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single-walled carbon nanotubes nanocomposite
Accepted Manuscript Title: An ultrasensitive electrochemical DNA biosensor based on a copper oxide nanowires/single-walled carbon nanotubes nanocomposite Author: Mei Chen Changjun Hou Danqun Huo Mei Yang Huanbao Fa PII: DOI: Reference:
S0169-4332(15)03208-0 http://dx.doi.org/doi:10.1016/j.apsusc.2015.12.203 APSUSC 32186
To appear in:
APSUSC
Received date: Revised date: Accepted date:
25-8-2015 23-12-2015 25-12-2015
Please cite this article as: M. Chen, C. Hou, D. Huo, M. Yang, H. Fa, An ultrasensitive electrochemical DNA biosensor based on a copper oxide nanowires/single-walled carbon nanotubes nanocomposite, Applied Surface Science (2015), http://dx.doi.org/10.1016/j.apsusc.2015.12.203 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1 An ultrasensitive electrochemical DNA biosensor based on a copper oxide nanowires/single-walled carbon nanotubes nanocomposite
Key Laboratory of Biorheology Science and Technology, Ministry of Education,
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Mei Chena, Changjun Houa,b*, Danqun Huoa,b, Mei Yanga, Huanbao Fac
National Key Laboratory of Fundamental Science of Micro/Nano-Device and System
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College of Bioengineering, Chongqing University, Chongqing 400044, China
Technology, Chongqing University, Chongqing, 400044, China College of Chemistry and Chemical Engineering, Chongqing University, Chongqing
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400044, China
*. Tel.:86 23 6511 2673; fax: 86 23 6510 2507, Email: [email protected]
Abstract
Here, we developed a novel and sensitive electrochemical biosensor to detect
specific-sequence target DNA. The biosensor was based on a hybrid nanocomposite consisting of copper oxide nanowires (CuO NWs) and carboxyl-functionalized single-walled carbon nanotubes (SWCNTs-COOH). The resulting CuO NWs/SWCNTs layers exhibited a good differential pulse voltammetry (DPV) current response for the target DNA sequences, which we attributed to the properties of CuO NWs and SWCNTs. CuO NWs and SWCNTs hybrid composites with highly conductive and biocompatible nanostructure were characterized by transmission 1 Page 1 of 20
electron microscopy (TEM), scanning electron microscopy (SEM), and cyclic voltammetry (CV). Immobilization of the probe DNA on the electrode surface was largely improved due to the unique synergetic effect of CuO NWs and SWCNTs.
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DPV was applied to monitor the DNA hybridization event, using adriamycin as an electrochemical indicator. Under optimal conditions, the peak currents of adriamycin
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were linear with the logarithm of target DNA concentrations (ranging from 1.0×10−14 to
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1.0×10−8 M), with a detection limit of 3.5×10−15 M (signal/noise ratio of 3). The biosensor also showed high selectivity to single-base mismatched target DNA.
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Compared with other electrochemical DNA biosensors, we showed that the of the
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Keywords
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proposed biosensor is simple to implement, with good stability and high sensitivity.
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Electrochemical, CuO nanowire, Single-walled carbon nanotube, Ariamycin, Differential pulse voltammetry, DNA biosensor 1. Introduction Identifying DNA sequences is of central importance to virus and gene detection and the early diagnosis of diseases[1, 2]. Many DNA detection techniques have been developed, such as radiochemistry, enzymatic activity, fluorescence, surface plasmon resonance spectroscopy, and quartz crystal microbalance[3-6]. The development of ultrasensitive methods to detect low-abundance DNA is particularly essential. Many ultrasensitive sequence-specific DNA detection methods have been reported, including polymerase chain reaction (PCR)[7], rolling circle amplification (RCA)[8-10], and nucleic acid sequence-based amplification[11]. However, these methods are complex and require costly polymerase, tedious labels, and dedicated instrumentation[12]. In the last 5 years, DNA electrochemical biosensors have attracted broad attention due to their low cost, fast response rate, high sensitivity, good selectivity, and instrument miniaturization[13-15]. To date, electrochemical DNA biosensors using a broad range of nanoparticles (NPs) to improve the sensitivity of the DNA biosensor have been reported, including Au NPs, graphene, and carbon nanotubes (CNTs). 2 Page 2 of 20
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Copper oxide (CuO) is an important transition metal oxide with potential application in batteries, sensors, and solar energy converters[16, 17], due to its enhanced catalytic, electrical, and magnetic properties[18, 19]. Single-walled CNTs (SWCNTs) are also popular for electrochemical studies, due to their unique structural and physical properties[20], including high electrical conductivity, chemical stability, large surface area, high surface/volume ratio, high mechanical strength, and chemically modifiable surface[21, 22]. In recent years, researchers have increasingly explored electrodes modified with composite materials through the integration of CNTs with copper and/or copper oxides such as Cu/MWCNTs [23], Cu NPs/SWCNTs [24], CuO NPs/MWCNTs [25, 26], and Cu2O/MWCNTs [27, 28]. These studies support the feasibility of developing high-performing electrochemical sensors using these materials. However, to our knowledge, no study has investigated DNA or nucleic acid hybridization by CuO NWs-SWCNTs nanocomposites. Here, we present a new electrochemical sensor for sensitive and selective DNA analysis. A hybrid nanocomposite of CuO NWs and SWCNTs-COOH was synthesized through a simple, scalable, and economical route. A sensitive, selective, and label-free electrochemical biosensing strategy for the detection of specific DNA sequences was constructed by assembling DNA probes on a CuO NWs/SWCNTs-COOH modified electrode with amine-tagged single-stranded DNA (NH2-ssDNA). First, ssDNA was immobilized on the CuO NWs/SWCNTs-COOH nanocomposite by amide bonds. The complementary target DNA was then hybridized with the DNA probes to form double-stranded structures on the biosensor surface. Then the target DNA was electrochemically detected by Adriamycin binding (Scheme 1). Compared with biosensors reported in the literature, our new nanocomposite shows a lower limit of detection (LOD), with a wide linear range and high selectivity. 2. Experiments 2.1. Reagents Copper nitrate (Cu(NO3)2), sodium hydroxide (NaOH), ethylenediamine (EDA, >99.9%), hydrazine (N2H4), sodium dodecyl sulfate (SDS), tris(hydroxymethyl)aminomethane (Tris-base), N,N-dimethylformamide (DMF), nitric acid, citric acid, ethanol, and acetone were purchased from Chongqing Chuan-dong Chemical Group (China). Other reagents included N-(3-dimethyl-amino-propyl)-N’-ethylcarbodiimide hydrochloride (EDC, Sigma, USA) and N-hydroxysuccinimide (NHS, Shanghai Medpep Ltd. Co., China). SWCNTs were bought from Unidym. All aqueous solutions were prepared with deionized (DI) water from a Millipore Direct-Q Water system. Synthetic DNA oligonucleotides (21 nucleotides) related to anthrax lethal factor were provided by Sangon Biotech Co., Ltd., (Shanghai, China). All oligonucleotide stock solutions were prepared with 0.01 M phosphate-buffered saline (PBS, pH 7.4) and stored at 4 °C. Their base sequences were as follows: Probe DNA: NH2- AAT GTA TAA TTG CGG GAC TCTAAT C-3ʹ . 3
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Complementary target DNA: 5ʹ -GAT TAG AGT CCC GCA ATT ATACAT T-3ʹ . Single-base mismatched target DNA: 5ʹ -GAT TAG AGT CNC GCAATT ATA CAT T-3ʹ (where N stands for A, T, or G). Non-complimentary target DNA: 5ʹ -AGC CCA CAC TGA TGG CGCCAC TGC A-3ʹ . 2.2. Instruments Transmission electron microscopy (TEM) images were obtained using a Hitachi H-600 (Japan) at an acceleration voltage of 100 kV, and scanning electron microscopy (SEM) images were collected on an XL30 ESEM-FEG (FEI, Netherlands) at an acceleration voltage of 15.0 kV. Fourier transform infrared spectroscopy (FT-IR) was measured on a Bruker-Tensor 27IR spectrophotometer. Electrochemical measurements were performed using a CHI 660D electrochemical workstation (Shanghai CH Instrument, China), with a conventional three-electrode system: a modified glassy carbon electrode (GCE) working electrode, a Ag/AgCl (3 M KCl) reference electrode, and a platinum wire counter electrode. 2.3. Fabrication of CuO NWs/SWCNTs-modified GCE CuO NWs were prepared by the thermal oxidation of Cu NWs, and were synthesized as we described previously [29]. In short, 20 mL 15 M NaOH solution was pre-heated to 60 °C, and 1 mL 0.1 M Cu(NO3)2, 0.16 mL EDA, and 25 µL N2H4 solution (35 wt%) were added sequentially. The reaction was maintained at 60 °C for 2 h. The reddish product (Cu NWs) was washed, collected by centrifugation, redispersed in DI water to remove soluble inorganic compounds, and washed in ethanol to remove residual EDA and N2H4. Afterwards, Cu NWs were kept in a high-temperature furnace for 5 h at 400 °C. The black product (CuO NWs) was suspended at 2 mg/mL in ethanol for further use. SWCNTs-COOH were prepared by mixing commercially available SWCNTs in concentrated nitric acid for 10 h[30, 31]. They were then suspended in DMF under sonication to obtain a suspension of SWCNTs-COOH. Before electrode modification, the 3-mm diameter GCE was polished with a 0.05 µm alumina slurry and ultrasonicated in DI water before drying at room temperature. Then, 15 µL of CuO NWs/SWCNTs suspension, prepared by mixing CuO NWs and SWCNTs suspensions at a ratio of 1:1 (v/v), was dropped onto the clean electrode and air dried to form the modified electrode. 2.4. Immobilization of probe ssDNA on CuO NWs/SWCNTs-modified GCE The CuO NWs/SWCNTs/GCE was immersed in a solution of 5.0 mM EDC and 8.0 mM NHS for 30 min to activate the carboxyl groups on the electrode interface. Then 5.0 µL 1.0 µM probe ssDNA in 50.0 mM PBS, pH 7.0 were dropped onto the electrode surface and immobilized onto the CuO NWs/SWCNTs/GCE surface by the formation of covalent amide bonds between the carboxyl group of SWCNTs and the 5ʹ -amine of ssDNA. Finally, the electrode was washed with double distilled water to remove unbound probe ssDNA, and the modified electrode was denoted ssDNA/CuO NWs/SWCNTs/GCE. 2.5. Hybridization and electrochemical measurements of the biosensor 4
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The hybridization reaction was performed by applying 5.0 µL of different concentrations of target ssDNA in 50.0 mM PBS for 50 min at 30 °C. After application, the electrode was rinsed with 0.1% SDS solution and double distilled water to remove the non-hybridized adsorbed DNA. Finally, the biosensor was incubated in 20 µM Adriamycin for 20 min. The electrode was immersed in PBS (pH 7.0), and the electrochemical response was measured by differential pulse voltammetry (DPV) with 0.008 V pulse amplitude, 0.05 s pulse width, and 0.2 s pulse period.
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3. Results and discussion 3.1. Characterization of CuO NWs/SWCNTs hybrid composite Fig. 1A shows a typical TEM image of the as-prepared CuO NWs characterized by a rough surface with abundant nano-featured protuberances resulting from the interaction with oxygen during thermal oxidation. This rugged surface provides a larger contact area for electrochemical analyte detection and hybridization with SWCNTs. The SAED (inset in Fig. 1A) exhibits “polymorphs” pattern, which consists of several sets of single-crystal diffraction spots in the monoclinic structured CuO NWs. In comparison, Fig. 1B shows that the CuO NWs-SWCNTs hybrid nanocomposite constitutes a highly dense network structure with CuO NWs, intertwined by a large amount of filamentous SWCNTs bundles. This structure indicates the good interaction/contact between CuO NWs and SWCNTs. The HRTEM (Fig. 1C) further reveals the structure of CuO NWs-SWCNTs. FT-IR analysis was performed between 500 and 4000 cm-1 to identify the bending and stretching vibrations of functional group in the samples (Fig. 1D). Curve “a” shows a peak around 539 cm-1 due to CuO, indicating the successful preparation of CuO NWs. The carboxylic stretching frequency in the SWCNT-COOH spectrum (curve “b”) sharply peaks at 1716 cm-1 (C=O stretching), in addition to a broad peak at 3418 cm−1 (O-H stretching). In the composite material (curve “c”), characteristic peaks of both components occur, further confirming the attachment of SWCNT-COOH to the surface of CuO NWs. 3.2. Electrochemical characterization of the different modified electrodes In this work, cyclic voltammograms (CVs) of different modified electrodes in 1.0 mM [Fe(CN)6]3-/4− solution containing 0.1 M KCl were recorded at a scan rate of 100 mV s−1, and the results are shown in Fig. 2. The CV of the bare GCE (curve “a”) shows the well-defined reversible redox behavior of [Fe(CN)6]3-/4−. Modification with CuO NWs increased the peak current (curve “b”), indicating an increased effective surface area of the electrode. Thus, these results demonstrate that CuO NWs are a kind of better conducting material, accelerating the electron transfer of [Fe(CN)6]3-/4−. Attachment of the CuO NWs/SWCNTs hybrid significantly increased the peak CV current (curve “c”), demonstrating good catalytic activity of the composites. The magnitude of increase for the oxidation peak current was greatest for CuO NWs/SWCNTs/GCE. This result was probably due to the good electronic performance of SWCNTs and the formation of entangled CuO NWs/SWCNTs porous and mesh structures. 5
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Subsequently, the capture probe was assembled on the surface of the CuO NWs/SWCNTs, and hybridized to the target DNA. The peak currents continuously decreased (curves “d” and “e”) because the negatively charged probe DNA blocked the electron transfer of [Fe(CN)6]3-/4-, resulting in the peak current noticeably decreasing. 3.3. Optimization of assay conditions To obtain excellent analytical performance, we investigated the effects of hybridization time and temperature of the capture probe DNA and the target DNA on the electrochemical signal. Many studies have demonstrated that adriamycin intercalates in the DNA double helix [32, 33], and it has been widely used in DNA hybridization detection as an electrochemical indicator [34]. Here, the peak current of adriamycin was recorded as a detectable signal. When the hybridization temperature was fixed at 25 °C, the peak current of adriamycin increased with increasing hybridization time from 20 to 70 min and plateaued after 50 min, indicating that hybridization was complete within 50 min (Fig. 3A). The effect of hybridization temperature was tested from 15–45 °C, with the largest peak current occurring at 30 °C. Therefore, the hybridization temperature was chosen as 30 °C (Fig. 3B). The effect of adriamycin concentration and incubation time was also studied. The current response increased with increasing adriamycin concentration from 5.0×10−6 to 3.0×10−5 M (Fig. 3C). Therefore, we employed 2.0×10−5 M adriamycin as the electrochemical indicator. The peak current of adriamycin increased significantly with increasing incubation time from 5 to 25 min (Fig. 3D) and plateaued after 20 min. Thus, 20 min was chosen as the optimal incubation time for adriamycin. 3.4. Sensitivity for target detection Under the optimal conditions, the analytical performance of the DNA biosensor was investigated using the probe DNA to hybridize with different concentrations of target DNA sequences. We observed that the DPV peak current increased gradually as the concentration of complementary DNA increased in the solution (shown in Fig. 4A). The calibration plot (Fig. 4B) showed a good linear relationship between the peak currents and the logarithm of the concentration of complementary DNA in the range of 1.0×10−14 to 1.0×10−8 M, with a correlation coefficient of 0.9931. The linear regression equation was ∆I = 69.51 + 4.736 log C, and the detection limit was 3.5×10−15 M (S/N = 3). We compared the performance of the fabricated electrochemical DNA biosensor with those reported in the literature using nanostructured materials for the DNA immobilization layer (Table 1). Our novel biosensor shows a much lower limit of detection, which we attributed to the integration of CuO NWs and SWCNTs producing a larger surface-to-volume ratio derived from the uneven surface of CuO NWs and highly dense network structures between the CuO NWs and SWCNTs. In particular, CuO NWs were entangled with the SWCNTs, and formed a mesh structure of CuO NWs/SWCNTs with good conductivity. 3.5. Selectivity of the biosensor The selectivity of the DNA biosensor was investigated using non-complementary, single-base mismatch (T, G, and A), and complementary DNA 6
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sequences (Fig. 5A). The electrochemical responses of the non-complementary and single-base mismatch sequences were only 5% and 30% that of the complementary sequence, respectively. These data suggest that this biosensor has good selectivity and can readily distinguish single-base mismatched target DNA from the non-complementary sequence and complementary sequence DNA. To assess the selectivity of this biosensor further, we used a different approach to investigate the selectivity of the biosensor. Four types of DNA sequences were selected for evaluation, including single-base mismatch target (1MT), two-base mismatch target (2MT), three-base mismatch target (3MT), and non-complementary sequence. Fig. 5B shows the peak currents for the target DNA and the four types of DNA sequences. The differences among 1MT, 2MT, and 3MT indicated that the base mismatch prevented the completion of hybridization, and a similar result was obtained for the non-complementary sequence. All results showed that the biosensor effectively discriminated different mismatched target DNA, suggesting that this biosensor possesses excellent selectivity. 3.6. Reproducibility and stability of the DNA sensor To characterize its reproducibility, the DNA sensor was tested in 0.1 M PBS (pH 7.0). Five parallel DNA biosensors were fabricated and hybridized with 1.0×10−10 M target DNA sequence, respectively. An acceptable relative standard deviation (R.S.D.) of 8.3% (n = 5) for the DPV current value demonstrates the high reproducibility of DNA detection. When stored in PBS (pH 7.0) at 4 °C for more than two weeks, the current response for the sensor decreased quickly (~71% of its initial value) in the first five days and, thereafter, remained stable from 6–14 days (Fig. 6). These results demonstrate that the proposed DNA biosensor has good stability. 4. Conclusions We developed an ultrasensitive DNA electrochemical biosensor by modifying the GCE with a CuO NWs/ SWCNTs hybrid nanocomposite. Excellent integration of CuO NWs and SWCNTs with good morphologic appearance and structural properties offered a large surface area, fast electron transfer, and good electrical conductivity for DNA detection. Compared with other electrochemical DNA biosensors, the proposed biosensor has many advantages, including simplicity, selectivity, stability, and high sensitivity. Thus, this work establishes a methodology for the development of DNA biosensors with good analytical properties. Furthermore, our novel DNA biosensor has great potential for use in the analysis of real samples. Acknowledgements The authors would like to acknowledge the financial support from Chongqing Graduate Student Research Innovation Project (CYB14028), National Natural Science Foundation of China (NSFC) (81271930), Key Technologies R&D Program of China (2012BAI19B03, 2014BAD07B02), Sichuan Key Technologies R&D Program (No. 2010NZ0093) , Key Laboratory Program of Sichuan Province, China (No. NJ2014-03) and sharing fund of Chongqing university’s large equipments. 7
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Fig. 1 (A) TEM image of CuO NWs, (B) SEM image of CuO NWs/SWCNTs-COOH
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hybrid composite, (C) HR-TEM image of CuO NWs/SWCNTs-COOH, (D) FT-IR
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spectra of (a) CuO nanowires, (b) SWCNTs-COOH, and (c) CuO NWs/SWCNTs-COOH
Fig. 2 Cyclic voltammograms of 1.0 mM [Fe(CN)6]3-/4− and 0.1 M KCl solution at different modified electrodes. “a to e” are bare GCE, CuO NWs/ GCE, CuO NWs-SWCNTs/ GCE, ssDNA/ CuO NWs-SWCNTs/ GCE, and dsDNA/ CuO NWs-SWCNTs/ GCE, respectively.
Fig. 3 Optimization of the experimental conditions: (A) hybridization time, (B) hybridization temperature , (C) on DPV signals of the peak current of adriamycin; the effect of adriamycin concentration (D) and incubation time of dsDNA/ CuO NWs-SWCNTs/ GCE on the DPV signals, Cadriamycin = 2.0 × 10−5M.
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Fig. 4 (A) DPV curves of the biosensor hybridized with increasing concentrations of complementary DNA: 0 M (a); 1.0 × 10−14M (b);1.0 ×10−13M (c); 1.0 × 10−12M (d); 1.0 × 10−11M (e); 1.0 × 10−10M (f); 1.0 × 10−9M (g); 1.0 × 10−8M(h). (B) Logarithmic
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plot of peak current of adriamycin (∆I) vs. the concentration of complementary DNA Fig. 5 Normalized histograms of the signals (∆I/ ∆ICOM) of the biosensor recognized
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different DNA sequences. (A) Non-complementary DNA (Non), single-base T, G, A
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mismatched DNA (T),(G),(A) and complementary DNA (Com), (B) Comparison of DPV responses of the blank and the four different DNA sequences. (1MT; two-base
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mismatch target: 2MT; three-base mismatch target: 3MT).
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Fig. 6 Signal intensities of the biosensor obtained over 14 days.
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Scheme 1 Schematic showing the fabrication procedure of DNA biosensor
Table 1 Comparison of the linear range and detection limit of electrochemical DNA sensors. Modified electrodes
Detection
Linear range (M)
Detection
technique
MWCNTs-COOH
DPV CV
CTS/V2O5/MWCNTs
limit (M) −9
1.6×10 -4.8×10 1.0×10
−11 −14
−8
-1.0×10
−6
-1.0×10
−8
Fe@AuNPs-AETGO
SWV
1.0×10
Ag NPS/PPAA/MWCNTs
DPV
9.0×10−12-9.0×10−9
Au NPs/TB-GO
DPV
GO/PANIw
DPV
MPA/GO-PAMAM/Au GA/Th-G/GA/Cys Ph-NH2/GO
DPV DPV DPV
CuO NWs/SWCNTs
DPV
References
1.0×10
−11
2.12×10
-1.0×10
−12
1.0×10
−12
1.0×10
−12
1.0×10
−12
1.0×10
−12
3.8×10−11 1.76×10
-2.12×10
-1.0×10
−6
-1.0×10
−7
-1.0×10
−6
-1.0×10
−6
[35] [36]
3.2×10−12
[37]
2.9 ×10 −6
[14]
−15
2.0×10
−9
−12
−12
3.25×10 1.0×10
−13
−12
1.26×10
−13
[38] [39] [40] [41]
1.1×10
−13
[42]
3.5×10
−15
This work
Highlights: •
An ultrasensitive DNA electrochemical biosensor was developed 11 Page 11 of 20
• CuO NWs entangled with the SWCNTs formed a mesh structure with good conductivity • It’s the first time use of CuONWs-SWCNTs hybridnanocompositefor DNA detection • The biosensor is simple, selective, stable, and sensitive • The biosensor has great potential for use in analysis of real samples
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Scheme.1.
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Fig. 1.
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Fig. 2.
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Fig. 3.
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Fig. 4.
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A novel and sensitive electrochemical biosensor based on hybrid nanocomposite consisting of copper oxide nanowires (CuO NWs) and carboxyl-functionalized singlewalled carbon nanotubes (SWCNTs-COOH) was first developed for the detection of the specific-sequence target DNA.This schematic represents the fabrication procedure of our DNA biosensor.
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S0169-4332(15)03208-0 http://dx.doi.org/doi:10.1016/j.apsusc.2015.12.203 APSUSC 32186
To appear in:
APSUSC
Received date: Revised date: Accepted date:
25-8-2015 23-12-2015 25-12-2015
Please cite this article as: M. Chen, C. Hou, D. Huo, M. Yang, H. Fa, An ultrasensitive electrochemical DNA biosensor based on a copper oxide nanowires/single-walled carbon nanotubes nanocomposite, Applied Surface Science (2015), http://dx.doi.org/10.1016/j.apsusc.2015.12.203 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1 An ultrasensitive electrochemical DNA biosensor based on a copper oxide nanowires/single-walled carbon nanotubes nanocomposite
Key Laboratory of Biorheology Science and Technology, Ministry of Education,
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Mei Chena, Changjun Houa,b*, Danqun Huoa,b, Mei Yanga, Huanbao Fac
National Key Laboratory of Fundamental Science of Micro/Nano-Device and System
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College of Bioengineering, Chongqing University, Chongqing 400044, China
Technology, Chongqing University, Chongqing, 400044, China College of Chemistry and Chemical Engineering, Chongqing University, Chongqing
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400044, China
*. Tel.:86 23 6511 2673; fax: 86 23 6510 2507, Email: [email protected]
Abstract
Here, we developed a novel and sensitive electrochemical biosensor to detect
specific-sequence target DNA. The biosensor was based on a hybrid nanocomposite consisting of copper oxide nanowires (CuO NWs) and carboxyl-functionalized single-walled carbon nanotubes (SWCNTs-COOH). The resulting CuO NWs/SWCNTs layers exhibited a good differential pulse voltammetry (DPV) current response for the target DNA sequences, which we attributed to the properties of CuO NWs and SWCNTs. CuO NWs and SWCNTs hybrid composites with highly conductive and biocompatible nanostructure were characterized by transmission 1 Page 1 of 20
electron microscopy (TEM), scanning electron microscopy (SEM), and cyclic voltammetry (CV). Immobilization of the probe DNA on the electrode surface was largely improved due to the unique synergetic effect of CuO NWs and SWCNTs.
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DPV was applied to monitor the DNA hybridization event, using adriamycin as an electrochemical indicator. Under optimal conditions, the peak currents of adriamycin
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were linear with the logarithm of target DNA concentrations (ranging from 1.0×10−14 to
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1.0×10−8 M), with a detection limit of 3.5×10−15 M (signal/noise ratio of 3). The biosensor also showed high selectivity to single-base mismatched target DNA.
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Compared with other electrochemical DNA biosensors, we showed that the of the
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Keywords
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proposed biosensor is simple to implement, with good stability and high sensitivity.
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Electrochemical, CuO nanowire, Single-walled carbon nanotube, Ariamycin, Differential pulse voltammetry, DNA biosensor 1. Introduction Identifying DNA sequences is of central importance to virus and gene detection and the early diagnosis of diseases[1, 2]. Many DNA detection techniques have been developed, such as radiochemistry, enzymatic activity, fluorescence, surface plasmon resonance spectroscopy, and quartz crystal microbalance[3-6]. The development of ultrasensitive methods to detect low-abundance DNA is particularly essential. Many ultrasensitive sequence-specific DNA detection methods have been reported, including polymerase chain reaction (PCR)[7], rolling circle amplification (RCA)[8-10], and nucleic acid sequence-based amplification[11]. However, these methods are complex and require costly polymerase, tedious labels, and dedicated instrumentation[12]. In the last 5 years, DNA electrochemical biosensors have attracted broad attention due to their low cost, fast response rate, high sensitivity, good selectivity, and instrument miniaturization[13-15]. To date, electrochemical DNA biosensors using a broad range of nanoparticles (NPs) to improve the sensitivity of the DNA biosensor have been reported, including Au NPs, graphene, and carbon nanotubes (CNTs). 2 Page 2 of 20
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Copper oxide (CuO) is an important transition metal oxide with potential application in batteries, sensors, and solar energy converters[16, 17], due to its enhanced catalytic, electrical, and magnetic properties[18, 19]. Single-walled CNTs (SWCNTs) are also popular for electrochemical studies, due to their unique structural and physical properties[20], including high electrical conductivity, chemical stability, large surface area, high surface/volume ratio, high mechanical strength, and chemically modifiable surface[21, 22]. In recent years, researchers have increasingly explored electrodes modified with composite materials through the integration of CNTs with copper and/or copper oxides such as Cu/MWCNTs [23], Cu NPs/SWCNTs [24], CuO NPs/MWCNTs [25, 26], and Cu2O/MWCNTs [27, 28]. These studies support the feasibility of developing high-performing electrochemical sensors using these materials. However, to our knowledge, no study has investigated DNA or nucleic acid hybridization by CuO NWs-SWCNTs nanocomposites. Here, we present a new electrochemical sensor for sensitive and selective DNA analysis. A hybrid nanocomposite of CuO NWs and SWCNTs-COOH was synthesized through a simple, scalable, and economical route. A sensitive, selective, and label-free electrochemical biosensing strategy for the detection of specific DNA sequences was constructed by assembling DNA probes on a CuO NWs/SWCNTs-COOH modified electrode with amine-tagged single-stranded DNA (NH2-ssDNA). First, ssDNA was immobilized on the CuO NWs/SWCNTs-COOH nanocomposite by amide bonds. The complementary target DNA was then hybridized with the DNA probes to form double-stranded structures on the biosensor surface. Then the target DNA was electrochemically detected by Adriamycin binding (Scheme 1). Compared with biosensors reported in the literature, our new nanocomposite shows a lower limit of detection (LOD), with a wide linear range and high selectivity. 2. Experiments 2.1. Reagents Copper nitrate (Cu(NO3)2), sodium hydroxide (NaOH), ethylenediamine (EDA, >99.9%), hydrazine (N2H4), sodium dodecyl sulfate (SDS), tris(hydroxymethyl)aminomethane (Tris-base), N,N-dimethylformamide (DMF), nitric acid, citric acid, ethanol, and acetone were purchased from Chongqing Chuan-dong Chemical Group (China). Other reagents included N-(3-dimethyl-amino-propyl)-N’-ethylcarbodiimide hydrochloride (EDC, Sigma, USA) and N-hydroxysuccinimide (NHS, Shanghai Medpep Ltd. Co., China). SWCNTs were bought from Unidym. All aqueous solutions were prepared with deionized (DI) water from a Millipore Direct-Q Water system. Synthetic DNA oligonucleotides (21 nucleotides) related to anthrax lethal factor were provided by Sangon Biotech Co., Ltd., (Shanghai, China). All oligonucleotide stock solutions were prepared with 0.01 M phosphate-buffered saline (PBS, pH 7.4) and stored at 4 °C. Their base sequences were as follows: Probe DNA: NH2- AAT GTA TAA TTG CGG GAC TCTAAT C-3ʹ . 3
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Complementary target DNA: 5ʹ -GAT TAG AGT CCC GCA ATT ATACAT T-3ʹ . Single-base mismatched target DNA: 5ʹ -GAT TAG AGT CNC GCAATT ATA CAT T-3ʹ (where N stands for A, T, or G). Non-complimentary target DNA: 5ʹ -AGC CCA CAC TGA TGG CGCCAC TGC A-3ʹ . 2.2. Instruments Transmission electron microscopy (TEM) images were obtained using a Hitachi H-600 (Japan) at an acceleration voltage of 100 kV, and scanning electron microscopy (SEM) images were collected on an XL30 ESEM-FEG (FEI, Netherlands) at an acceleration voltage of 15.0 kV. Fourier transform infrared spectroscopy (FT-IR) was measured on a Bruker-Tensor 27IR spectrophotometer. Electrochemical measurements were performed using a CHI 660D electrochemical workstation (Shanghai CH Instrument, China), with a conventional three-electrode system: a modified glassy carbon electrode (GCE) working electrode, a Ag/AgCl (3 M KCl) reference electrode, and a platinum wire counter electrode. 2.3. Fabrication of CuO NWs/SWCNTs-modified GCE CuO NWs were prepared by the thermal oxidation of Cu NWs, and were synthesized as we described previously [29]. In short, 20 mL 15 M NaOH solution was pre-heated to 60 °C, and 1 mL 0.1 M Cu(NO3)2, 0.16 mL EDA, and 25 µL N2H4 solution (35 wt%) were added sequentially. The reaction was maintained at 60 °C for 2 h. The reddish product (Cu NWs) was washed, collected by centrifugation, redispersed in DI water to remove soluble inorganic compounds, and washed in ethanol to remove residual EDA and N2H4. Afterwards, Cu NWs were kept in a high-temperature furnace for 5 h at 400 °C. The black product (CuO NWs) was suspended at 2 mg/mL in ethanol for further use. SWCNTs-COOH were prepared by mixing commercially available SWCNTs in concentrated nitric acid for 10 h[30, 31]. They were then suspended in DMF under sonication to obtain a suspension of SWCNTs-COOH. Before electrode modification, the 3-mm diameter GCE was polished with a 0.05 µm alumina slurry and ultrasonicated in DI water before drying at room temperature. Then, 15 µL of CuO NWs/SWCNTs suspension, prepared by mixing CuO NWs and SWCNTs suspensions at a ratio of 1:1 (v/v), was dropped onto the clean electrode and air dried to form the modified electrode. 2.4. Immobilization of probe ssDNA on CuO NWs/SWCNTs-modified GCE The CuO NWs/SWCNTs/GCE was immersed in a solution of 5.0 mM EDC and 8.0 mM NHS for 30 min to activate the carboxyl groups on the electrode interface. Then 5.0 µL 1.0 µM probe ssDNA in 50.0 mM PBS, pH 7.0 were dropped onto the electrode surface and immobilized onto the CuO NWs/SWCNTs/GCE surface by the formation of covalent amide bonds between the carboxyl group of SWCNTs and the 5ʹ -amine of ssDNA. Finally, the electrode was washed with double distilled water to remove unbound probe ssDNA, and the modified electrode was denoted ssDNA/CuO NWs/SWCNTs/GCE. 2.5. Hybridization and electrochemical measurements of the biosensor 4
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The hybridization reaction was performed by applying 5.0 µL of different concentrations of target ssDNA in 50.0 mM PBS for 50 min at 30 °C. After application, the electrode was rinsed with 0.1% SDS solution and double distilled water to remove the non-hybridized adsorbed DNA. Finally, the biosensor was incubated in 20 µM Adriamycin for 20 min. The electrode was immersed in PBS (pH 7.0), and the electrochemical response was measured by differential pulse voltammetry (DPV) with 0.008 V pulse amplitude, 0.05 s pulse width, and 0.2 s pulse period.
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3. Results and discussion 3.1. Characterization of CuO NWs/SWCNTs hybrid composite Fig. 1A shows a typical TEM image of the as-prepared CuO NWs characterized by a rough surface with abundant nano-featured protuberances resulting from the interaction with oxygen during thermal oxidation. This rugged surface provides a larger contact area for electrochemical analyte detection and hybridization with SWCNTs. The SAED (inset in Fig. 1A) exhibits “polymorphs” pattern, which consists of several sets of single-crystal diffraction spots in the monoclinic structured CuO NWs. In comparison, Fig. 1B shows that the CuO NWs-SWCNTs hybrid nanocomposite constitutes a highly dense network structure with CuO NWs, intertwined by a large amount of filamentous SWCNTs bundles. This structure indicates the good interaction/contact between CuO NWs and SWCNTs. The HRTEM (Fig. 1C) further reveals the structure of CuO NWs-SWCNTs. FT-IR analysis was performed between 500 and 4000 cm-1 to identify the bending and stretching vibrations of functional group in the samples (Fig. 1D). Curve “a” shows a peak around 539 cm-1 due to CuO, indicating the successful preparation of CuO NWs. The carboxylic stretching frequency in the SWCNT-COOH spectrum (curve “b”) sharply peaks at 1716 cm-1 (C=O stretching), in addition to a broad peak at 3418 cm−1 (O-H stretching). In the composite material (curve “c”), characteristic peaks of both components occur, further confirming the attachment of SWCNT-COOH to the surface of CuO NWs. 3.2. Electrochemical characterization of the different modified electrodes In this work, cyclic voltammograms (CVs) of different modified electrodes in 1.0 mM [Fe(CN)6]3-/4− solution containing 0.1 M KCl were recorded at a scan rate of 100 mV s−1, and the results are shown in Fig. 2. The CV of the bare GCE (curve “a”) shows the well-defined reversible redox behavior of [Fe(CN)6]3-/4−. Modification with CuO NWs increased the peak current (curve “b”), indicating an increased effective surface area of the electrode. Thus, these results demonstrate that CuO NWs are a kind of better conducting material, accelerating the electron transfer of [Fe(CN)6]3-/4−. Attachment of the CuO NWs/SWCNTs hybrid significantly increased the peak CV current (curve “c”), demonstrating good catalytic activity of the composites. The magnitude of increase for the oxidation peak current was greatest for CuO NWs/SWCNTs/GCE. This result was probably due to the good electronic performance of SWCNTs and the formation of entangled CuO NWs/SWCNTs porous and mesh structures. 5
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Subsequently, the capture probe was assembled on the surface of the CuO NWs/SWCNTs, and hybridized to the target DNA. The peak currents continuously decreased (curves “d” and “e”) because the negatively charged probe DNA blocked the electron transfer of [Fe(CN)6]3-/4-, resulting in the peak current noticeably decreasing. 3.3. Optimization of assay conditions To obtain excellent analytical performance, we investigated the effects of hybridization time and temperature of the capture probe DNA and the target DNA on the electrochemical signal. Many studies have demonstrated that adriamycin intercalates in the DNA double helix [32, 33], and it has been widely used in DNA hybridization detection as an electrochemical indicator [34]. Here, the peak current of adriamycin was recorded as a detectable signal. When the hybridization temperature was fixed at 25 °C, the peak current of adriamycin increased with increasing hybridization time from 20 to 70 min and plateaued after 50 min, indicating that hybridization was complete within 50 min (Fig. 3A). The effect of hybridization temperature was tested from 15–45 °C, with the largest peak current occurring at 30 °C. Therefore, the hybridization temperature was chosen as 30 °C (Fig. 3B). The effect of adriamycin concentration and incubation time was also studied. The current response increased with increasing adriamycin concentration from 5.0×10−6 to 3.0×10−5 M (Fig. 3C). Therefore, we employed 2.0×10−5 M adriamycin as the electrochemical indicator. The peak current of adriamycin increased significantly with increasing incubation time from 5 to 25 min (Fig. 3D) and plateaued after 20 min. Thus, 20 min was chosen as the optimal incubation time for adriamycin. 3.4. Sensitivity for target detection Under the optimal conditions, the analytical performance of the DNA biosensor was investigated using the probe DNA to hybridize with different concentrations of target DNA sequences. We observed that the DPV peak current increased gradually as the concentration of complementary DNA increased in the solution (shown in Fig. 4A). The calibration plot (Fig. 4B) showed a good linear relationship between the peak currents and the logarithm of the concentration of complementary DNA in the range of 1.0×10−14 to 1.0×10−8 M, with a correlation coefficient of 0.9931. The linear regression equation was ∆I = 69.51 + 4.736 log C, and the detection limit was 3.5×10−15 M (S/N = 3). We compared the performance of the fabricated electrochemical DNA biosensor with those reported in the literature using nanostructured materials for the DNA immobilization layer (Table 1). Our novel biosensor shows a much lower limit of detection, which we attributed to the integration of CuO NWs and SWCNTs producing a larger surface-to-volume ratio derived from the uneven surface of CuO NWs and highly dense network structures between the CuO NWs and SWCNTs. In particular, CuO NWs were entangled with the SWCNTs, and formed a mesh structure of CuO NWs/SWCNTs with good conductivity. 3.5. Selectivity of the biosensor The selectivity of the DNA biosensor was investigated using non-complementary, single-base mismatch (T, G, and A), and complementary DNA 6
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sequences (Fig. 5A). The electrochemical responses of the non-complementary and single-base mismatch sequences were only 5% and 30% that of the complementary sequence, respectively. These data suggest that this biosensor has good selectivity and can readily distinguish single-base mismatched target DNA from the non-complementary sequence and complementary sequence DNA. To assess the selectivity of this biosensor further, we used a different approach to investigate the selectivity of the biosensor. Four types of DNA sequences were selected for evaluation, including single-base mismatch target (1MT), two-base mismatch target (2MT), three-base mismatch target (3MT), and non-complementary sequence. Fig. 5B shows the peak currents for the target DNA and the four types of DNA sequences. The differences among 1MT, 2MT, and 3MT indicated that the base mismatch prevented the completion of hybridization, and a similar result was obtained for the non-complementary sequence. All results showed that the biosensor effectively discriminated different mismatched target DNA, suggesting that this biosensor possesses excellent selectivity. 3.6. Reproducibility and stability of the DNA sensor To characterize its reproducibility, the DNA sensor was tested in 0.1 M PBS (pH 7.0). Five parallel DNA biosensors were fabricated and hybridized with 1.0×10−10 M target DNA sequence, respectively. An acceptable relative standard deviation (R.S.D.) of 8.3% (n = 5) for the DPV current value demonstrates the high reproducibility of DNA detection. When stored in PBS (pH 7.0) at 4 °C for more than two weeks, the current response for the sensor decreased quickly (~71% of its initial value) in the first five days and, thereafter, remained stable from 6–14 days (Fig. 6). These results demonstrate that the proposed DNA biosensor has good stability. 4. Conclusions We developed an ultrasensitive DNA electrochemical biosensor by modifying the GCE with a CuO NWs/ SWCNTs hybrid nanocomposite. Excellent integration of CuO NWs and SWCNTs with good morphologic appearance and structural properties offered a large surface area, fast electron transfer, and good electrical conductivity for DNA detection. Compared with other electrochemical DNA biosensors, the proposed biosensor has many advantages, including simplicity, selectivity, stability, and high sensitivity. Thus, this work establishes a methodology for the development of DNA biosensors with good analytical properties. Furthermore, our novel DNA biosensor has great potential for use in the analysis of real samples. Acknowledgements The authors would like to acknowledge the financial support from Chongqing Graduate Student Research Innovation Project (CYB14028), National Natural Science Foundation of China (NSFC) (81271930), Key Technologies R&D Program of China (2012BAI19B03, 2014BAD07B02), Sichuan Key Technologies R&D Program (No. 2010NZ0093) , Key Laboratory Program of Sichuan Province, China (No. NJ2014-03) and sharing fund of Chongqing university’s large equipments. 7
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Fig. 1 (A) TEM image of CuO NWs, (B) SEM image of CuO NWs/SWCNTs-COOH
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hybrid composite, (C) HR-TEM image of CuO NWs/SWCNTs-COOH, (D) FT-IR
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spectra of (a) CuO nanowires, (b) SWCNTs-COOH, and (c) CuO NWs/SWCNTs-COOH
Fig. 2 Cyclic voltammograms of 1.0 mM [Fe(CN)6]3-/4− and 0.1 M KCl solution at different modified electrodes. “a to e” are bare GCE, CuO NWs/ GCE, CuO NWs-SWCNTs/ GCE, ssDNA/ CuO NWs-SWCNTs/ GCE, and dsDNA/ CuO NWs-SWCNTs/ GCE, respectively.
Fig. 3 Optimization of the experimental conditions: (A) hybridization time, (B) hybridization temperature , (C) on DPV signals of the peak current of adriamycin; the effect of adriamycin concentration (D) and incubation time of dsDNA/ CuO NWs-SWCNTs/ GCE on the DPV signals, Cadriamycin = 2.0 × 10−5M.
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Fig. 4 (A) DPV curves of the biosensor hybridized with increasing concentrations of complementary DNA: 0 M (a); 1.0 × 10−14M (b);1.0 ×10−13M (c); 1.0 × 10−12M (d); 1.0 × 10−11M (e); 1.0 × 10−10M (f); 1.0 × 10−9M (g); 1.0 × 10−8M(h). (B) Logarithmic
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plot of peak current of adriamycin (∆I) vs. the concentration of complementary DNA Fig. 5 Normalized histograms of the signals (∆I/ ∆ICOM) of the biosensor recognized
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different DNA sequences. (A) Non-complementary DNA (Non), single-base T, G, A
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mismatched DNA (T),(G),(A) and complementary DNA (Com), (B) Comparison of DPV responses of the blank and the four different DNA sequences. (1MT; two-base
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mismatch target: 2MT; three-base mismatch target: 3MT).
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Fig. 6 Signal intensities of the biosensor obtained over 14 days.
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Scheme 1 Schematic showing the fabrication procedure of DNA biosensor
Table 1 Comparison of the linear range and detection limit of electrochemical DNA sensors. Modified electrodes
Detection
Linear range (M)
Detection
technique
MWCNTs-COOH
DPV CV
CTS/V2O5/MWCNTs
limit (M) −9
1.6×10 -4.8×10 1.0×10
−11 −14
−8
-1.0×10
−6
-1.0×10
−8
Fe@AuNPs-AETGO
SWV
1.0×10
Ag NPS/PPAA/MWCNTs
DPV
9.0×10−12-9.0×10−9
Au NPs/TB-GO
DPV
GO/PANIw
DPV
MPA/GO-PAMAM/Au GA/Th-G/GA/Cys Ph-NH2/GO
DPV DPV DPV
CuO NWs/SWCNTs
DPV
References
1.0×10
−11
2.12×10
-1.0×10
−12
1.0×10
−12
1.0×10
−12
1.0×10
−12
1.0×10
−12
3.8×10−11 1.76×10
-2.12×10
-1.0×10
−6
-1.0×10
−7
-1.0×10
−6
-1.0×10
−6
[35] [36]
3.2×10−12
[37]
2.9 ×10 −6
[14]
−15
2.0×10
−9
−12
−12
3.25×10 1.0×10
−13
−12
1.26×10
−13
[38] [39] [40] [41]
1.1×10
−13
[42]
3.5×10
−15
This work
Highlights: •
An ultrasensitive DNA electrochemical biosensor was developed 11 Page 11 of 20
• CuO NWs entangled with the SWCNTs formed a mesh structure with good conductivity • It’s the first time use of CuONWs-SWCNTs hybridnanocompositefor DNA detection • The biosensor is simple, selective, stable, and sensitive • The biosensor has great potential for use in analysis of real samples
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Scheme.1.
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Fig. 1.
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Fig. 2.
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A novel and sensitive electrochemical biosensor based on hybrid nanocomposite consisting of copper oxide nanowires (CuO NWs) and carboxyl-functionalized singlewalled carbon nanotubes (SWCNTs-COOH) was first developed for the detection of the specific-sequence target DNA.This schematic represents the fabrication procedure of our DNA biosensor.
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