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graphite hybrid microfiber structure
Bioelectrochemistry 128 (2019) 126–132
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Bioelectrochemistry journal homepage: www.elsevier.com/locate/bioelechem
Electrochemical detection of DNA hybridization based on threedimensional ZnO nanowires/graphite hybrid microfiber structure Jian Zhang a,b, Dong Han b, Ruiqi Yang a, Yanchen Ji a, Jing Liu c, Xin Yu a,⁎ a b c
Institute for Advanced Interdisciplinary Research (iAIR), University of Jinan, Jinan 250022, China Université de Lyon, Ecole Centrale de Lyon, UMR CNRS 5270, Institut des Nanotechnologies de Lyon, Ecully 69130, France The College of Life Sciences, Northwest University, Xi'an 710069, China
a r t i c l e
i n f o
Article history: Received 11 January 2019 Received in revised form 3 April 2019 Accepted 3 April 2019 Available online 06 April 2019 Keywords: DNA hybridization Electrochemical impedance spectroscopy Zinc oxide Graphite fibers Hybrids electrodes
a b s t r a c t In the present work, zinc oxide (ZnO) nanowire (NWs)/graphite microfiber hybrid electrodes were used for the detection of DNA hybridization. ZnO NWs were in-situ synthesized onto graphite fibers using the hydrothermal technique, and the result was characterized by X-ray diffraction (XRD), scanning electron microscope (SEM) as well as transmission electron microscope (TEM). An electrochemical sensor for the analysis of DNA hybridization was developed based on immobilizing DNA probes onto the ZnO NWs/graphite microfiber electrodes surface via electrostatic interaction. Cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and X-ray photoelectron spectroscopy (XPS) were used to corroborate the fabrication of the sensor. Based on the results, ZnO NWs/graphite microfiber hybrid electrodes showed a great hybridization capacity for the determination of target DNA. A wide dynamic detection range from 1.0 × 10−14 M to 1.0 × 10−7 M with an ultralow detection limit of 3.3 × 10−15 M was achieved for detecting the target DNA. Moreover, it showed good selectivity and stability during the detection process. © 2019 Elsevier B.V. All rights reserved.
1. Introduction DNA hybridization reaction has important applications in biologyrelated fields such as genetic detection, medicial bioengineering and clinical diagnostics, and has attracted considerable interest [1–3]. Various techniques have been employed for DNA analysis [4–7]. Among them, electrochemical method is a popular one because of its lowcost, high sensitivity and selectivity. In addition, electrochemical biosensors are suitable for miniaturization and are fully compatible with nanoelectronics. Among the various electroanalytical techniques, electrochemical impedance spectroscopy (EIS) is particularly suitable for probing the features of surface-modified electrodes. Molecular immobilization or biochemical recognition at electrode surfaces changes the capacitance and interfacial electron transfer resistance of electrodes. Relying on the charge-transfer kinetics of a redox couple, impedance spectroscopy can be described by a simple equivalent circuit model. The EIS technique is simple, sensitive, and label free. It has been widely used to monitor DNA hybridization analysis in many electrochemical biosensors [8–10]. To develop a high performance electrochemical DNA biosensors, the most important issue is how to immobilize DNA probes effectively, enabling fast hybridization. Immobilization activity is a critical element in ⁎ Corresponding author. E-mail address: [email protected] (X. Yu).
https://doi.org/10.1016/j.bioelechem.2019.04.003 1567-5394/© 2019 Elsevier B.V. All rights reserved.
the selection and development of the sensing materials [11,12]. With the development of nanotechnology, several nanomaterials have already been employed for the fabrication of DNA biosensor such as metal nanoparticles, carbon nanomaterials, semiconductor metal oxide, and two-dimensional transition metal sulfide, etc. [13–17]. Nanomaterials could provide large surface area to volume ratio for DNA immobilization and enhanced electron-transfer property. It can provide suitable microenvironment of high biocompatibility, nontoxicity and chemical stability. Various approaches of immobilizing DNA onto nanomaterial-modified electrodes have been developed. Au nanoparticles modified electrodes could immobilize thiolated DNA by the covalent Au-S bond [18]. Two dimensional nanosheets such as graphene and WS2 can bind single stranded DNA due to the noncovalent π-π stacking interactions, although they have less affinity towards double stranded DNA [19]. In addition, DNA oligonucleotides can bind onto metal oxide nanostructure surfaces via certain covalent linkers [20,21]. Zinc oxide (ZnO) is a wide band gap (3.37 eV) semiconductor, which has recently attracted more and more attention in solar cells, piezoelectric devices and ultraviolet laser applications [22–24]. ZnO also has high stability and good biocompatibility. In addition, ZnO nanostructures have the advantages of high specific surface area and high electron communication features, therefore gaining broad application as a receptor in biosensor designs [25–27]. It has quite high isoelectric point (IEP 9.5) and strong adsorption ability, which make it suitable for immobilization
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Scheme 1. Scheme of DNA probes immobilization onto ZnO NWs/graphite microfibers electrodes and their hybridization with the target DNA sequence.
of molecules of low isoelectric point (IEP 4.2) such as DNAs, and proteins, through electrostatic interaction. Previous reports have also mentioned the fabrication of ZnO nanostructure-modified electrodes as a biosensor utilizing the strong adsorption ability of ZnO for immobilizing molecules of low isoelectric point at neutral physiological pH [28–35]. Graphite fiber electrodes have been used as biosensor substrate recently due to their special one dimensional structure and high conductivity, which are particularly suitable for integrating with other devices and can even afford implantable devices for in vivo detection. Various nanomaterial hybrid structures synthesized in situ on the fiber surface have been developed for biosensing applications [36–39]. Fibrous electrodes with ZnO nanostructures have gained much attention in the applications of photodetector, photoelectrochemical cell, solar cells and photocatalysts [40–43]. To the best of our knowledge, there is currently no report on DNA hybridization detection using fibrous electrodes with ZnO nanostructure. In our work, ZnO nanowires (NWs)/graphite microfiber hybrid electrodes were designed and experiments have been conducted for the detection of DNA hybridization with improved performance. ZnO NWs were fabricated in situ on the surface immobilized graphite microfibers through a hydrothermal method. XRD (X-ray diffraction), SEM (Scanning Electron Microscope) and TEM (Transmission Electron Microscope) were used to study the crystal structure and morphology of the ZnO NWs/graphite microfiber hybrid structure. A fibroid electrochemical DNA biosensor was then fabricated by immobilizing DNA probes through electrostatic interaction. By XPS, CV and EIS measurements, the effect of pH value on DNA immobilization onto the ZnO hybrid electrodes were studied for the first time. As a result, the high DNA immobilization ability of ZnO NWs and the high sensitivity of EIS technique allows DNA detection with high sensitivity and selectivity.
2. Experimental section
2.2. Preparation of ZnO NWs/graphite microfibers Graphite microfibers were treated with oxygen plasma for cleaning, enhancing the hydrophilic of the surface. ZnO NWs were synthesized on graphite microfibers by a two-step seeding and hydrothermal method [44,45]. ZnO seeds were deposited on the surface of the graphite microfibers by thermally decomposing zinc acetate solution at 350 °C. Briefly, the graphite microfibers were immersed in 0.02 M zinc acetate in ethanol for 10 min, rinsed with clean ethanol, and then dried in the air. This step is repeated for five times, followed by thermal decomposition at 350 °C for 30 min. Then, ZnO NWs were grown on the seedcoated graphite microfibers by immersing in an aqueous solution of 16 mM Zn(NO3)2 and 25 mM HMTA. The temperature was maintained at 95 °C in an oven for 4 h. The prepared ZnO NWs/graphite microfibers were rinsed with double distilled water and dried at 40 °C in vacuum. In this work, the graphite microfibers were obtained from Toray (M40-JB12 K) (Japan). Each fiber bundle was composed of 12,000 microfibers. After pretreatment, ZnO NWs grew well on each microfiber. Even if the fibrous material with individual roots was not very well grown, it would not affect the overall property of the electrode surface. The electrodes were fabricated as follows: as-prepared ZnO NWs/graphite fibers were fixed on one side of a clean fluorine-doped tin oxide (FTO) glass, while a copper wire was fixed on the other side of the glass. Conductive silver adhesives were used to connect them and polydimethylsiloxane was used to encapsulate the FTO glass.
2.3. DNA immobilization and hybridization onto ZnO NWs/graphite microfibers electrode The ZnO NWs/graphite microfibers electrodes were incubated in 0.01 M PBS (pH 5, 7, 9, containing 0.1 M KCl) solution containing 60 × 10−6 M DNA probe at 25 °C for 8 h. Several washing steps with
2.1. Reagents and materials Zinc acetate dehydrate, hexamethylenetetramine, zinc nitrate hexahydrate, potassium chloride (KCl), potassium hexacyanoferrate (K4[Fe (CN)6]), potassium ferricyanide (K3[Fe(CN)6]), sodium dihydrogen phosphate anhydrous (NaH2PO4), and dibasic sodium phosphate (Na2HPO4) were obtained from China National Medicines Corporation Ltd. Graphite fibers were purchased from Toray (Japan). Phosphate buffer solutions (PBS, 0.1 M) with various pH values were prepared with Na2HPO4 and NaH2PO4 and buffed with 0.1 M H3PO4 or 0.1 M NaOH solution. Double distilled ion-free Water was obtained from Milli-Q academic systerm (Millipore, USA) and used to prepare all solutions. DNA strands were purchased from SBS Genetech Ltd. Company (Beijing, China). DNA sequences employed in this work were listed as follows: Probe DNA: 5′-HS-AAG CGG AGG ATT GAC GAC TA-3′. Complementary DNA: 5′-TAG TCG TCA ATC CTC CGC TT-3′. Non-complementary DNA: 5′-AAG CGG AGG ATT GAC GAC TA-3′. Single-base mismatched DNA: 5′-AAG TCG TCA ATC CTC CGC TT-3′.
Fig. 1. XRD patterns of (a) ZnO (JCPDS 36–1451), (b) graphite microfiber (JCPDS 65–6212) and (c) ZnO NWs/graphite microfiber.
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Fig. 2. SEM images of graphite microfiber (A and B), ZnO NWs/graphite microfiber (C and D), high resolution image of ZnO NWs (E), TEM image of ZnO NWs (F) (insets of HRTEM).
PBS buffer were repeated to remove the unbound DNA probes from the electrode. Hybridization reaction between the probe DNA and the target DNA was carried out by incubating the prepared probe DNA/ZnO NWs/ graphite microfibers electrode with the complementary target DNA, or with the single-base mismatched DNA, or else with a noncomplementary DNA for 120 s (Fig. S1). Several rounds of washing were then conducted on the hybridized microfibers electrodes after hybridization, for removing unbound target DNA. The electrochemical sensing strategy for DNA sensing mechanism is shown in the Scheme 1.
as the reference electrode. The working electrodes were the as-prepared microfiber electrode. Electrochemical impedance spectroscopy (EIS) measurements were performed in 0.01 M PBS (pH 7.4, containing 0.1 M KCl) containing 5 mM (K4[Fe(CN)6])/(K3[Fe(CN)6]) (1:1), scanning in the frequency range from 100 mHz to 100 kHz applying a 5 mV voltage amplitude. The electrical parameters of the system were fitted with a software package embedded in Gamry Reference 3000. All the data was taken from five independent experiments. Error bars represent the standard errors.
2.4. Surface characterization and electrochemical measurements
3. Results and discussion
The crystalline phase characteristics of the prepared ZnO NWs/ graphite microfibers were examined using X-ray Diffraction (XRD) (D8-advance, Bruker AXS, Germany). Morphology and structure of the prepared ZnO NWs/graphite microfibers were studied using a field emission scanning electron microscope (FESEM) (HITACHI S8020, Japan) and transmission electron microscopy (TEM) (JEM 2100F, Japan). Electrochemical measurements were performed on a Gamry 3000 electrochemical workstation (USA), in a standard threeelectrode cell system. There were a Pt plate (area, 1 cm × 1 cm) electrode as the counter electrode and a standard Ag/AgCl (saturated KCl)
3.1. Characterizations of ZnO NWs/graphite microfibers The XRD patterns for ZnO NWs/graphite microfiber is shown in Fig. 1. The diffraction peak of graphite microfiber (curve b) centered at 2θ =25.7 can be referenced as graphite (JCPDS 65–6212) [45]. On curve c, the peaks corresponding to (100), (002), (101) and (102), (110), (103) and (112) planes reveal that ZnO NWs grown on graphite microfibers have a hexagonal wurtzite structure (curve a). No peak corresponding to any other phase or compound appears, which indicates the existence of single phase ZnO NWs on graphite microfiber [46].
Fig. 3. Nyquist diagrams spectra (A) and CV response (B) for the graphite microfiber electrodes (a), ZnO NWs/graphite microfiber electrodes (b), probe DNA immobilized graphite fiber electrodes at pH 7.0 (c), probe DNA immobilized ZnO NWs/graphite microfiber electrodes at different pH: 7.0 (d), 5.0 (e) and 9.0 (f) recorded in 0.01 M PBS containing 5 mM [Fe(CN)6]3−/4− and 0.1 M KCl (pH 7.4).
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Fig. 4. XPS spectra of (A) N 1 s and (B) P 2p for the ZnO NWs/graphite microfiber electrodes (a), probe DNA immobilized ZnO NWs/graphite microfiber electrodes at different pH: 5.0 (b), 9.0 (c) and 7.0 (d).
Morphology of the non-modified graphite microfiber and the ZnO NWs/graphite microfiber have been examined and compared with SEM. Graphite microfibers are about 6 μm in diameter and have smooth surfaces (Fig. 2A, B). Images of the ZnO NWs/graphite microfiber image show that the graphite microfiber is densely buried by ZnO NWs with a size of about 100–150 nm (Fig. 2C, D, E). A high-resolution TEM (HRTEM) image (inset of Fig. 2F) taken from the edge of ZnO NWs indicates that the ZnO NWs grow along the [002] direction [46]. 3.2. Probe DNA immobilization on ZnO nanowires/graphite microfibers EIS and CV were used to characterize the immobilization of the DNA probes on the ZnO NWs/graphite microfiber electrodes, using [Fe(CN) 3−/4− as a redox probe. The immobilization time and concentration 6] of DNA probe were optimized (Fig. S2 and S3). The impedance of spectra includes a semicircle portion at higher frequencies corresponding to the electro transfer resistance (Rct) and a linear portion at lower frequencies corresponding to the diffusion process at the interface of the electrode and solution (Fig. 3A). Compared with the bare microfiber electrode (curve a), the ZnO NWs/graphite microfiber electrodes (curve b) shows a lower Rct due to the high surface area and accelerated electron transfer of ZnO nanowires. It is worth nothing that after immobilization of probe DNA onto the graphite microfiber electrode, the Rct had hardly changed, which indicates that the graphite microfiber electrode has no DNA adsorption ability (curve c). However, upon immobilization of probe DNA onto the ZnO NWs /graphite microfiber electrodes at a set of different pH values, the Rct changed differently. At pH 7.0, Rct increased significantly (curve d). Because the negative charges of DNA repel the negatively charged redox probes [Fe(CN)6]3−/4−. The steric hindrance introduced with the formation of DNA layer on the ZnO NWs/graphite microfiber electrode surface also prevents transfer of electrons [47,48]. At this physiological pH value, ZnO having a relatively high isoelectric point (IEP 9.5) is positively charged, and DNA having a relatively low isoelectric point (IEP 4.2) is negatively charged, which is suitable for the immobilization of DNA onto the ZnO NWs/graphite microfiber electrodes via the strong electrostatic interaction. However, at pH 5.0 and 9.0, there is no significant increase of Rct value (curve e and f). A small increase may result from some weak adsorption of DNA onto the ZnO NWs/graphite microfiber electrodes. It demonstrates that pH value affects the assembly of DNA on the ZnO NWs/graphite microfiber electrodes. If the selected pH is close to the isoelectric point of DNA or ZnO, DNA cannot be assembled efficiently on the ZnO NWs/ graphite microfiber electrode surface. At the physiological pH value, DNA can be assembled efficiently on the ZnO NWs/graphite microfiber electrode surface via strong electrostatic interaction. EIS has also been
used to study the ZnO NWs/graphite microfiber electrodes immersed in 0.01 M PBS (pH 5.0, 7.0, 9.0, containing 0.1 M KCl) solution without the DNA probe (Fig. S4). After incubation in the pure PBS solution, EIS response has hardly changed, which reveals that the significant increase of Rct value of probe DNA immobilized ZnO NWs/graphite microfiber electrodes is indeed resulted from the introduction of DNA molecules. Similar DNA probes without the thiol group was used to immobilize the ZnO NWs/graphite fibers electrode surface, the EIS response has hardly changed compared with the DNA probe with the thiol group (Fig. S5). It seems that the electrostatic interaction occurs between the positively charged ZnO NWs and the negatively charged phosphate backbone of the DNA [34]. CV response of the DNA assembly process on ZnO NWs/graphite microfiber electrodes has also been studied (Fig. 3B), which is consistent with that of the EIS measurements. X-ray photoelectron spectroscopic (XPS) has been used to study the immobilization of DNA molecules on the electrode surface because of the nitrogen and phosphorus elements having evident XPS response. As shown in Fig. 4A, compared with the ZnO NWs/graphite microfiber electrodes (curve a), an intensive and obvious peak of N1 s can be detected after the immobilization of DNA probe at pH 7.0, indicating that the DNA probes were successfully assembled on the ZnO NWs/graphite microfiber electrodes surface (curve d). When immobilizing the DNA
Fig. 5. Nyquist diagrams of probe DNA immobilized ZnO NWs/graphite microfiber electrodes before (a) and after (b) hybridization with target DNA (1.0 × 10−11 M) recorded in 0.01 M PBS containing 5 mM [Fe(CN)6]3−/4− and 0.1 M KCl (pH 7.4). Inset: equivalent circuit used to fit the impedance data.
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Fig. 6. (A) Nyquist diagrams at probe DNA immobilized ZnO NWs/graphite microfiber electrodes after hybridization with complementary target DNA of different concentrations in 0.01 M PBS containing 5 mM [Fe(CN)6]3−/4− and 0.1 M KCl (pH 7.4): a–h 1.0 × 10−14, 1.0 × 10−13, 1.0 × 10−12, 1.0 × 10−11, 1.0 × 10−10, 1.0 × 10−9, 1.0 × 10−8, 1.0 × 10−7 M. (B) The plot of Δ Rct (Δ Rct = |Rct, target − Rct, probe|) versus complementary target DNA concentration.
probe at pH 5.0 and 9.0 (curve b, c), the XPS spectrum shows a weak signal of N1 s, resulting from the weak adsorption of DNA on the ZnO NWs/ graphite microfiber electrodes. On the P2p spectra, the increase of peak intensity is consistent with the increase in phosphorus content (Fig. 4B). The XPS results also confirmed the successful immobilization of DNA onto the ZnO NWs/graphite microfiber electrodes surface. 3.3. EIS detection of target DNA Fig. 5 shows the impedance diagrams of DNA probe modified ZnO NWs/graphite microfiber electrodes before and after hybridization with 1.0 × 10−11 M complementary target DNA. After hybridization reaction, double-stranded helix between the probe and target DNA are formed on electrodes surface and will block the electron transfer process of [Fe(CN)6]3−/4− through the electrostatic repulsion and the steric hindrance effect. It leads to the increase of Rct [47,48]. An equivalent circuit (inset Fig. 5) was used to fit the EIS data. Rs representing the solution-phase resistance. Rct represents the charge-transfer resistance, which is inversely proportional to the rate of electron transfer. Zw corresponds to the Warburg impedance, which results from mass-transfer limitations. CPE (constant phase element) corresponds to the capacitance of the double layer. Fig. 6A shows the Nyquist plots of DNA probe modified ZnO NWs/ graphite microfiber electrodes hybridizing with the complementary target DNA at different concentrations from 1.0 × 10−14 M to 1.0 × 10−7 M. It can be seen that the Rct value increases with the increasing
concentration of target DNA. The variation of the Rct value (Δ Rct) before and after each hybridization reaction is used as the sensing readout signal, having a linear relationship with the logarithmic of the concentration of the target DNA, in the range from 1.0 × 10−14 M to 1.0 × 10−7 M (Fig. 6B). The observed dependence of the Rct component on the logarithm of analyte concentration directly reflects the fact that more and more DNA strands have been captured on the electrodesolution interface, therefore impairing the efficiency of interface charge transfer, resulting in larger and larger Rct component as we have observed. The specific logarithm dependence is mostly possible resulted from gradual saturation of the surface-immobilized DNA binding sites. The detection limit is estimated to be 3.3 × 10−15 M at a signal-tonoise (S/N) ratio of 3, which indicates that the label-free electrochemical strategy based on ZnO NWs/graphite microfiber electrodes exhibits high sensitivity. The selectivity of this DNA biosensor based on ZnO NWs/graphite microfiber electrodes is further assessed by hybridization with control target DNA sequences (fully complementary, single-base mismatched, and non-complementary sequences), as shown in Fig. 7. A significant increase of Δ Rct is observed for the fully complementary sequence (curve c), suggesting that the hybridization reaction works well in this case. A less evident increase of Δ Rct is obtained after hybridization with singlebase mismatched sequences (curve b), while there is no remarkable Δ Rct change after hybridization with the non-complementary sequence (curve a). It demonstrates that the developed ZnO NWs/graphite microfiber electrodes biosensor displays a high selectivity for DNA
Fig. 7. (A) Nyquist diagrams at probe DNA immobilized ZnO NWs/graphite microfiber electrodes after hybridization with 1.0 × 10−11 M DNA sequences: non-complementary DNA (a), single-mismatched DNA (b) and complementary target DNA (c) in 0.01 M PBS containing 5 mM [Fe(CN)6]3−/4− and 0.1 M KCl (pH 7.4. (B) The plot of Δ Rct (Δ Rct = |Rct, target − Rct, probe|) corresponding to hybridization with 1.0 × 10−11 M different DNA sequences: non-complementary DNA (a), single-mismatched DNA (b) and complementary target DNA (c).
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hybridization detection. Moreover, the DNA probe immobilized ZnO NWs/graphite microfiber electrode has also immersed in bovine serum albumin (BSA) solution (2.5 g/L) for 120 s at room temperature before DNA detection. As shown in Fig. S6, compared with the DNA probe immobilized ZnO NWs/graphite microfiber electrode (curve a), Rct slightly changed when BSA is used (curve b), which suggested that BSA is hardly adsorbed by ZnO NWs. However, after hybridization with the target complementary DNA, Rct can be seen enhanced significantly. These results reveal that the non-specific adsorption has no significant effect on the DNA hybridization process. In addition, the biosensor shows good reproducibility and stability for the detection of DNA (Fig. S7 and S8). Furthermore, SEM images of ZnO NWs/graphite microfiber electrodes after DNA probe immobilization and after ten cycles of usage have been recorded (Fig. S9). The electrode surface has hardly changed, which reveals good stability of ZnO NWs/graphite microfiber structure. 4. Conclusion In this work, a novel, simple, sensitive and label-free DNA hybridization detection platform based on ZnO NWs/graphite microfiber electrodes was constructed. ZnO nanowires were in situ synthesized on the graphite microfibers to form a three-dimensional electrode structure for immobilization of DNA probe via strong electrostatic interaction. XPS, CV and EIS studies conformed the successful immobilization of the DNA probe onto the ZnO nanowires under neutral condition. The developed biosensor was successfully applied to the impedance detection of DNA hybridization and demonstrated good selectivity and remarkable detection limit. In summary, the proposed DNA hybridization detection platform based on the ZnO NWs/graphite microfiber electrodes presents a promising strategy for DNA detection in the future. Moreover, because of its fiber morphology and flexibility, it may become an excellent DNA monitoring candidate for cellular or even in vivo applications in the future. Acknowledgments The work was supported by the Major Program of Shandong Province Natural Science Foundation (No. ZR2018ZC0843), Natural Science Foundation of Shandong Province (No. ZR2018BEM010), the National Natural Science Foundation of China (No. 51802115). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.bioelechem.2019.04.003. References [1] J. Guo, J. Wang, J. Zhao, Z. Guo, Y, Zhang, ultrasensitive multiplexed immunoassay for tumor biomarkers based on DNA hybridization chain reaction amplifying signal, ACS Appl. Mater. Interfaces 8 (2016) 6898–6904. [2] Z. Cheglakov, T.M. Cronin, C. He, Y. Weizmann, Live cell microRNA imaging using cascade hybridization reaction, J. Am. Chem. Soc. 137 (2015) 6116–6119. [3] S.X. Chen, G. Seelig, An engineered kinetic amplification mechanism for single nucleotide variant discrimination by DNA hybridization probes, J. Am. Chem. Soc. 138 (2016) 5076–5086. [4] D. Xia, J. Yan, S. Hou, Fabrication of nanofluidic biochips with nanochannels for applications in DNA analysis, Small 8 (2012) 2787–2801. [5] F. Wallet, S. Nseir, L. Baumann, S. Herwegh, B. Sendid, M. Boulo, M. RousselDelvallez, A.V. Durocher, R.J. Courcol, Preliminary clinical study using a multiplex real-time PCR test for the detection of bacterial and fungal DNA directly in blood, Clin. Microbiol. Infec. 16 (2010) 774–779. [6] C. Zhu, Z. Zeng, H. Li, F. Li, C. Fan, H. Zhang, Single-layer MoS2-based nanoprobes for homogeneous detection of biomolecules, J. Am. Chem. Soc. 135 (2013) 5998–6001. [7] Y. Zhang, B. Zheng, C. Zhu, X. Zhang, C. Tan, H. Li, B. Chen, J. Yang, J. Chen, Y. Huang, L. Wang, H. Zhang, Single-layer transition metal dichalcogenide nanosheets-based nanosensors for rapid, sensitive, and multiplexed detection of DNA, Adv. Mater. 27 (2015) 935–939. [8] S.Z. Mousavisani, J.B. Raoof, R. Ojani, Z. Bagheryan, An impedimetric biosensor for DNA damage detection and study of the protective effect of deferoxamine against DNA damage, Bioelectrochemistry 122 (2018) 142–148.
131
[9] A. Hájková, J. Barek, V. Vyskočil, Electrochemical DNA biosensor for detection of DNA damage induced by hydroxyl radicals, Bioelectrochemistry 116 (1–9) (2017). [10] A.A. Ensafi, N. Kazemnadi, M. Amini, B. Rezaei, Impedimetric DNA-biosensor for the study of dopamine induces DNA damage and investigation of inhibitory and repair effects of some antioxidants, Bioelectrochemistry 104 (2015) 71–78. [11] V.C. Diculescu, A.M. Chiorcea-Paquim, A.M. Oliveira-Brett, Applications of a DNAelectrochemical biosensor, Trac-Trend. Anal. Chem. 79 (2016) 23–36. [12] M.A. Tabrizi, M. Shamsipur, A label-free electrochemical DNA biosensor based on covalent immobilization of salmonella DNA sequences on the nanoporous glassy carbon electrode, Biosens. Bioelectron. 69 (2015) 100–105. [13] J. Lee, M. Morita, K. Takemura, E.Y. Park, A multi-functional gold/iron-oxide nanoparticle-CNT hybrid nanomaterial as virus DNA sensing platform, Biosens. Bioelectron. 102 (2018) 425–431. [14] M. Fojta, A. Daňhel, L. Havran, V. Vyskočil, Recent progress in electrochemical sensors and assays for DNA damage and repair, Trac-Trend. Anal. Chem. 79 (2016) 160–167. [15] H. Huang, W. Bai, C. Dong, R. Guo, Z. Liu, An ultrasensitive electrochemical DNA biosensor based on graphene/au nanorod/polythionine for human papillomavirus DNA detection, Biosens. Bioelectron. 68 (2015) 442–446. [16] H. Gao, M. Sun, C. Lin, S. Wang, Electrochemical DNA biosensor based on graphene and TiO2 nanorods composite film for the detection of transgenic soybean gene sequence of MON89788, Electroanalysis 24 (2012) 2283–2290. [17] M.R. Saidur, A.A. Aziz, W.J. Basirun, Recent advances in DNA-based electrochemical biosensors for heavy metal ion detection: a review, Biosens. Bioelectron. 90 (2017) 125–139. [18] J. Wang, A. Shi, X. Fang, X. Han, Y. Zhang, An ultrasensitive super sandwich electrochemical DNA biosensor based on gold nanoparticles decorated reduced graphene oxide, Anal. Biochem. 469 (2015) 71–75. [19] G. Yang, C. Zhu, D. Du, J. Zhu, Y. Lin, Graphene-like two-dimensional layered nanomaterials: applications in biosensors and nanomedicine, Nanoscale 7 (2015) 14217–14231. [20] C. Wang, N. Huang, H. Zhuang, X. Jiang, Enhanced performance of nanocrystalline ZnO DNA biosensor via introducing electrochemical covalent biolinkers, ACS Appl. Mater. Inter. 7 (2015) 7605–7612. [21] A. Hájková, J. Barek, V. Vyskočil, Voltammetric determination of 2-Aminofluoren-9one and investigation of its interaction with DNA on a glassy carbon electrode, Electroanal. 27 (2015) 101–110. [22] D.Y. Son, K.H. Bae, H.S. Kim, N.G. Park, Effects of seed layer on growth of ZnO nanorod and performance of perovskite solar cell, J. Phys. Chem. C 119 (2015) 10321–10328. [23] H. Li, N. Liu, X. Zhang, J. Su, L. Li, Y. Gao, Z.L. Wang, Piezotronic and piezo-phototronic logic computations using au decorated ZnO microwires, Nano Energy 27 (2016) 587–594. [24] P.S. Shewale, Y.S. Yu, Structural, surface morphological and UV photodetection properties of pulsed laser deposited Mg-doped ZnO nanorods: effect of growth time, J. Alloy. Compd. 654 (2016) 79–86. [25] X. Cao, X. Cao, H. Guo, T. Li, Y. Jie, N. Wang, Z.L. Wang, Piezotronic effect enhanced label-free detection of DNA using a schottky-contacted ZnO nanowire biosensor, ACS Nano 10 (2016) 8038–8044. [26] T. Kong, Y. Chen, Y. Ye, K. Zhang, Z. Wang, X. Wang, An amperometric glucose biosensor based on the immobilization of glucose oxidase on the ZnO nanotubes, Sensor. Actuat. B-Chem. 138 (2009) 344–350. [27] A. Tereshchenko, M. Bechelany, R. Viter, V. Khranovskyy, V. Smyntyna, N. Starodub, R. Yakimova, Optical biosensors based on ZnO nanostructures: advantages and perspectives. A review, Sensor. Actuat. B-Chem. 229 (2016) 664–677. [28] S.P. Singh, S.K. Arya, P. Pandey, B.D. Malhotra, S. Saha, K. Sreenivas, V. Gupta, Cholesterol biosensor based on rf sputtered zinc oxide nanoporous thin film, Appl. Phys. Lett. 91 (2007), 063901. [29] S. Saha, V. Gupta, K. Sreenivas, H.H. Tan, C. Jagadish, Third generation biosensing matrix based on Fe-implanted ZnO thin film, Appl. Phys. Lett. 97 (2010), 133704. [30] A. Wei, X.W. Sun, J.X. Wang, Y. Lei, X.P. Cai, C.M. Li, Z.L. Dong, W. Huang, Enzymatic glucose biosensor based on ZnO nanorod array grown by hydrothermal decomposition, Appl. Phys. Lett. 89 (2006), 123902. [31] J.X. Wang, X.W. Sun, A. Wei, Y. Lei, X.P. Cai, C.M. Li, Z.L. Dong, Zinc oxide nanocomb biosensor for glucose detection, Appl. Phys. Lett. 88 (2006), 233106. [32] M. Das, G. Sumana, R. Nagarajan, B.D. Malhotra, Application of nanostructured ZnO films for electrochemical DNA biosensor, Thin Solid Films 519 (2010) 1196–1201. [33] A.A. Ansari, R. Singh, G. Sumana, B.D. Malhotra, Sol–gel derived nano-structured zinc oxide film for sexually transmitted disease sensor, Analyst 134 (2009) 997–1002. [34] M. Tak, V. Gupta, M. Tomar, Flower-like ZnO nanostructure based electrochemical DNA biosensor for bacterial meningitis detection, Biosens. Bioelectron. 59 (2014) 200–207. [35] M. Tak, V. Gupta, M. Tomar, An electrochemical DNA biosensor based on Ni doped ZnO thin film for meningitis detection, J. Electroanaly. Chem. 792 (2017) 8–14. [36] J. Zhang, A. Li, X. Yu, W. Guo, Z. Zhao, J. Qiu, X. Mou, J.P. Claverie, H. Liu, Scaly graphene oxide/graphite fiber hybrid electrodes for DNA biosensors, Adv. Mater. Interfaces 2 (2015), 1500072. [37] J. Zhang, X. Yu, W. Guo, J. Qiu, X. Mou, A. Li, H. Liu, Construction of titanium dioxide nanorod/graphite microfiber hybrid electrodes for a high performance electrochemical glucose biosensor, Nanoscale 8 (2016) 9382–9389. [38] M. Kang, Y. Lee, H. Jung, J.H. Shim, N.S. Lee, J.M. Baik, C.S. Lee, C. Lee, Y. Lee, M.H. Kim, Single carbon fiber decorated with RuO2 nanorods as a highly electrocatalytic sensing element, Anal. Chem. 84 (2012) 9485–9491. [39] J. Du, R. Yue, F. Ren, Z. Yao, F. Jiang, P. Yang, Y. Du, Novel graphene flowers modified carbon fibers for simultaneous determination of ascorbic acid, dopamine and uric acid, Biosens. Bioelectron. 53 (2014) 220–224.
132
J. Zhang et al. / Bioelectrochemistry 128 (2019) 126–132
[40] H.E. Unalan, D. Wei, K. Suzuki, S. Dalal, P. Hiralal, H. Matsumoto, S. Imaizumi, M. Minagawa, A. Tanioka, A.J. Flewitt, W.I. Milne, G.A.J. Amaratunga, Photoelectrochemical cell using dye sensitized zinc oxide nanowires grown on carbon fibers, Appl. Phys. Lett. 93 (2008), 133116. [41] X. Xue, W. Zang, P. Deng, Q. Wang, L. Xing, Y. Zhang, Z. L. piezo-potential enhanced photocatalytic degradation of organic dye using ZnO nanowires, Nano Energy 13 (2015) 414–422. [42] F. Zhang, S. Niu, W. Guo, G. Zhu, Y. Liu, X. Zhang, Z.L. Wang, Piezo-phototronic effect enhanced visible/UV photodetector of a carbon-fiber/ZnO-CdS double-shell microwire, ACS Nano 7 (2013) 4537–4544. [43] I. Shakir, M. Shahid, U.A. Rana, I.M. Al Nashef, R. Hussain, Nickel-cobalt layered double hydroxide anchored zinc oxide nanowires grown on carbon fiber cloth for highperformance flexible pseudocapacitive energy storage devices, Electrochim. Acta 129 (2014) 28–32.
[44] L.E. Greene, M. Law, D.H. Tan, M. Montano, J. Goldberger, G. Somorjai, P. Yang, General route to vertical ZnO nanowire arrays using textured ZnO seeds, Nano Lett. 5 (2005) 1231–1236. [45] J. Zhang, D. Han, S. Wang, X. Zhang, R. Yang, Y. Ji, X. Yu, Electrochemical detection of adenine and guanine based on a three-dimensional WS2 nanosheet/ graphite microfiber hybrid electrode, Electrochem. Commun. 99 (2019) 75–80. [46] H. Chen, L. Zhu, H. Liu, W. Li, Growth of ZnO nanowires on fibers for onedimensional flexible quantum dot-sensitized solar cells, Nanotechnology 23 (2012), 075402. [47] A. Bonanni, M. Pumera, Graphene platform for hairpin-DNA-based impedimetric genosensing, ACS Nano 5 (2011) 2356–2361. [48] A. Li, J. Zhang, J. Qiu, Z. Zhao, C. Wang, C. Zhao, H. Liu, A novel aptameric biosensor based on the self-assembled DNA-WS2 nanosheet architecture, Talanta 163 (2017) 78–84.
Contents lists available at ScienceDirect
Bioelectrochemistry journal homepage: www.elsevier.com/locate/bioelechem
Electrochemical detection of DNA hybridization based on threedimensional ZnO nanowires/graphite hybrid microfiber structure Jian Zhang a,b, Dong Han b, Ruiqi Yang a, Yanchen Ji a, Jing Liu c, Xin Yu a,⁎ a b c
Institute for Advanced Interdisciplinary Research (iAIR), University of Jinan, Jinan 250022, China Université de Lyon, Ecole Centrale de Lyon, UMR CNRS 5270, Institut des Nanotechnologies de Lyon, Ecully 69130, France The College of Life Sciences, Northwest University, Xi'an 710069, China
a r t i c l e
i n f o
Article history: Received 11 January 2019 Received in revised form 3 April 2019 Accepted 3 April 2019 Available online 06 April 2019 Keywords: DNA hybridization Electrochemical impedance spectroscopy Zinc oxide Graphite fibers Hybrids electrodes
a b s t r a c t In the present work, zinc oxide (ZnO) nanowire (NWs)/graphite microfiber hybrid electrodes were used for the detection of DNA hybridization. ZnO NWs were in-situ synthesized onto graphite fibers using the hydrothermal technique, and the result was characterized by X-ray diffraction (XRD), scanning electron microscope (SEM) as well as transmission electron microscope (TEM). An electrochemical sensor for the analysis of DNA hybridization was developed based on immobilizing DNA probes onto the ZnO NWs/graphite microfiber electrodes surface via electrostatic interaction. Cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and X-ray photoelectron spectroscopy (XPS) were used to corroborate the fabrication of the sensor. Based on the results, ZnO NWs/graphite microfiber hybrid electrodes showed a great hybridization capacity for the determination of target DNA. A wide dynamic detection range from 1.0 × 10−14 M to 1.0 × 10−7 M with an ultralow detection limit of 3.3 × 10−15 M was achieved for detecting the target DNA. Moreover, it showed good selectivity and stability during the detection process. © 2019 Elsevier B.V. All rights reserved.
1. Introduction DNA hybridization reaction has important applications in biologyrelated fields such as genetic detection, medicial bioengineering and clinical diagnostics, and has attracted considerable interest [1–3]. Various techniques have been employed for DNA analysis [4–7]. Among them, electrochemical method is a popular one because of its lowcost, high sensitivity and selectivity. In addition, electrochemical biosensors are suitable for miniaturization and are fully compatible with nanoelectronics. Among the various electroanalytical techniques, electrochemical impedance spectroscopy (EIS) is particularly suitable for probing the features of surface-modified electrodes. Molecular immobilization or biochemical recognition at electrode surfaces changes the capacitance and interfacial electron transfer resistance of electrodes. Relying on the charge-transfer kinetics of a redox couple, impedance spectroscopy can be described by a simple equivalent circuit model. The EIS technique is simple, sensitive, and label free. It has been widely used to monitor DNA hybridization analysis in many electrochemical biosensors [8–10]. To develop a high performance electrochemical DNA biosensors, the most important issue is how to immobilize DNA probes effectively, enabling fast hybridization. Immobilization activity is a critical element in ⁎ Corresponding author. E-mail address: [email protected] (X. Yu).
https://doi.org/10.1016/j.bioelechem.2019.04.003 1567-5394/© 2019 Elsevier B.V. All rights reserved.
the selection and development of the sensing materials [11,12]. With the development of nanotechnology, several nanomaterials have already been employed for the fabrication of DNA biosensor such as metal nanoparticles, carbon nanomaterials, semiconductor metal oxide, and two-dimensional transition metal sulfide, etc. [13–17]. Nanomaterials could provide large surface area to volume ratio for DNA immobilization and enhanced electron-transfer property. It can provide suitable microenvironment of high biocompatibility, nontoxicity and chemical stability. Various approaches of immobilizing DNA onto nanomaterial-modified electrodes have been developed. Au nanoparticles modified electrodes could immobilize thiolated DNA by the covalent Au-S bond [18]. Two dimensional nanosheets such as graphene and WS2 can bind single stranded DNA due to the noncovalent π-π stacking interactions, although they have less affinity towards double stranded DNA [19]. In addition, DNA oligonucleotides can bind onto metal oxide nanostructure surfaces via certain covalent linkers [20,21]. Zinc oxide (ZnO) is a wide band gap (3.37 eV) semiconductor, which has recently attracted more and more attention in solar cells, piezoelectric devices and ultraviolet laser applications [22–24]. ZnO also has high stability and good biocompatibility. In addition, ZnO nanostructures have the advantages of high specific surface area and high electron communication features, therefore gaining broad application as a receptor in biosensor designs [25–27]. It has quite high isoelectric point (IEP 9.5) and strong adsorption ability, which make it suitable for immobilization
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Scheme 1. Scheme of DNA probes immobilization onto ZnO NWs/graphite microfibers electrodes and their hybridization with the target DNA sequence.
of molecules of low isoelectric point (IEP 4.2) such as DNAs, and proteins, through electrostatic interaction. Previous reports have also mentioned the fabrication of ZnO nanostructure-modified electrodes as a biosensor utilizing the strong adsorption ability of ZnO for immobilizing molecules of low isoelectric point at neutral physiological pH [28–35]. Graphite fiber electrodes have been used as biosensor substrate recently due to their special one dimensional structure and high conductivity, which are particularly suitable for integrating with other devices and can even afford implantable devices for in vivo detection. Various nanomaterial hybrid structures synthesized in situ on the fiber surface have been developed for biosensing applications [36–39]. Fibrous electrodes with ZnO nanostructures have gained much attention in the applications of photodetector, photoelectrochemical cell, solar cells and photocatalysts [40–43]. To the best of our knowledge, there is currently no report on DNA hybridization detection using fibrous electrodes with ZnO nanostructure. In our work, ZnO nanowires (NWs)/graphite microfiber hybrid electrodes were designed and experiments have been conducted for the detection of DNA hybridization with improved performance. ZnO NWs were fabricated in situ on the surface immobilized graphite microfibers through a hydrothermal method. XRD (X-ray diffraction), SEM (Scanning Electron Microscope) and TEM (Transmission Electron Microscope) were used to study the crystal structure and morphology of the ZnO NWs/graphite microfiber hybrid structure. A fibroid electrochemical DNA biosensor was then fabricated by immobilizing DNA probes through electrostatic interaction. By XPS, CV and EIS measurements, the effect of pH value on DNA immobilization onto the ZnO hybrid electrodes were studied for the first time. As a result, the high DNA immobilization ability of ZnO NWs and the high sensitivity of EIS technique allows DNA detection with high sensitivity and selectivity.
2. Experimental section
2.2. Preparation of ZnO NWs/graphite microfibers Graphite microfibers were treated with oxygen plasma for cleaning, enhancing the hydrophilic of the surface. ZnO NWs were synthesized on graphite microfibers by a two-step seeding and hydrothermal method [44,45]. ZnO seeds were deposited on the surface of the graphite microfibers by thermally decomposing zinc acetate solution at 350 °C. Briefly, the graphite microfibers were immersed in 0.02 M zinc acetate in ethanol for 10 min, rinsed with clean ethanol, and then dried in the air. This step is repeated for five times, followed by thermal decomposition at 350 °C for 30 min. Then, ZnO NWs were grown on the seedcoated graphite microfibers by immersing in an aqueous solution of 16 mM Zn(NO3)2 and 25 mM HMTA. The temperature was maintained at 95 °C in an oven for 4 h. The prepared ZnO NWs/graphite microfibers were rinsed with double distilled water and dried at 40 °C in vacuum. In this work, the graphite microfibers were obtained from Toray (M40-JB12 K) (Japan). Each fiber bundle was composed of 12,000 microfibers. After pretreatment, ZnO NWs grew well on each microfiber. Even if the fibrous material with individual roots was not very well grown, it would not affect the overall property of the electrode surface. The electrodes were fabricated as follows: as-prepared ZnO NWs/graphite fibers were fixed on one side of a clean fluorine-doped tin oxide (FTO) glass, while a copper wire was fixed on the other side of the glass. Conductive silver adhesives were used to connect them and polydimethylsiloxane was used to encapsulate the FTO glass.
2.3. DNA immobilization and hybridization onto ZnO NWs/graphite microfibers electrode The ZnO NWs/graphite microfibers electrodes were incubated in 0.01 M PBS (pH 5, 7, 9, containing 0.1 M KCl) solution containing 60 × 10−6 M DNA probe at 25 °C for 8 h. Several washing steps with
2.1. Reagents and materials Zinc acetate dehydrate, hexamethylenetetramine, zinc nitrate hexahydrate, potassium chloride (KCl), potassium hexacyanoferrate (K4[Fe (CN)6]), potassium ferricyanide (K3[Fe(CN)6]), sodium dihydrogen phosphate anhydrous (NaH2PO4), and dibasic sodium phosphate (Na2HPO4) were obtained from China National Medicines Corporation Ltd. Graphite fibers were purchased from Toray (Japan). Phosphate buffer solutions (PBS, 0.1 M) with various pH values were prepared with Na2HPO4 and NaH2PO4 and buffed with 0.1 M H3PO4 or 0.1 M NaOH solution. Double distilled ion-free Water was obtained from Milli-Q academic systerm (Millipore, USA) and used to prepare all solutions. DNA strands were purchased from SBS Genetech Ltd. Company (Beijing, China). DNA sequences employed in this work were listed as follows: Probe DNA: 5′-HS-AAG CGG AGG ATT GAC GAC TA-3′. Complementary DNA: 5′-TAG TCG TCA ATC CTC CGC TT-3′. Non-complementary DNA: 5′-AAG CGG AGG ATT GAC GAC TA-3′. Single-base mismatched DNA: 5′-AAG TCG TCA ATC CTC CGC TT-3′.
Fig. 1. XRD patterns of (a) ZnO (JCPDS 36–1451), (b) graphite microfiber (JCPDS 65–6212) and (c) ZnO NWs/graphite microfiber.
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Fig. 2. SEM images of graphite microfiber (A and B), ZnO NWs/graphite microfiber (C and D), high resolution image of ZnO NWs (E), TEM image of ZnO NWs (F) (insets of HRTEM).
PBS buffer were repeated to remove the unbound DNA probes from the electrode. Hybridization reaction between the probe DNA and the target DNA was carried out by incubating the prepared probe DNA/ZnO NWs/ graphite microfibers electrode with the complementary target DNA, or with the single-base mismatched DNA, or else with a noncomplementary DNA for 120 s (Fig. S1). Several rounds of washing were then conducted on the hybridized microfibers electrodes after hybridization, for removing unbound target DNA. The electrochemical sensing strategy for DNA sensing mechanism is shown in the Scheme 1.
as the reference electrode. The working electrodes were the as-prepared microfiber electrode. Electrochemical impedance spectroscopy (EIS) measurements were performed in 0.01 M PBS (pH 7.4, containing 0.1 M KCl) containing 5 mM (K4[Fe(CN)6])/(K3[Fe(CN)6]) (1:1), scanning in the frequency range from 100 mHz to 100 kHz applying a 5 mV voltage amplitude. The electrical parameters of the system were fitted with a software package embedded in Gamry Reference 3000. All the data was taken from five independent experiments. Error bars represent the standard errors.
2.4. Surface characterization and electrochemical measurements
3. Results and discussion
The crystalline phase characteristics of the prepared ZnO NWs/ graphite microfibers were examined using X-ray Diffraction (XRD) (D8-advance, Bruker AXS, Germany). Morphology and structure of the prepared ZnO NWs/graphite microfibers were studied using a field emission scanning electron microscope (FESEM) (HITACHI S8020, Japan) and transmission electron microscopy (TEM) (JEM 2100F, Japan). Electrochemical measurements were performed on a Gamry 3000 electrochemical workstation (USA), in a standard threeelectrode cell system. There were a Pt plate (area, 1 cm × 1 cm) electrode as the counter electrode and a standard Ag/AgCl (saturated KCl)
3.1. Characterizations of ZnO NWs/graphite microfibers The XRD patterns for ZnO NWs/graphite microfiber is shown in Fig. 1. The diffraction peak of graphite microfiber (curve b) centered at 2θ =25.7 can be referenced as graphite (JCPDS 65–6212) [45]. On curve c, the peaks corresponding to (100), (002), (101) and (102), (110), (103) and (112) planes reveal that ZnO NWs grown on graphite microfibers have a hexagonal wurtzite structure (curve a). No peak corresponding to any other phase or compound appears, which indicates the existence of single phase ZnO NWs on graphite microfiber [46].
Fig. 3. Nyquist diagrams spectra (A) and CV response (B) for the graphite microfiber electrodes (a), ZnO NWs/graphite microfiber electrodes (b), probe DNA immobilized graphite fiber electrodes at pH 7.0 (c), probe DNA immobilized ZnO NWs/graphite microfiber electrodes at different pH: 7.0 (d), 5.0 (e) and 9.0 (f) recorded in 0.01 M PBS containing 5 mM [Fe(CN)6]3−/4− and 0.1 M KCl (pH 7.4).
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Fig. 4. XPS spectra of (A) N 1 s and (B) P 2p for the ZnO NWs/graphite microfiber electrodes (a), probe DNA immobilized ZnO NWs/graphite microfiber electrodes at different pH: 5.0 (b), 9.0 (c) and 7.0 (d).
Morphology of the non-modified graphite microfiber and the ZnO NWs/graphite microfiber have been examined and compared with SEM. Graphite microfibers are about 6 μm in diameter and have smooth surfaces (Fig. 2A, B). Images of the ZnO NWs/graphite microfiber image show that the graphite microfiber is densely buried by ZnO NWs with a size of about 100–150 nm (Fig. 2C, D, E). A high-resolution TEM (HRTEM) image (inset of Fig. 2F) taken from the edge of ZnO NWs indicates that the ZnO NWs grow along the [002] direction [46]. 3.2. Probe DNA immobilization on ZnO nanowires/graphite microfibers EIS and CV were used to characterize the immobilization of the DNA probes on the ZnO NWs/graphite microfiber electrodes, using [Fe(CN) 3−/4− as a redox probe. The immobilization time and concentration 6] of DNA probe were optimized (Fig. S2 and S3). The impedance of spectra includes a semicircle portion at higher frequencies corresponding to the electro transfer resistance (Rct) and a linear portion at lower frequencies corresponding to the diffusion process at the interface of the electrode and solution (Fig. 3A). Compared with the bare microfiber electrode (curve a), the ZnO NWs/graphite microfiber electrodes (curve b) shows a lower Rct due to the high surface area and accelerated electron transfer of ZnO nanowires. It is worth nothing that after immobilization of probe DNA onto the graphite microfiber electrode, the Rct had hardly changed, which indicates that the graphite microfiber electrode has no DNA adsorption ability (curve c). However, upon immobilization of probe DNA onto the ZnO NWs /graphite microfiber electrodes at a set of different pH values, the Rct changed differently. At pH 7.0, Rct increased significantly (curve d). Because the negative charges of DNA repel the negatively charged redox probes [Fe(CN)6]3−/4−. The steric hindrance introduced with the formation of DNA layer on the ZnO NWs/graphite microfiber electrode surface also prevents transfer of electrons [47,48]. At this physiological pH value, ZnO having a relatively high isoelectric point (IEP 9.5) is positively charged, and DNA having a relatively low isoelectric point (IEP 4.2) is negatively charged, which is suitable for the immobilization of DNA onto the ZnO NWs/graphite microfiber electrodes via the strong electrostatic interaction. However, at pH 5.0 and 9.0, there is no significant increase of Rct value (curve e and f). A small increase may result from some weak adsorption of DNA onto the ZnO NWs/graphite microfiber electrodes. It demonstrates that pH value affects the assembly of DNA on the ZnO NWs/graphite microfiber electrodes. If the selected pH is close to the isoelectric point of DNA or ZnO, DNA cannot be assembled efficiently on the ZnO NWs/ graphite microfiber electrode surface. At the physiological pH value, DNA can be assembled efficiently on the ZnO NWs/graphite microfiber electrode surface via strong electrostatic interaction. EIS has also been
used to study the ZnO NWs/graphite microfiber electrodes immersed in 0.01 M PBS (pH 5.0, 7.0, 9.0, containing 0.1 M KCl) solution without the DNA probe (Fig. S4). After incubation in the pure PBS solution, EIS response has hardly changed, which reveals that the significant increase of Rct value of probe DNA immobilized ZnO NWs/graphite microfiber electrodes is indeed resulted from the introduction of DNA molecules. Similar DNA probes without the thiol group was used to immobilize the ZnO NWs/graphite fibers electrode surface, the EIS response has hardly changed compared with the DNA probe with the thiol group (Fig. S5). It seems that the electrostatic interaction occurs between the positively charged ZnO NWs and the negatively charged phosphate backbone of the DNA [34]. CV response of the DNA assembly process on ZnO NWs/graphite microfiber electrodes has also been studied (Fig. 3B), which is consistent with that of the EIS measurements. X-ray photoelectron spectroscopic (XPS) has been used to study the immobilization of DNA molecules on the electrode surface because of the nitrogen and phosphorus elements having evident XPS response. As shown in Fig. 4A, compared with the ZnO NWs/graphite microfiber electrodes (curve a), an intensive and obvious peak of N1 s can be detected after the immobilization of DNA probe at pH 7.0, indicating that the DNA probes were successfully assembled on the ZnO NWs/graphite microfiber electrodes surface (curve d). When immobilizing the DNA
Fig. 5. Nyquist diagrams of probe DNA immobilized ZnO NWs/graphite microfiber electrodes before (a) and after (b) hybridization with target DNA (1.0 × 10−11 M) recorded in 0.01 M PBS containing 5 mM [Fe(CN)6]3−/4− and 0.1 M KCl (pH 7.4). Inset: equivalent circuit used to fit the impedance data.
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Fig. 6. (A) Nyquist diagrams at probe DNA immobilized ZnO NWs/graphite microfiber electrodes after hybridization with complementary target DNA of different concentrations in 0.01 M PBS containing 5 mM [Fe(CN)6]3−/4− and 0.1 M KCl (pH 7.4): a–h 1.0 × 10−14, 1.0 × 10−13, 1.0 × 10−12, 1.0 × 10−11, 1.0 × 10−10, 1.0 × 10−9, 1.0 × 10−8, 1.0 × 10−7 M. (B) The plot of Δ Rct (Δ Rct = |Rct, target − Rct, probe|) versus complementary target DNA concentration.
probe at pH 5.0 and 9.0 (curve b, c), the XPS spectrum shows a weak signal of N1 s, resulting from the weak adsorption of DNA on the ZnO NWs/ graphite microfiber electrodes. On the P2p spectra, the increase of peak intensity is consistent with the increase in phosphorus content (Fig. 4B). The XPS results also confirmed the successful immobilization of DNA onto the ZnO NWs/graphite microfiber electrodes surface. 3.3. EIS detection of target DNA Fig. 5 shows the impedance diagrams of DNA probe modified ZnO NWs/graphite microfiber electrodes before and after hybridization with 1.0 × 10−11 M complementary target DNA. After hybridization reaction, double-stranded helix between the probe and target DNA are formed on electrodes surface and will block the electron transfer process of [Fe(CN)6]3−/4− through the electrostatic repulsion and the steric hindrance effect. It leads to the increase of Rct [47,48]. An equivalent circuit (inset Fig. 5) was used to fit the EIS data. Rs representing the solution-phase resistance. Rct represents the charge-transfer resistance, which is inversely proportional to the rate of electron transfer. Zw corresponds to the Warburg impedance, which results from mass-transfer limitations. CPE (constant phase element) corresponds to the capacitance of the double layer. Fig. 6A shows the Nyquist plots of DNA probe modified ZnO NWs/ graphite microfiber electrodes hybridizing with the complementary target DNA at different concentrations from 1.0 × 10−14 M to 1.0 × 10−7 M. It can be seen that the Rct value increases with the increasing
concentration of target DNA. The variation of the Rct value (Δ Rct) before and after each hybridization reaction is used as the sensing readout signal, having a linear relationship with the logarithmic of the concentration of the target DNA, in the range from 1.0 × 10−14 M to 1.0 × 10−7 M (Fig. 6B). The observed dependence of the Rct component on the logarithm of analyte concentration directly reflects the fact that more and more DNA strands have been captured on the electrodesolution interface, therefore impairing the efficiency of interface charge transfer, resulting in larger and larger Rct component as we have observed. The specific logarithm dependence is mostly possible resulted from gradual saturation of the surface-immobilized DNA binding sites. The detection limit is estimated to be 3.3 × 10−15 M at a signal-tonoise (S/N) ratio of 3, which indicates that the label-free electrochemical strategy based on ZnO NWs/graphite microfiber electrodes exhibits high sensitivity. The selectivity of this DNA biosensor based on ZnO NWs/graphite microfiber electrodes is further assessed by hybridization with control target DNA sequences (fully complementary, single-base mismatched, and non-complementary sequences), as shown in Fig. 7. A significant increase of Δ Rct is observed for the fully complementary sequence (curve c), suggesting that the hybridization reaction works well in this case. A less evident increase of Δ Rct is obtained after hybridization with singlebase mismatched sequences (curve b), while there is no remarkable Δ Rct change after hybridization with the non-complementary sequence (curve a). It demonstrates that the developed ZnO NWs/graphite microfiber electrodes biosensor displays a high selectivity for DNA
Fig. 7. (A) Nyquist diagrams at probe DNA immobilized ZnO NWs/graphite microfiber electrodes after hybridization with 1.0 × 10−11 M DNA sequences: non-complementary DNA (a), single-mismatched DNA (b) and complementary target DNA (c) in 0.01 M PBS containing 5 mM [Fe(CN)6]3−/4− and 0.1 M KCl (pH 7.4. (B) The plot of Δ Rct (Δ Rct = |Rct, target − Rct, probe|) corresponding to hybridization with 1.0 × 10−11 M different DNA sequences: non-complementary DNA (a), single-mismatched DNA (b) and complementary target DNA (c).
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hybridization detection. Moreover, the DNA probe immobilized ZnO NWs/graphite microfiber electrode has also immersed in bovine serum albumin (BSA) solution (2.5 g/L) for 120 s at room temperature before DNA detection. As shown in Fig. S6, compared with the DNA probe immobilized ZnO NWs/graphite microfiber electrode (curve a), Rct slightly changed when BSA is used (curve b), which suggested that BSA is hardly adsorbed by ZnO NWs. However, after hybridization with the target complementary DNA, Rct can be seen enhanced significantly. These results reveal that the non-specific adsorption has no significant effect on the DNA hybridization process. In addition, the biosensor shows good reproducibility and stability for the detection of DNA (Fig. S7 and S8). Furthermore, SEM images of ZnO NWs/graphite microfiber electrodes after DNA probe immobilization and after ten cycles of usage have been recorded (Fig. S9). The electrode surface has hardly changed, which reveals good stability of ZnO NWs/graphite microfiber structure. 4. Conclusion In this work, a novel, simple, sensitive and label-free DNA hybridization detection platform based on ZnO NWs/graphite microfiber electrodes was constructed. ZnO nanowires were in situ synthesized on the graphite microfibers to form a three-dimensional electrode structure for immobilization of DNA probe via strong electrostatic interaction. XPS, CV and EIS studies conformed the successful immobilization of the DNA probe onto the ZnO nanowires under neutral condition. The developed biosensor was successfully applied to the impedance detection of DNA hybridization and demonstrated good selectivity and remarkable detection limit. In summary, the proposed DNA hybridization detection platform based on the ZnO NWs/graphite microfiber electrodes presents a promising strategy for DNA detection in the future. Moreover, because of its fiber morphology and flexibility, it may become an excellent DNA monitoring candidate for cellular or even in vivo applications in the future. Acknowledgments The work was supported by the Major Program of Shandong Province Natural Science Foundation (No. ZR2018ZC0843), Natural Science Foundation of Shandong Province (No. ZR2018BEM010), the National Natural Science Foundation of China (No. 51802115). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.bioelechem.2019.04.003. References [1] J. Guo, J. Wang, J. Zhao, Z. Guo, Y, Zhang, ultrasensitive multiplexed immunoassay for tumor biomarkers based on DNA hybridization chain reaction amplifying signal, ACS Appl. Mater. Interfaces 8 (2016) 6898–6904. [2] Z. Cheglakov, T.M. Cronin, C. He, Y. Weizmann, Live cell microRNA imaging using cascade hybridization reaction, J. Am. Chem. Soc. 137 (2015) 6116–6119. [3] S.X. Chen, G. Seelig, An engineered kinetic amplification mechanism for single nucleotide variant discrimination by DNA hybridization probes, J. Am. Chem. Soc. 138 (2016) 5076–5086. [4] D. Xia, J. Yan, S. Hou, Fabrication of nanofluidic biochips with nanochannels for applications in DNA analysis, Small 8 (2012) 2787–2801. [5] F. Wallet, S. Nseir, L. Baumann, S. Herwegh, B. Sendid, M. Boulo, M. RousselDelvallez, A.V. Durocher, R.J. Courcol, Preliminary clinical study using a multiplex real-time PCR test for the detection of bacterial and fungal DNA directly in blood, Clin. Microbiol. Infec. 16 (2010) 774–779. [6] C. Zhu, Z. Zeng, H. Li, F. Li, C. Fan, H. Zhang, Single-layer MoS2-based nanoprobes for homogeneous detection of biomolecules, J. Am. Chem. Soc. 135 (2013) 5998–6001. [7] Y. Zhang, B. Zheng, C. Zhu, X. Zhang, C. Tan, H. Li, B. Chen, J. Yang, J. Chen, Y. Huang, L. Wang, H. Zhang, Single-layer transition metal dichalcogenide nanosheets-based nanosensors for rapid, sensitive, and multiplexed detection of DNA, Adv. Mater. 27 (2015) 935–939. [8] S.Z. Mousavisani, J.B. Raoof, R. Ojani, Z. Bagheryan, An impedimetric biosensor for DNA damage detection and study of the protective effect of deferoxamine against DNA damage, Bioelectrochemistry 122 (2018) 142–148.
131
[9] A. Hájková, J. Barek, V. Vyskočil, Electrochemical DNA biosensor for detection of DNA damage induced by hydroxyl radicals, Bioelectrochemistry 116 (1–9) (2017). [10] A.A. Ensafi, N. Kazemnadi, M. Amini, B. Rezaei, Impedimetric DNA-biosensor for the study of dopamine induces DNA damage and investigation of inhibitory and repair effects of some antioxidants, Bioelectrochemistry 104 (2015) 71–78. [11] V.C. Diculescu, A.M. Chiorcea-Paquim, A.M. Oliveira-Brett, Applications of a DNAelectrochemical biosensor, Trac-Trend. Anal. Chem. 79 (2016) 23–36. [12] M.A. Tabrizi, M. Shamsipur, A label-free electrochemical DNA biosensor based on covalent immobilization of salmonella DNA sequences on the nanoporous glassy carbon electrode, Biosens. Bioelectron. 69 (2015) 100–105. [13] J. Lee, M. Morita, K. Takemura, E.Y. Park, A multi-functional gold/iron-oxide nanoparticle-CNT hybrid nanomaterial as virus DNA sensing platform, Biosens. Bioelectron. 102 (2018) 425–431. [14] M. Fojta, A. Daňhel, L. Havran, V. Vyskočil, Recent progress in electrochemical sensors and assays for DNA damage and repair, Trac-Trend. Anal. Chem. 79 (2016) 160–167. [15] H. Huang, W. Bai, C. Dong, R. Guo, Z. Liu, An ultrasensitive electrochemical DNA biosensor based on graphene/au nanorod/polythionine for human papillomavirus DNA detection, Biosens. Bioelectron. 68 (2015) 442–446. [16] H. Gao, M. Sun, C. Lin, S. Wang, Electrochemical DNA biosensor based on graphene and TiO2 nanorods composite film for the detection of transgenic soybean gene sequence of MON89788, Electroanalysis 24 (2012) 2283–2290. [17] M.R. Saidur, A.A. Aziz, W.J. Basirun, Recent advances in DNA-based electrochemical biosensors for heavy metal ion detection: a review, Biosens. Bioelectron. 90 (2017) 125–139. [18] J. Wang, A. Shi, X. Fang, X. Han, Y. Zhang, An ultrasensitive super sandwich electrochemical DNA biosensor based on gold nanoparticles decorated reduced graphene oxide, Anal. Biochem. 469 (2015) 71–75. [19] G. Yang, C. Zhu, D. Du, J. Zhu, Y. Lin, Graphene-like two-dimensional layered nanomaterials: applications in biosensors and nanomedicine, Nanoscale 7 (2015) 14217–14231. [20] C. Wang, N. Huang, H. Zhuang, X. Jiang, Enhanced performance of nanocrystalline ZnO DNA biosensor via introducing electrochemical covalent biolinkers, ACS Appl. Mater. Inter. 7 (2015) 7605–7612. [21] A. Hájková, J. Barek, V. Vyskočil, Voltammetric determination of 2-Aminofluoren-9one and investigation of its interaction with DNA on a glassy carbon electrode, Electroanal. 27 (2015) 101–110. [22] D.Y. Son, K.H. Bae, H.S. Kim, N.G. Park, Effects of seed layer on growth of ZnO nanorod and performance of perovskite solar cell, J. Phys. Chem. C 119 (2015) 10321–10328. [23] H. Li, N. Liu, X. Zhang, J. Su, L. Li, Y. Gao, Z.L. Wang, Piezotronic and piezo-phototronic logic computations using au decorated ZnO microwires, Nano Energy 27 (2016) 587–594. [24] P.S. Shewale, Y.S. Yu, Structural, surface morphological and UV photodetection properties of pulsed laser deposited Mg-doped ZnO nanorods: effect of growth time, J. Alloy. Compd. 654 (2016) 79–86. [25] X. Cao, X. Cao, H. Guo, T. Li, Y. Jie, N. Wang, Z.L. Wang, Piezotronic effect enhanced label-free detection of DNA using a schottky-contacted ZnO nanowire biosensor, ACS Nano 10 (2016) 8038–8044. [26] T. Kong, Y. Chen, Y. Ye, K. Zhang, Z. Wang, X. Wang, An amperometric glucose biosensor based on the immobilization of glucose oxidase on the ZnO nanotubes, Sensor. Actuat. B-Chem. 138 (2009) 344–350. [27] A. Tereshchenko, M. Bechelany, R. Viter, V. Khranovskyy, V. Smyntyna, N. Starodub, R. Yakimova, Optical biosensors based on ZnO nanostructures: advantages and perspectives. A review, Sensor. Actuat. B-Chem. 229 (2016) 664–677. [28] S.P. Singh, S.K. Arya, P. Pandey, B.D. Malhotra, S. Saha, K. Sreenivas, V. Gupta, Cholesterol biosensor based on rf sputtered zinc oxide nanoporous thin film, Appl. Phys. Lett. 91 (2007), 063901. [29] S. Saha, V. Gupta, K. Sreenivas, H.H. Tan, C. Jagadish, Third generation biosensing matrix based on Fe-implanted ZnO thin film, Appl. Phys. Lett. 97 (2010), 133704. [30] A. Wei, X.W. Sun, J.X. Wang, Y. Lei, X.P. Cai, C.M. Li, Z.L. Dong, W. Huang, Enzymatic glucose biosensor based on ZnO nanorod array grown by hydrothermal decomposition, Appl. Phys. Lett. 89 (2006), 123902. [31] J.X. Wang, X.W. Sun, A. Wei, Y. Lei, X.P. Cai, C.M. Li, Z.L. Dong, Zinc oxide nanocomb biosensor for glucose detection, Appl. Phys. Lett. 88 (2006), 233106. [32] M. Das, G. Sumana, R. Nagarajan, B.D. Malhotra, Application of nanostructured ZnO films for electrochemical DNA biosensor, Thin Solid Films 519 (2010) 1196–1201. [33] A.A. Ansari, R. Singh, G. Sumana, B.D. Malhotra, Sol–gel derived nano-structured zinc oxide film for sexually transmitted disease sensor, Analyst 134 (2009) 997–1002. [34] M. Tak, V. Gupta, M. Tomar, Flower-like ZnO nanostructure based electrochemical DNA biosensor for bacterial meningitis detection, Biosens. Bioelectron. 59 (2014) 200–207. [35] M. Tak, V. Gupta, M. Tomar, An electrochemical DNA biosensor based on Ni doped ZnO thin film for meningitis detection, J. Electroanaly. Chem. 792 (2017) 8–14. [36] J. Zhang, A. Li, X. Yu, W. Guo, Z. Zhao, J. Qiu, X. Mou, J.P. Claverie, H. Liu, Scaly graphene oxide/graphite fiber hybrid electrodes for DNA biosensors, Adv. Mater. Interfaces 2 (2015), 1500072. [37] J. Zhang, X. Yu, W. Guo, J. Qiu, X. Mou, A. Li, H. Liu, Construction of titanium dioxide nanorod/graphite microfiber hybrid electrodes for a high performance electrochemical glucose biosensor, Nanoscale 8 (2016) 9382–9389. [38] M. Kang, Y. Lee, H. Jung, J.H. Shim, N.S. Lee, J.M. Baik, C.S. Lee, C. Lee, Y. Lee, M.H. Kim, Single carbon fiber decorated with RuO2 nanorods as a highly electrocatalytic sensing element, Anal. Chem. 84 (2012) 9485–9491. [39] J. Du, R. Yue, F. Ren, Z. Yao, F. Jiang, P. Yang, Y. Du, Novel graphene flowers modified carbon fibers for simultaneous determination of ascorbic acid, dopamine and uric acid, Biosens. Bioelectron. 53 (2014) 220–224.
132
J. Zhang et al. / Bioelectrochemistry 128 (2019) 126–132
[40] H.E. Unalan, D. Wei, K. Suzuki, S. Dalal, P. Hiralal, H. Matsumoto, S. Imaizumi, M. Minagawa, A. Tanioka, A.J. Flewitt, W.I. Milne, G.A.J. Amaratunga, Photoelectrochemical cell using dye sensitized zinc oxide nanowires grown on carbon fibers, Appl. Phys. Lett. 93 (2008), 133116. [41] X. Xue, W. Zang, P. Deng, Q. Wang, L. Xing, Y. Zhang, Z. L. piezo-potential enhanced photocatalytic degradation of organic dye using ZnO nanowires, Nano Energy 13 (2015) 414–422. [42] F. Zhang, S. Niu, W. Guo, G. Zhu, Y. Liu, X. Zhang, Z.L. Wang, Piezo-phototronic effect enhanced visible/UV photodetector of a carbon-fiber/ZnO-CdS double-shell microwire, ACS Nano 7 (2013) 4537–4544. [43] I. Shakir, M. Shahid, U.A. Rana, I.M. Al Nashef, R. Hussain, Nickel-cobalt layered double hydroxide anchored zinc oxide nanowires grown on carbon fiber cloth for highperformance flexible pseudocapacitive energy storage devices, Electrochim. Acta 129 (2014) 28–32.
[44] L.E. Greene, M. Law, D.H. Tan, M. Montano, J. Goldberger, G. Somorjai, P. Yang, General route to vertical ZnO nanowire arrays using textured ZnO seeds, Nano Lett. 5 (2005) 1231–1236. [45] J. Zhang, D. Han, S. Wang, X. Zhang, R. Yang, Y. Ji, X. Yu, Electrochemical detection of adenine and guanine based on a three-dimensional WS2 nanosheet/ graphite microfiber hybrid electrode, Electrochem. Commun. 99 (2019) 75–80. [46] H. Chen, L. Zhu, H. Liu, W. Li, Growth of ZnO nanowires on fibers for onedimensional flexible quantum dot-sensitized solar cells, Nanotechnology 23 (2012), 075402. [47] A. Bonanni, M. Pumera, Graphene platform for hairpin-DNA-based impedimetric genosensing, ACS Nano 5 (2011) 2356–2361. [48] A. Li, J. Zhang, J. Qiu, Z. Zhao, C. Wang, C. Zhao, H. Liu, A novel aptameric biosensor based on the self-assembled DNA-WS2 nanosheet architecture, Talanta 163 (2017) 78–84.