was lower than 4.7%. These results demonstrate that the complementary target DNA sequence could form double-stranded DNA with the DNA aptamer, which produces a significant increase in signal. The method allowed selective recognition of the DNA sequences with transduction. 3.4. Electrochemically quantitative recognition detection In this study, the sensitivity and selectivity of the sensor under the optimized detection conditions was tested using DPV. The upper inset of Fig. 5 shows an increasing oxidation peak current of the indicator with increasing the complementary target DNA concentration. The lower inset of Fig. 5 indicates the plotting of the current response with increasing the complementary target DNA concentration using a general curvilinear coordinate system. As shown in the insert, the complementary target DNA calibration followed a Langmuir-like adsorption curve [36]. The oxidation peak current of the indicator varies approximately linearly with the log(concentration) of the complementary target DNA over the range of 5.0 × 10−12 to 1.0 × 10−9 M with a correlation coefficient of 0.9863 (main panel, Fig. 5). The regression equation of the complementary target DNA was i (A) = 5.01828 log c(pM) + 1.48193. The detection limit (S/N = 3 compared with Table 1 The performance comparison of current fabricated DNA biosensor with reported in the literatures. Layers for DNA immobilization
Detection methods
Detection limit (mol L−1 )
References
Au NPs/CdS NPs GCE/MWNT-COOH Au NPs Chit and Au NPs Ppy Ag NPs MWNT/Au NPs PPy/ssDNA/MWNT paste Pt NPs and MWNT CPE CdS Nps and Ppy film MWNT/PPy/nano-Au
DPV DPV DPV DPV DPV ASV DPV DPV DPV PSA EIS DPV
2.0 × 10−11 1.0 × 10−10 5.0 × 10−10 5.0 × 10−11 1.0 × 10−10 5.0 × 10−13 6.2 × 10−12 8.5 × 10−11 1.0 × 10−11 4 × 10−9 1 × 10−9 4.3 × 10−13
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Abbreviations: NPs, nanoparticles; Ppy, polypyrrole; EIS, electrochemical impedance spectroscopy; DPV, differential pulse voltammetry; GCE: glassy carbon electrode; Chit: Chitson; CPE: carbon paste electrode; PSA: Chronopotentiometric Stripping Analysis.
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a hybrid nanomaterial-modified electrode exposed to the noncomplementary sequence) [37] was 4.3 × 10−13 M. This sensitivity is as good as Pd-nanoparticles/MWNT modified electrode [38] and much better than hollow gold nanospheres (HGP) [39] and other similar nanomaterial-based electrodes (Table 1). 4. Conclusion A sensitive electrochemical method for the detection of avian influenza virus genotype using a DNA aptamer immobilized hybrid nanomaterial-modified electrode was developed. The modified electrode was assembled with MWNT, PPNWs and GNPs. The detection of target DNA sequences was accomplished not only via hybridization but also via dimensional preferred orientation. The biosensor has high recognition and selectivity for H5N1 specific sequence, and the signal of the indicator was approximately linear to log(concentration) of the H5N1 specific sequence from 5.0 × 10−12 to 1.0 × 10−9 M (R = 0.9863) with a detection limit of 4.3 × 10−13 M. Acknowledgements This work was supported by the National Natural Science Foundation of China (No. 21075078) and the Natural Science Foundation of Shandong province, China (No. ZR2010BM005). References [1] C. Qi, X.S. Tian, S. Chen, J.H. Yan, Z. Cao, K.G. Tian, G.F. Gao, G. Jin, Biosens. Bioelectron. 25 (2010) 1530. [2] T.H. Chua, T.M. Ellis, C.W. Wong, Y. Guan, S.X. Ge, G. Peng, C. Lamichhane, C. Maliadis, S.W. Tan, P. Selleck, J. Parkinson, Avian Dis. 51 (2007) 96. [3] W.J. Chen, B. He, C.G. Li, X.W. Zhang, W.L. Wu, X.Y. Yin, B.X. Fan, X.L. Fan, J. Wang, J. Med. Microbiol. 56 (2007) 603. [4] K. Tsukamoto, H. Ashizawa, K. Nakanishi, N. Kaji, K. Suzuki, M. Okamatsu, S. Yamaguchi, M. Mase, J. Clin. Microbiol. 46 (2008) 3048. [5] J.H. Chen, J. Zhang, K. Wang, L.Y. Huang, X.H. Lin, G.N. Chen, Electrochem. Commun. 10 (2008) 1448. [6] J.M. Picuri, B.M. Frezza, M.R. Ghadiri, J. Am. Chem. Soc. 131 (2009) 9368. [7] A.D. Ellington, J.W. Szostac, Nature 346 (1990) 818. [8] R.D. Jenison, S.C. Gill, A. Pardi, B. Polisky, Science 263 (1994) 1425. [9] C. Mannironi, A.D. Nardo, P. Fruscoloni, G.P. Tocchini-Valentini, Biochemistry 36 (1997) 9726. [10] M. Sassanfar, J.W. Szostak, Nature 364 (1993) 550.
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Electrochemical detection of avian influenza virus H5N1 gene sequence using a DNA aptamer immobilized onto a hybrid nanomaterial-modified electrode Xianggang Liu a , Ziqiang Cheng b,∗ , Hai Fan a , Shiyun Ai a,∗ , Ruixia Han a a b
College of Chemistry and Material Science, Shandong Agricultural University, Taian 271018, Shandong, PR China College of Animal Science and Technology, Shandong Agricultural University, Taian 271018, Shandong, PR China
a r t i c l e
i n f o
Article history: Received 23 February 2011 Received in revised form 9 May 2011 Accepted 17 May 2011 Available online 23 May 2011 Keywords: DNA aptamer Avian influenza virus H5N1 gene sequence Electrochemical detection DNA recognition Hybrid nanomaterial
a b s t r a c t A sensitive electrochemical method for the detection of avian influenza virus (AIV) H5N1 gene sequence using a DNA aptamer immobilized onto a hybrid nanomaterial-modified electrode was developed. To enhance the selectivity and sensitivity, the modified electrode was assembled with multi-wall carbon nanotubes (MWNT), polypyrrole nanowires (PPNWs) and gold nanoparticles (GNPs). This electrode offered a porous structure with a large effective surface area, highly electrocatalytic activities and electronic conductivity. Therefore, the amount of DNA aptamer immobilized onto the electrode was increased while the accessibility of the detection target was maintained. The biosensor is based on the hybridization and preferred orientation of a DNA aptamer immobilized onto a modified electrode surface with its target (H5N1 specific sequence) present in solution. It is selective for the H5N1 specific sequence, and the signal of the indicator was approximately linear to log(concentration) of the H5N1 specific sequence from 5.0 × 10−12 to 1.0 × 10−9 M (R = 0.9863) with a detection limit of 4.3 × 10−13 M. These studies showed that the new hybrid nanomaterial (MWNT/PPNWs/GNPs) and the DNA aptamer could be used to fabricate an electrochemical biosensor for gene sequence detection. Furthermore, this design strategy is expected to have extensive applications in other biosensors. © 2011 Elsevier Ltd. All rights reserved.
1. Introduction AIV has become an increasingly severe threat to human health. Until September 2009, 442 cases of the AIV H5N1 directly crossing barriers and infecting humans occurred and lead to 262 deaths [1]. Obviously, an effective method for monitoring highly pathogenic AIV is urgently needed. Previously, several previously developed methods have been used for the detection and identification of AIV. Such as enzyme-linked immunosorbent assays (ELISA) [2] and the polymerase chain reaction (PCR) [3,4]. However, these methods are labor-intensive and time-consuming for large numbers of clinical samples. Recently, increasing interest has been focused on the development of electrochemical DNA biosensors for the simple diagnosis of infectious agents [5,6]. Most of these biosensors are based on the hybridization of a DNA-specific sequence (probe) previously immobilized onto an electrode surface with its complementary sequence (target) present in solution. The DNA-specific sequence is based on the target sequence and is synthesized using chemical methods. It has dimensional structural differences with spatial
∗ Corresponding author. Tel.: +86 538 8247660; fax: +86 538 8242251. E-mail addresses: [email protected] (Z. Cheng), [email protected], [email protected] (S. Ai). 0013-4686/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.electacta.2011.05.055
recognition sites of the target sequence. Therefore, the preferred orientation is not well defined. In this case, hybridization is the main mode of binding to the target DNA sequence. Aptamers are a new class of single-stranded DNA or RNA oligonucleotides obtained using the “systematic evolution of ligands by exponential enrichment (SELEX)” from random RNA or DNA libraries [7]. This process is different from classical chemical synthesis method in that DNA aptamers have a uniform dimensional structure with spatial recognition sites of target sequence. The aptamers can discriminate targets on the basis of subtle structural differences, such as the presence or absence of chemical groups [8,9] and the dimensional structure [10,11]. Therefore, they have two different modes of binding to target DNA sequences: (i) hybridization and (ii) dimensional preferred orientation. They have been used in a wide range of applications that involve proteins [12], peptides, amino acids, nucleotides, drugs, carbohydrates and other small organic and inorganic compounds [13,14]. Many aptamer-based protocols have been proposed for electrochemical monitoring of DNA [15,16]. The key issues with any electrochemical DNA biosensor include the enhancement of aptamer immobilization amount, maintenance of target accessibility [17,18], improvement of the catalytic capabilities and conductivity of the modified electrode, and the demonstration of useful detection signals [19]. To solve these problems, various types of strategies have been developed [20,21] and nanomaterials have
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Fig. 1. A schematic diagram depicting the experimental procedures.
attracted significant attention in research and practical applications. MWNT has attracted substantial interest due to its unique properties such as fast electron transfer ability, mechanical strength, chemical stability and catalysis effect. They have had a significant impact on the development of new methods and instrumentation for chemical analysis [22]. Conducting polymeric nanomaterials are very effective substrates for biomaterial immobilization. Some of these nanomaterials are doped and/or covalently or physically modified by bio-nanomaterials, especially proteins and nucleic acids, which exhibit unique catalytic [23] or affinity properties that can be easily employed in the design of biosensors. Nanomaterials, such as PPNWs are easily deposited [24] and are electroactive in neutral environments. Moreover, they provide a suitable interface on electrode surfaces due to their numerous attractive properties, including high surface free energy, good ion-exchange capacity, small cross-sectional dimension and high electronic conductivity [25]. Noble metal (gold) nanoparticles (GNPs) have also been extensively utilized in recent years because of their good conductivity, useful electrocatalytic ability and biocompatibility [26]. Several research groups have fabricated electrochemical sensors and biosensors [27–30]. However, for many applications, greater flexibility and control of the properties and functionalities of nanomaterials is desired. One approach is to use hybrid nanomaterials that have properties that differ from those of single component nanomaterials. The use of multiple components offers a higher degree of flexibility for altering and controlling the properties and functionalities of nanomaterials. Multiple components are often used in electrode surface modification for DNA biosensor fabrication, but the vast majority of approaches are based on mixed coatings and do not provide a large effective surface area on electrode surfaces. To the best of our knowledge, there are no prior reports on the electrochemical detection of target sequence using this type of porous hybrid nanomaterial (MWNT/PPNWs/GNPs) modified electrode. In this paper, a sensitive electrochemical method for the detection of AIV H5N1 gene sequences using a DNA aptamer immobilized hybrid nanomaterial-modified electrode was developed, as shown in Fig. 1. The hybrid nanomaterial (MWNT/PPNWs/GNPs) provides a porous structure with a large effective surface area, highly electrocatalytic activities and electronic conductivity. In order to enhance the recognition and selectivity of the biosensor, DNA aptamers were used as capture probes. The results indicate that this strategy may provide a sensitive, selective, simple, and inexpensive detection method for the AIV genotype.
2. Experimental 2.1. Apparatus and chemicals Cyclic voltammetry (CV) and DPV measurements were conducted with a BAS Epsilon Electrochemical Analyzer (Bioanalytical Systems, Inc. USA). Electrochemical impedance spectroscopy (EIS) was obtained with a CHI 660 C electrochemical analyzer (Shanghai Chenhua Co., China). A three-electrode set-up was used and consisted of a platinum auxiliary electrode, a saturated calomel (SCE) reference electrode and an Au working electrode (1.6 mm diameter). All potentials were referenced to the SCE reference electrode, and all measurements were performed at room temperature. The target sequence of H5N1 was selected based on the past research [6]. All oligonucleotides were prepared and purified by Takara Biotechnology (Dalian) CO., Ltd. (China). Their base sequences are as follows: 5 -HS-(CH2 )6 -TTG-CCC-GTT-TAC-TTTGGG-TCT-3 (thiol-terminated DNA aptamer (HS-DNA)), 5 -AGACCC-AAA-GTA-AAC-GGG-CAA-3 (target DNA), 5 -AGA-CCC-AAAGTA-AAC-GCG-CAA-3 (one-base mismatch DNA), 5 -AGA-CACAAA-GAA-AAC-GCG-CAA-3 (three-base mismatch DNA), 5 -CCTAGG-GCT-TGC-GCG-ATA-GAT-3 (non-complementary DNA). All synthetic oligonucleotide stock solutions were prepared using TE (10 mM Tris–HCl, 1 mM EDTA, pH 8.00) and were kept frozen. The tris(1,10-phenanthroline) cobalt (III) perchlorate indicator was synthesized as previously described [31]. All other chemicals were of analytical grade. Doubly distilled water (DDW) was used throughout the experiments. 2.2. Preparation of hybrid nanomaterials modified electrodes Before each use, the Au electrode was polished with a wet slurry of 0.05 m Al2 O3 on a felt pad. Then the electrode was successively ultrasonic rinsed with Piranha (a mixture with 3:7 (v/v) of 30% H2 O2 and 98% H2 SO4 ), ethanol, and DDW for 5 min each. The electrode was then dried under a stream of high purity nitrogen. Finally, 2 L of 1.5 mg/mL MWNT was placed on a previously conditioned Au electrode that was held upside-down and was exposed to the air for 1.0 h at room temperature. The PPNWs were fabricated according to previous work [26]. They were electrochemically deposited by repeatedly cycling the potential from 0.0 to 0.80 V in 0.1 M PBS (pH 7.4) solution containing 0.1 M LiClO4 and 0.015 M pyrrole at a sweep rate of 50 mV/s for 20 cycles. The GNPs were electrochemically deposited on the prepared electrode by CV scanning from 0.2 V to −1.0 V in 0.1 M
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Fig. 2. (A) CV responses of various modified electrodes in a 0.1 M KCl solution containing 2.0 mM K3 [Fe(CN)6 ]. Scan rate: 50 mV/s. (B) Nyquist relationships of EIS data performed in a 0.1 M KCl solution containing 5.0 mM K3 [Fe(CN)6 ]/K4 [Fe(CN)6 ] (1:1) for various modified electrodes. The amplitude and potential were 0.005 and 0.165 V, respectively. (a) Bare Au, (b) Au/MWNT/PPNWs, (c) Au/MWNT/PPNWs/GNPs, (d) Au/MWNT/PPNWs/GNPs/DNA-aptamer.
KCl solution containing 0.5 mM HAuCl4 at a sweep rate of 50 mV/s for 20 cycles. The modified electrode was obtained and denoted as Au/MWNT/PPNWs/GNPs. 2.3. Preparation of the DNA aptamer monolayer For this process, 4 L of the DNA aptamer (9.6 M HS-DNA) was placed on a previously conditioned Au electrode that was held upside down, and the end of the electrode was fitted with a plastic cup to protect the solution from evaporation. The assembly was kept standing for 3.0 h at room temperature. After rinsing, the modified electrode was treated with 1.0 mM 6-mercapto-1-hexanol (MCH) solution for 1 h to mask the unmodified Au sites. 2.4. Recognition and transduction After modification with the DNA aptamer monolayer, the modified electrode was used to recognize various concentrations of different signal-stranded DNA diluted with 1× SSC solution by incubation at room temperature for 1 h. After incubation, the modified electrode was then rinsed with PBS and DDW to remove the free DNA oligonucleotides. The electrode was then immersed in a 10 M solution of indicator for 20 min to perform electrochemical detection. 2.5. Electrochemical measurements CV measurements were performed in 2 mM K3 [Fe(CN)6 ] solution containing 0.1 M KCl with a scan rate of 50 mV/s. DPV measurements were performed in 0.1 M PBS by scanning from 0 to +0.40 V with an pulse amplitude of 10 mV, pulse width of 0.05 s, and pulse period of 0.2 s. EIS was performed in a 0.1 M KCl solution within the frequency range of 10−2 to 105 Hz using a 5.0 mM K3 [Fe(CN)6 ]/K4 [Fe(CN)6 ] (1:1) mixture as an electroactive probe. The amplitude and potential were 0.005 and 0.165 V, respectively.
peak current at the Au/MWNT/PPNWs and a 3.9-fold increase at the Au/MWNT/PPNWs/GNPs were observed compared to that of bare Au. A significant acceleration of the probe redox reaction occurred due to the presence of the assembly (MWNT/PPNWs and MWNT/PPNWs/GNPs). As shown in Fig. 2A-d, it was apparent that the oxidation peak currents (ip ) decreased dramatically to approximately 27 A. This was due to the DNA aptamer attached to the surface of the modified electrode, which led to an increase in the negative charge on the surface of the electrode and prevented access of negatively charged probe ions (e.g., the redox couple Fe(CN)6 ]3−/4− ) to the surface due to electrostatic repulsion. EIS is also a highly effective method for probing the features of a surface-modified electrode [32]. In EIS, the semicircle diameter of EIS reflects the electron transfer resistance, which controls the electron transfer kinetics of the redox-probe at the electrode interface. In this study, EIS was conducted to investigate the changes of electron transfer resistance that originated from each surface modification step as shown in Fig. 2B. These results were consistent with those obtained in CVs. The superior characteristics of the MWNT/PPNWs/GNPmodified electrode are illustrated by the CV and EIS results. This electrode had highly electrocatalytic activities and electronic conductivity and increased the electron transfer rate between electrode surface and redox probe. 3.2. Response studies of various modified electrodes The influences of MWNT, PPNWs and GNPs on the electrochemical detection of DNA were investigated using DPV measurements. As illustrated in Fig. 3, the bare Au (Fig. 3a) showed a very small DPV
3. Results and discussion 3.1. Electrochemical characterization of the modified electrodes CV is an effective method for probing the features of a surface-modified electrode using the redox probe Fe(CN)6 ]3−/4− . The CV curves of various modified electrodes were shown in Fig. 2A. It is clear from Fig. 2A-a that Fe(CN)6 ]3−/4− has a few well-defined redox peaks at the bare Au. However, the peak obviously increased after the electrode was modified with MWNT/PPNWs and MWNT/PPNWs/GNPs, respectively (curves Fig. 2A-b and c). An approximately 3.5-fold increase in the
Fig. 3. (A) DPV responses of indicator oxidation signals at different DNA aptamer modified electrodes after recognition with 1 M complementary target DNA sequence. (a) Bare Au, (b) Au/GNPs, (c) Au/PPNWs/GNPs, (d) Au/MWNT/ PPNWs/GNPs.
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Fig. 4. (A) The corresponding DPV responses of indicator at Au/MWNT/ PPNWs/GNPs/DNA-aptamer before recognition (a) and after recognition with the non-complementary DNA sequence (b), three-base mismatch DNA sequence (c), one-base mismatch DNA sequence (d) and complementary target DNA sequence (e). (B) The corresponding histograms. Error bars are ±RSD. Experimental conditions as described in the text.
peak current of the indicator whereas a large DPV peak current was observed after immobilizing GNPs onto the bare Au electrode (Fig. 3b). An approximately 4-fold increased peak current was obtained in comparison with that of bare Au. The current increase can be attributed mainly to the increased DNA aptamer immobilization on the GNPs modified layer. The Au/PPNWs/GNPs (Fig. 3c) resulted in excellent amplification of the indicator oxidation response with a large peak current value. An approximately 35-fold increased peak current was obtained in comparison with that of bare Au. This increase was because the PPNWs provided a porous structure with a large effective surface area. The indicator’s largest oxidation peak was observed at Au/MWNT/PPNWs/GNPs (Fig. 3d). The large current increase was mainly ascribed to the catalytic and conductive effects of MWNT. The response was also associated with the MWNT’s ability to improve the effective surface area of the modified electrode. This phenomenon clearly showed that the novel modified electrode provided a good environment for DNA aptamer immobilization and promoted the hybridization and preferred orientation of the DNA aptamer with the target sequence. 3.3. Sequence-specific recognition studies The DPV technique can provide better peak resolution and current sensitivity [33]. In addition, the charging current contribution to the background current, which is a limiting factor in the analytical determination, is negligible in DPV mode [34,35]. To test whether the method was sufficiently selective to distinguish complementary target DNA, the prepared electrode was reacted with the target in the buffer. The recognition of different ss-DNA using the modified electrodes was conducted with DPV using 10 M tris(1,10-phenanthroline) cobalt (III) perchlorate PBS solution (pH 7.4) as shown in Fig. 4A. It was apparent that similar responses were acquired at Au/MWNT/PPNWs/GNPs/HS-DNA before and after treatment with the non-complementary sequence (Fig. 4A-a and b). Almost no recognition occurred with the noncomplementary sequence. The signal increased slightly when the DNA aptamer modified GNPs were treated with the three-base mismatch sequence (Fig. 4A-c) suggesting that the recognition efficiency was not high. However, the signal increased substantially after the DNA aptamer modified GNPs were treated with a one-base mismatch (Fig. 4A-d) and complementary target sequence (Fig. 4Ae), respectively. This was due to the fact that the indicator cannot bind to the single-stranded DNA aptamer before recognition. After recognition, the response was associated with the redox process involving intercalation of the indicator into the double-stranded DNA duplex. The resulting histograms of the relevant experiments are displayed in Fig. 4B. Each sample was tested five times to verify the reproducibility. The relative standard deviation (RSD) of the
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Fig. 5. Semi-log calibration curve for complementary DNA with concentrations varying from 5.0 × 10−12 to 1.0 × 10−9 M. Error bars are ±RSD and n = 3. Upper inset shows DPV responses of the indicator at Au/MWNT/PPNWs/GNPs/DNA-aptamer after recognition with the following increasing concentration of the complementary target DNA: (a) 0, (b) 0.5, (c) 1, (d) 2.5, (e) 5, (f) 10, (g) 25, (h) 50, (i) 100, (j) 250, (k) 500, and (l) 1000 pM. Lower inset indicates the general curvilinear coordinate system for target DNA with concentrations varying from 5.0 × 10−12 to 1.0 × 10−9 M.
DNA aptamer modified electrode treated with complementary target DNA sequence was 5.0% (Fig. 4B-e), and the other recognition situation was lower than 4.7%. These results demonstrate that the complementary target DNA sequence could form double-stranded DNA with the DNA aptamer, which produces a significant increase in signal. The method allowed selective recognition of the DNA sequences with transduction. 3.4. Electrochemically quantitative recognition detection In this study, the sensitivity and selectivity of the sensor under the optimized detection conditions was tested using DPV. The upper inset of Fig. 5 shows an increasing oxidation peak current of the indicator with increasing the complementary target DNA concentration. The lower inset of Fig. 5 indicates the plotting of the current response with increasing the complementary target DNA concentration using a general curvilinear coordinate system. As shown in the insert, the complementary target DNA calibration followed a Langmuir-like adsorption curve [36]. The oxidation peak current of the indicator varies approximately linearly with the log(concentration) of the complementary target DNA over the range of 5.0 × 10−12 to 1.0 × 10−9 M with a correlation coefficient of 0.9863 (main panel, Fig. 5). The regression equation of the complementary target DNA was i (A) = 5.01828 log c(pM) + 1.48193. The detection limit (S/N = 3 compared with Table 1 The performance comparison of current fabricated DNA biosensor with reported in the literatures. Layers for DNA immobilization
Detection methods
Detection limit (mol L−1 )
References
Au NPs/CdS NPs GCE/MWNT-COOH Au NPs Chit and Au NPs Ppy Ag NPs MWNT/Au NPs PPy/ssDNA/MWNT paste Pt NPs and MWNT CPE CdS Nps and Ppy film MWNT/PPy/nano-Au
DPV DPV DPV DPV DPV ASV DPV DPV DPV PSA EIS DPV
2.0 × 10−11 1.0 × 10−10 5.0 × 10−10 5.0 × 10−11 1.0 × 10−10 5.0 × 10−13 6.2 × 10−12 8.5 × 10−11 1.0 × 10−11 4 × 10−9 1 × 10−9 4.3 × 10−13
[40] [41] [42] [43] [44] [45] [46] [47] [48] [49] [50] This work
Abbreviations: NPs, nanoparticles; Ppy, polypyrrole; EIS, electrochemical impedance spectroscopy; DPV, differential pulse voltammetry; GCE: glassy carbon electrode; Chit: Chitson; CPE: carbon paste electrode; PSA: Chronopotentiometric Stripping Analysis.
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a hybrid nanomaterial-modified electrode exposed to the noncomplementary sequence) [37] was 4.3 × 10−13 M. This sensitivity is as good as Pd-nanoparticles/MWNT modified electrode [38] and much better than hollow gold nanospheres (HGP) [39] and other similar nanomaterial-based electrodes (Table 1). 4. Conclusion A sensitive electrochemical method for the detection of avian influenza virus genotype using a DNA aptamer immobilized hybrid nanomaterial-modified electrode was developed. The modified electrode was assembled with MWNT, PPNWs and GNPs. The detection of target DNA sequences was accomplished not only via hybridization but also via dimensional preferred orientation. The biosensor has high recognition and selectivity for H5N1 specific sequence, and the signal of the indicator was approximately linear to log(concentration) of the H5N1 specific sequence from 5.0 × 10−12 to 1.0 × 10−9 M (R = 0.9863) with a detection limit of 4.3 × 10−13 M. Acknowledgements This work was supported by the National Natural Science Foundation of China (No. 21075078) and the Natural Science Foundation of Shandong province, China (No. ZR2010BM005). References [1] C. Qi, X.S. Tian, S. Chen, J.H. Yan, Z. Cao, K.G. Tian, G.F. Gao, G. Jin, Biosens. Bioelectron. 25 (2010) 1530. [2] T.H. Chua, T.M. Ellis, C.W. Wong, Y. Guan, S.X. Ge, G. Peng, C. Lamichhane, C. Maliadis, S.W. Tan, P. Selleck, J. Parkinson, Avian Dis. 51 (2007) 96. [3] W.J. Chen, B. He, C.G. Li, X.W. Zhang, W.L. Wu, X.Y. Yin, B.X. Fan, X.L. Fan, J. Wang, J. Med. Microbiol. 56 (2007) 603. [4] K. Tsukamoto, H. Ashizawa, K. Nakanishi, N. Kaji, K. Suzuki, M. Okamatsu, S. Yamaguchi, M. Mase, J. Clin. Microbiol. 46 (2008) 3048. [5] J.H. Chen, J. Zhang, K. Wang, L.Y. Huang, X.H. Lin, G.N. Chen, Electrochem. Commun. 10 (2008) 1448. [6] J.M. Picuri, B.M. Frezza, M.R. Ghadiri, J. Am. Chem. Soc. 131 (2009) 9368. [7] A.D. Ellington, J.W. Szostac, Nature 346 (1990) 818. [8] R.D. Jenison, S.C. Gill, A. Pardi, B. Polisky, Science 263 (1994) 1425. [9] C. Mannironi, A.D. Nardo, P. Fruscoloni, G.P. Tocchini-Valentini, Biochemistry 36 (1997) 9726. [10] M. Sassanfar, J.W. Szostak, Nature 364 (1993) 550.
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