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chitosan-modified electrodes
Analytica Chimica Acta 584 (2007) 268–274
Electrical detection of deoxyribonucleic acid hybridization based on carbon-nanotubes/nano zirconium dioxide/chitosan-modified electrodes Yunhui Yang a,∗ , Zhijie Wang b , Minghui Yang b , Jishan Li b , Fang Zheng b , Guoli Shen b , Ruqin Yu b a
College of Chemistry and Chemical Engineering, Yunnan Normal University, Jianshe Road, Kunming, Yunnan 650092, China b State Key Laboratory for Chemo/Biosensing and Cheometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, China Received 15 August 2006; received in revised form 19 November 2006; accepted 23 November 2006 Available online 28 November 2006
Abstract A novel and sensitive electrochemical DNA biosensor based on nanoparticles ZrO2 and multi-walled carbon nanotubes (MWNTs) for DNA immobilization and enhanced hybridization detection is described. The MWNTs/nano ZrO2 /chitosan-modified glassy carbon electrode (GCE) was fabricated and oligonucleotides were immobilized to the GCE. The hybridization reaction on the electrode was monitored by differential pulse voltammetry (DPV) analysis using electroactive daunomycin as an indicator. Compared with previous DNA sensors with oligonucleotides directly incorporated on carbon electrodes, this carbon nanotube-based assay with its large surface area and good charge-transport characteristics increased DNA attachment quantity and complementary DNA detection sensitivity. The response signal increases linearly with the increase of the logarithm of the target DNA concentration in the range of 1.49 × 10−10 to 9.32 × 10−8 mol L−1 with the detection limit of 7.5 × 10−11 mol L−1 (S/N = 3). The linear regression equation is I = 32.62 + 3.037 log CDNA (mol L−1 ) with a correlation coefficient value of 0.9842. This is the first application of carbon nanotubes combined with nano ZrO2 to the fabrication of an electrochemical DNA biosensor with a favorable performance for the rapid detection of specific hybridization. © 2006 Elsevier B.V. All rights reserved. Keywords: Nanoparticles zirconium dioxide; Multi-walled carbon nanotubes; Hybridization; Electrochemical deoxyribonucleic acid biosensor; Daunomycin
1. Introduction Carbon nanotubes (CNTs) represent an important group of nanomaterials with attractive geometrical, electronic and chemical properties [1,2]. Since the discovery of CNTs in 1991 [3], considerable efforts have been made to study the application of this new material. The unique electrical properties, high chemical stability and high surface-to-volume ratio of CNTs have been intensively researched for electrocatalytic and sensing applications [4–6]. Various chemical sensors and biosensors based on CNTs have been developed to detect some important species that are related to human health, such as glucose, NADH, ascorbic acid, and cytochrome c [4,7–9]. CNTs have also been utilized in development of electrochemical DNA hybridization biosensors. Various configurations of
∗
Corresponding author. Tel.: +86 871 5515367; fax: +86 871 5516061. E-mail address: [email protected] (Y. Yang).
0003-2670/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2006.11.055
such biosensors have been described in recent years. Most of DNA biosensors rely on the immobilization of single-stranded oligonucleotides onto the electrode surface labeled with an electrochemical indicator to recognize its complementary target sequence. A biosensor based on chitosan doped with CNTs was fabricated to detect salmon sperm DNA. Methylene Blue (MB) was employed as a DNA indicator [10]. In an assay based on magnetic particles, CNTs can be used as carriers for enzyme tags and simultaneously the ␣-naphthol products of enzymatic reaction [11]. In another biosensor, based on magnetic particles for monitoring DNA hybridization, sensing was carried out with a GCE modified with carboxylated Multiwalled carbon nanotubes (MWNTs) and a polypyrrole layer [12]. Most CNTs-sensing research has focused on the utilizing of only CNTs to promote electron-transfer reactions with electroactive species. Recently, composite materials based on integration of CNTs and some other materials to possess properties of the individual components with a synergistic effect have gained growing interest. Materials for such purposes include
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conducting polymers, redox mediators and metal nanoparticles [13–15]. For example, coupling CNTs with Toluidine Blue resulted in the remarkable improvement of the electroactivity of the composite materials toward -nicotinamide adenine dinucleotide (NADH) through synergistic effect [14]. Yang et al. have utilized carbon nanotubes/cobalt hexacyanoferrate nanoparticle (CoNP)-biopolymer system for the fabrication of biosensors [16]. The CoNP–CNTs–chitosan (CHIT) system formed shows synergy between CNTs and CoNP with the significant improvement of redox activity of CoNP due to the excellent electron-transfer ability of CNTs. A series of nanoelectrode arrays prepared by embedding MWNTs in a SiO2 matrix have been developed for ultrasensitive determination of DNA/RNA [17–19]. Zirconia is an inorganic oxide with the thermal stability, chemical inertness, lack of toxicity [20] and affinity for the groups containing oxygen [21], so it is an ideal candidate of materials for immobilization of biomolecules with oxygen groups. Liu et al. [22] described a method of immobilizing DNA based on sol–gel technique. DNA was immobilized on ZrO2 gel casting on glassy carbon electrode, and the resulting DNAmodified electrode was characterized with the cyclic voltammetry. Despite of some advantages of sol–gel immobilization matrix, the brittleness of the sol–gel immobilization matrix confined their wide application in biomolecules immobilization. Zhu et al. have presented a simple and practical DNA hybridization detection based on Methylene Blue (MB) and zirconia (ZrO2 ) thin films modified gold electrode for literature [23]. However, the integration of MWNTs, ZrO2 and CHIT film for DNA hybridization detection has not been explored to enlarge the electrochemical signal of the DNA indicator and increase sensitivity for DNA detection previously. In this study, the MWNTs/ZrO2 /CHIT film modified glassy carbon electrode (GCE) provides a synergistic augmentation of the response current toward indicator of DNA hybridization (daunomycin) compared to MWNTs/CHIT or ZrO2 /CHIT modified GCE. The large amplification of the current could be due to the MWNTs/ZrO2 /CHIT synergy effect leading to improve the loading amount of DNA. The high sensitivity of the modified electrode toward daunomycin was used to immobilize DNA for the fabrication of DNA biosensor.
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Table 1 Oligonucleotide sequences used in this work Target DNA Mismatch DNA Probe DNA
5 -AAAACTTGTGGTAGTTGGAGCTGATGGCGTAGGCAAGAGTGCCC-3 5 -AAATCTTGTGGTAGTTGTAGCTGATGGCGCAGGCAAGAGTGCGC-3 5 -GGGCACTCTTGCCTACGCCATCAGCTCCAACTACCACAAGTTTT-3
otech. Port Co., Ltd. (Shenzhen, China). Daunomycin (DNR) was purchased from Shenzhen Main Luck Pharmaceutical Inc. (Shenzhen, China). Hybridization buffer solution was prepared with 0.05 mol L−1 NaCl, 0.01 mol L−1 Tris, 0.001 mol L−1 EDTA (pH 8.8, STE) in asepsis water and stored in a refrigerator. The supporting electrolyte for the determination of target DNA was 0.01 mol L−1 phosphate buffer solution containing 0.1 mol L−1 NaCl (pH 7.0, PBS). 1.0 mol L−1 DNR solution was prepared freshly with 0.01 mol L−1 phosphate buffer solution containing 0.3 mol L−1 NaCl. Other chemicals were of analytical reagent grade. All solutions were prepared with doubly distilled water. 2.2. Apparatus Cyclic voltammetry (CV) and differential pulse voltammetry (DVP) experiments were carried out using a CHI 760b Electrochemical Analyzer (Chen Hua Instrument Inc., Shanghai, China). A three-electrode cell (10 mL) with the modified glassy carbon (GC) electrode as the working electrode, a saturated calomel electrode (SCE) as reference electrode and a platinum foil electrode as counter electrode was used. All potentials were measured and reported versus the SCE. Scanning electron microscopy (SEM) image of the carbon nanotube/ZrO2 /chitosan film was obtained by using XL30ESEM-TMP (Philips, Holland). 2.3. Treatment of nanoparticles zirconia Nanoparticles zirconia can be treated by adding 0.5 g of nanoparticles zirconia into 100 mL 0.7% sodium dodecylbenzene sulfonate and adjusting pH to 4. After stirred for 6 h, the resulting solution was filtered. Washed with water and dried, the surface-treated nanoparticles zirconia was obtained [24].
2. Experimental 2.4. Treatment of MWNTs 2.1. Reagents and materials Oligonucleotides were received from Dalian Biotechnology Co., Ltd. (Dalian, China). The oligonucleotides sequences were shown in Table 1. The target DNA sequence is the gene sequence associated with colorectal cancer. The probe DNA is the sequence completely complementary with the target DNA. The mismatch DNA is shown with four-mismatched bases underlined. Nanoparticles ZrO2 was produced by Nano Material Application Engineering Technology Center (Zhejiang, China). Chitosan (CHIT, MW ∼ 1 × 106 ; ∼80% deacetylation) were purchased from Sigma (St. Louis, MO, USA). Multi-walled carbon nanotubes (MWNTs) were obtained from Shenzhen Nan-
The MWNTs were first treated with a 3:1 (v/v) mixture of concentrated sulfuric acid and nitric acid with the aid of ultrasonication for 6 h to introduce carboxylic acid groups on the nanotubes surface in order to improve its dispersibility in CHIT. Then, the oxidized carbon were filtrated and washed with distilled water until the pH of the solution was neutral, then dried at 100 ◦ C. 2.5. Preparation of ZrO2 /MWNTs/CHIT solution A 0.2 wt.% CHIT solution was prepared by dissolving appropriate amount of CHIT flakes into 0.05 mol L−1 acetic acid
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and stirring for 3 h at room temperature until complete dissolution. Appropriate amount of surface-treated nanoparticles zirconia and MWNTs dispersed in 0.1% of chitosan. The mass ratio of ZrO2 :MWNTs:chitosan was 1:2.5:100.The mixture was sonicated for 15 min after stirring 1 h. Finally, a high dispersed colloidal solution was formed. 2.6. Immobilization of ssDNA probe on MWNTs–ZrO2 -modified GC electrode A glass carbon (0.4 mm diameter) electrode was polished before each experiment with 0.05 m ␣-alumina powder, successively rinsed thoroughly with absolute alcohol and distilled water in ultrasonic bath and dried in air. Firstly, 10 l of MWNTs/ZrO2 /CHIT casting solution was coated on the glass carbon electrode surface and dried in air. Then, 10 L of probe DNA at 130 nmol L−1 was pipetted onto the surface of above modified electrode. The casting solution was allowed to absorb at room temperature for 1 h. Finally, the DNA electrode was rinsed vigorously with 0.01 mol L−1 PBS (pH 7.0) in order to wash out the unimmobilized probe DNA from the electrode surface before hybridization. 2.7. Determination of target DNA The ssDNA-modified electrode was immersed in hybridization buffer solution (STE, pH 8.8) containing the desired amount of target DNA for 30 min at 60 ◦ C. Subsequently, the electrode was soaked in a stirred PBS (pH 7.0) for removing nonspecifically absorbed target DNA. Similar procedures were also repeated by using the four-base-mismatched DNA sequence. Then, the electrode was immersed into the stirred solution of DNR (1.0 mol L−1 ) and allowed to react for 15 min at room temperature. After accumulation of DNR, the electrode was rinsed in a stirred PBS (pH 7.0) for 10 s. The response signal of DNR was measured by using differential pulse voltammetry and cyclic voltammetry in 0.01 mol L−1 PBS (pH 7.0) with three-electrode system consisted of a dsDNA-modified electrode with accumulated DNR as working electrode, a SCE as reference electrode and a Pt foil as counter electrode. The scan range was from −1.2 to 0.90 V with 50 mV s−1 of scan rate. DNR has an oxidation peak at 0.47 V under this experimental condition.
Fig. 1. SEM of MWNTs/ZrO2 /CHIT.
1.0 × 10−6 mol L−1 DNR of glass carbon electrodes modified with different films of ZrO2 /CHIT; MWNTs/CHIT and MWNTs/ZrO2 /CHIT were studied. Fig. 2 shows cyclic voltammetric recorded at film electrodes in 1.0 mol L−1 DNR solution at the potential scan rate of 50 mV s−1 . It can be seen that the CV response current increased gradually from ZrO2 /CHIT (a) and MWNTs/CHIT (b) to MWNTs/ZrO2 /CHIT (c) film. For the ZrO2 /CHIT film (a), no obvious voltammetric peak was observed in the solution, which indicated that ZrO2 has no catalysis to the oxidation of DNR. For the MWNTs/CHIT film (b), there is an increase of the peak current. The peak potentials of redox waves were at −650 and −710 mV. This is due to the unique catalysis feature and excellent electron-transfer ability of MWNTs. In particular, the introduction of MWNTs and nano ZrO2 in the biopolymer amplified the peak current of DNR by 1.36 times compared with MWNTs/CHIT film which revealed the presence of synergistic effects in MWNTs/ZrO2 /CHIT film leading to improved redox of DNR. The peak potentials of one couple of redox waves were at 474 and 260 mV, respectively, and the peak potentials of the other couple of redox waves
3. Results and discussion 3.1. Characterization of the MWNTS/ZrO2 /CHIT film The SEM image in Fig. 1 confirmed the co-existence of MWNTs and ZrO2 . The nanoparticle ZrO2 were dispersed among carbon nanotube. The diameter of the particles is around 100 nm, while the diameter of MWNTs is between 80 and 120 nm. 3.2. Electrochemical characterization of different film To discern the role of individual components and possible synergy between them, the cyclic voltammetry response to
Fig. 2. Cyclic voltammograms for different electrodes in 1.0 mol L−1 daunomycin solution. (a) ZrO2 /CHIT modified GC electrode; (b) MWNTs/CHIT modified GC electrode; (c) MWNTs/ZrO2 /CHIT modified GC electrode; scan rate was 50 mV s−1 .
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Fig. 3. Cyclic voltammograms of: (a) ZrO2 /CHIT film modified electrode, (b) MWNTs/CHIT film modified electrode and (c) MWNTs/ZrO2 /CHIT film modified electrode in 20 mM K3 Fe(CN)6 containing 0.2 M KCl. Scan rate, 100 mV s−1 .
were at −650 and −710 mV. The synergistic effects with the introduction of MWNTs and nano ZrO2 can be ascribed to the excellent electron-transfer ability of MWNTs and the increased effect surface area of electrode caused by nano ZrO2 . To further verify that peak current increase was really due to the synergistic effects, Fig. 3 shows the CVs of the ZrO2 /CHIT (a), MWNTs/CHIT (b) and ZrO2 /MWNTs/CHIT (c) films modified electrodes in 20 mM K3 Fe(CN)6 containing 0.2 M KCl at 100 mV s−1 . The well-defined oxidation and reduction peaks due to the Fe3+ /Fe2+ redox couple were noticeable at +0.243 and +0.169 V, respectively. The electroactive surface areas of the modified electrodes were determined according to the Randles–Sevcik equation [25]: Ip = 2.69 × 105 AD1/2 n3/2 γ 1/2 C where A represents the area of the electrode (cm2 ), n is the number of electrons participating in the reaction, D is the diffusion coefficient of the molecule in solution which is (6.70 ± 0.02) × 10−6 cm2 s−1 , C represents the concentration of the probe molecule in the solution and γ is the scan rate (0.1 V s−1 ). The average value of the electroactive surface area for ZrO2 /MWNTS /CHIT modified electrode was 0.487 ± 0.03 cm2 compared to 0.29 ± 0.03 cm2 for the MWNTs/CHIT modified electrode and 0.015 ± 0.001 cm2 for the ZrO2 /CHIT electrode. The electroactive surface area for ZrO2 /MWNTs/CHIT modified electrode was about 1.68 times larger than the MWNTs/CHIT modified electrode. DNA biosensor with DNR as hybridization indicator was ever reported in previous literatures [26,27]. Thus, the increased loading of ssDNA probe can enhance the signal response of DNR. Fig. 4 displays the DPV response of DNR in PBS (pH 7.0) obtained with above three different electrodes immobilized with ssDNA probe after incubated in STE containing 3.73 × 10−9 mol L−1 target DNA. The three electrodes were ssDNA/ZrO2 / CHIT/GC electrode (a), ssDNA/MWNTs/CHIT/GC electrode (b) and ssDNA/MWNTs/ZrO2 /CHIT/GC electrode (c). As can be seen, compared with the signal obtained with the
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Fig. 4. The DPV response of daunomycin as indicator in PBS (pH 7.0) recorded for: (a) ZrO2 /CHIT modified GC electrode; (b) MWNTs/CHIT modified GC electrode; (c) MWNTs/ZrO2 /CHIT modified GC electrode; after incubation in STE buffer solution containing 3.73 × 10−9 mol L−1 target DNA. Stirred in PBS (pH 7.0) containing 1.0 mol L−1 daunomycin solution for 5 min. Scan rate was 50 mV s−1 .
ssDNA/ZrO2 /CHIT/GC electrode and ssDNA/MWNTs/CHIT/ GC electrode, the ssDNA/MWNTs/ZrO2 /CHIT/GC electrode offered a maximal signal. This confirmed the synergistic effects of MWNTs/ZrO2 /CHIT composite film which can effectively increased the loading of the ssDNA probe, ascribed to the excellent electron-transfer ability of MWNTs and the high surface area of nano ZrO2 dispersed in CHIT film. 3.3. Optimization of experimental parameters 3.3.1. Hybridization time When the targets DNA in the hybridization buffer solution reach the probe DNA at the surface of the DNA sensor, it takes time for the contacting species to form dsDNA. The effect of hybridization time on DPV signals was also investigated. Fig. 5
Fig. 5. Effect of incubation time. The DNA biosensor was incubated in STE buffer solution containing 7.46 × 10−10 mol L−1 target DNA at 65 ◦ C.
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Fig. 6. The effect of daunomycin accumulation time. The hybridized-electrode was incubated in PBS (pH 7.0) containing 1.0 mol L−1 daunomycin.
demonstrates with the prolonging hybridization time from 0.25 to 1.5 h, the current increases dramatically and then tends to change only slightly. So an incubation time of 30 min was adopted in experiments. One would expect that most of the surface-exposed probes DNA are binding with the target DNA in the hybridization solution, forming compact complexes on the surface of the DNA sensor. 3.3.2. Hybridization temperature An additional parameter that affected the assay was the hybridization temperature. The effect of hybridization temperature on response current was examined from 20 to 65 ◦ C. It was found that the signal increases with an increase of temperature up to 60 ◦ C. The linear regression equation for the linear part is I = 0.0458 + 0.1108x, where I is the current (A) and x is the temperature (◦ C). At higher temperature, the dsDNA would untie. To obtain the maximum current, 60 ◦ C was selected in the subsequent work.
Fig. 7. The DPV response of the DNA biosensor was incubated in SET buffer solution of pH 8.8 containing: (a) 1.49 × 10−10 mol L−1 , (b) 3.72 × 10−10 mol L−1 , (c) 7.45 × 10−10 mol L−1 , (d) 1.86 × 10−8 mol L−1 and (e) 9.32 × 10−8 mol L−1 target DNA. The DPV response of daunomycin as indicator was measured in PBS (pH 7.0).
of the logarithm of the target DNA concentration in the range of 1.49 × 10−10 to 9.32 × 10−8 mol L−1 with a detection limit of 7.5 × 10−11 mol L−1 (S/N = 3). The linear regression equation is I = 32.62 + 3.037 log CDNA (mol L−1 ) with a correlation coefficient value of 0.9842. The detection limit was lower than the value using MWNTs or ZrO2 solely reported by Li et al. [10], Cai et al. [26] and Zhu et al. [23] due to the synergistic effects of MWNTs and nano ZrO2 . 3.5. The selectivity of the DNA sensor The selectivity of this assay was explored by using the ssDNA/MWNTs/ZrO2 /CHIT to hybridize with different kinds of DNA sequences. Fig. 9 shows DPV response of
3.3.3. Accumulation time The accumulation time of DNR was also investigated. The hybridized-electrode was incubated in PBS (pH 7.0) containing 1.0 mol L−1 DNR. With the increasing accumulation time from 5 to 15 min, the peak current of DNR increased significantly (Fig. 6). The response signal tended to stabilize when the accumulation time reached 15 min or above. Therefore, the optimum accumulation time was 15 min. 3.4. Calibration curve To obtain the calibration curve, the peak current values of DNR were measured by DPV under the optimum conditions after ssDNA probe hybridized with the target DNA sequences of different concentrations according to the procedure described. As can be seen from Fig. 7, the DPV peak current increased as target DNA concentration increased. Fig. 8 shows the calibration curve. The response signal increases linearly with the increase
Fig. 8. Calibration curve of the DNA biosensor. The DNA biosensor was incubated in SET buffer solution of pH 8.8 containing different amount of target DNA. The DPV response of daunomycin as indicator was measured in PBS (pH 7.0). Other conditions are optimal experimental conditions.
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Fig. 9. The DPV response of daunomycin as indicator in PBS (pH 7.0) using ssDNA probe-modified MWNTs/ZrO2 /CHIT/GC electrode (a), after exposure to 1.32 × 10−9 mol L−1 four-base-mismatched DNA sequence (b); the same after hybridization with the complementary target DNA sequence (7.45 × 10−10 mol L−1 ) (c).
the ssDNA-modified electrode (a), ssDNA-modified electrode hybridized with 1.32 × 10−9 mol L−1 of four-base-mismatched oligonucleotide (b), ssDNA-modified electrode hybridized with 7.45 × 10−10 mol L−1 of perfectly matched oligonucleotide (c). The sensor have much smaller responses when hybridization with four-base-mismatched oligonucleotide. The current ratio is only 0.66 when four-base mismatch sequence and complete complementary oligonucleotide concentration ratio is 1.77. As expected, only the complementary target sequence gave a significant increasing response, which shows the high selectivity of this hybridization assay. The high sequence-selectivity of this sensor is double-guaranteed by the complementary hybridization of target DNA with capture and detection probes. However, in Cai’s article [26], DPV response obtained with the colitoxin probe hybridized with same amount (1.25 × 10−8 mol L−1 ) of three-base mismatch sequence and complete complementary oligonucleotide are about 17.4 and 20.7 A. The current ratio is 0.84 while three-base mismatch sequence and complete complementary oligonucleotide concentration ratio is only 1.0. This comparison means that specificity of this DNA sensor is better than previous report [26]. 3.6. Reproducibility of the DNA sensor Reproducibility of the DNA sensor was tested by measuring 3.72 × 10−10 mol L−1 target DNA solution. Three DNA sensors, made independently, showed the response current values of 5.02, 4.94 and 5.39 A with an acceptable variation coefficient of 4.69% (n = 3). It indicated that a satisfactory reproducibility could be obtained by this system.
4. Conclusions In this article, an efficient DNA immobilization matrix based on a MWNTs/nano ZrO2 /chitosan composite film was firstly developed for the fabrication of DNA biosensor. The simple manipulation procedure, high selectivity, low-cost, fast response and broad linear range are the main features of proposed DNA sensor. Nano-porous ZrO2 and carbon nanotubes have synergistic effects on the redox of DNR. Compared with previous DNA sensors with oligonucleotides solely immobilized on MWNTs or ZrO2 film, this carbon nanotubes and nano ZrO2 based assay with its large surface area and good charge-transport characteristics increased ssDNA loading quantity and improved detection sensitivity for DNA hybridization. This is the first application of carbon nanotubes combined with nano ZrO2 to fabricate an electrochemical DNA biosensor with a favorable performance for the rapid detection of specific hybridization. Acknowledgements This work was supported by Foundation of Science Commission of Yunnan Province (No. 2006B0028M) and the National Natural Science Foundation of China (Grant Nos. 20435010, 20375012 and 20205005). References [1] R.H. Baughman, A. Zakhidov, W.A. De Heer, Science 297 (2002) 787. [2] C.N. Rao, B.C. Satishkumar, A. Govindaraj, M. Nath, Chem. Phys. Chem. 2 (2001) 78. [3] S. Iijima, Nature 354 (1991) 56.
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Electrical detection of deoxyribonucleic acid hybridization based on carbon-nanotubes/nano zirconium dioxide/chitosan-modified electrodes Yunhui Yang a,∗ , Zhijie Wang b , Minghui Yang b , Jishan Li b , Fang Zheng b , Guoli Shen b , Ruqin Yu b a
College of Chemistry and Chemical Engineering, Yunnan Normal University, Jianshe Road, Kunming, Yunnan 650092, China b State Key Laboratory for Chemo/Biosensing and Cheometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, China Received 15 August 2006; received in revised form 19 November 2006; accepted 23 November 2006 Available online 28 November 2006
Abstract A novel and sensitive electrochemical DNA biosensor based on nanoparticles ZrO2 and multi-walled carbon nanotubes (MWNTs) for DNA immobilization and enhanced hybridization detection is described. The MWNTs/nano ZrO2 /chitosan-modified glassy carbon electrode (GCE) was fabricated and oligonucleotides were immobilized to the GCE. The hybridization reaction on the electrode was monitored by differential pulse voltammetry (DPV) analysis using electroactive daunomycin as an indicator. Compared with previous DNA sensors with oligonucleotides directly incorporated on carbon electrodes, this carbon nanotube-based assay with its large surface area and good charge-transport characteristics increased DNA attachment quantity and complementary DNA detection sensitivity. The response signal increases linearly with the increase of the logarithm of the target DNA concentration in the range of 1.49 × 10−10 to 9.32 × 10−8 mol L−1 with the detection limit of 7.5 × 10−11 mol L−1 (S/N = 3). The linear regression equation is I = 32.62 + 3.037 log CDNA (mol L−1 ) with a correlation coefficient value of 0.9842. This is the first application of carbon nanotubes combined with nano ZrO2 to the fabrication of an electrochemical DNA biosensor with a favorable performance for the rapid detection of specific hybridization. © 2006 Elsevier B.V. All rights reserved. Keywords: Nanoparticles zirconium dioxide; Multi-walled carbon nanotubes; Hybridization; Electrochemical deoxyribonucleic acid biosensor; Daunomycin
1. Introduction Carbon nanotubes (CNTs) represent an important group of nanomaterials with attractive geometrical, electronic and chemical properties [1,2]. Since the discovery of CNTs in 1991 [3], considerable efforts have been made to study the application of this new material. The unique electrical properties, high chemical stability and high surface-to-volume ratio of CNTs have been intensively researched for electrocatalytic and sensing applications [4–6]. Various chemical sensors and biosensors based on CNTs have been developed to detect some important species that are related to human health, such as glucose, NADH, ascorbic acid, and cytochrome c [4,7–9]. CNTs have also been utilized in development of electrochemical DNA hybridization biosensors. Various configurations of
∗
Corresponding author. Tel.: +86 871 5515367; fax: +86 871 5516061. E-mail address: [email protected] (Y. Yang).
0003-2670/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2006.11.055
such biosensors have been described in recent years. Most of DNA biosensors rely on the immobilization of single-stranded oligonucleotides onto the electrode surface labeled with an electrochemical indicator to recognize its complementary target sequence. A biosensor based on chitosan doped with CNTs was fabricated to detect salmon sperm DNA. Methylene Blue (MB) was employed as a DNA indicator [10]. In an assay based on magnetic particles, CNTs can be used as carriers for enzyme tags and simultaneously the ␣-naphthol products of enzymatic reaction [11]. In another biosensor, based on magnetic particles for monitoring DNA hybridization, sensing was carried out with a GCE modified with carboxylated Multiwalled carbon nanotubes (MWNTs) and a polypyrrole layer [12]. Most CNTs-sensing research has focused on the utilizing of only CNTs to promote electron-transfer reactions with electroactive species. Recently, composite materials based on integration of CNTs and some other materials to possess properties of the individual components with a synergistic effect have gained growing interest. Materials for such purposes include
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conducting polymers, redox mediators and metal nanoparticles [13–15]. For example, coupling CNTs with Toluidine Blue resulted in the remarkable improvement of the electroactivity of the composite materials toward -nicotinamide adenine dinucleotide (NADH) through synergistic effect [14]. Yang et al. have utilized carbon nanotubes/cobalt hexacyanoferrate nanoparticle (CoNP)-biopolymer system for the fabrication of biosensors [16]. The CoNP–CNTs–chitosan (CHIT) system formed shows synergy between CNTs and CoNP with the significant improvement of redox activity of CoNP due to the excellent electron-transfer ability of CNTs. A series of nanoelectrode arrays prepared by embedding MWNTs in a SiO2 matrix have been developed for ultrasensitive determination of DNA/RNA [17–19]. Zirconia is an inorganic oxide with the thermal stability, chemical inertness, lack of toxicity [20] and affinity for the groups containing oxygen [21], so it is an ideal candidate of materials for immobilization of biomolecules with oxygen groups. Liu et al. [22] described a method of immobilizing DNA based on sol–gel technique. DNA was immobilized on ZrO2 gel casting on glassy carbon electrode, and the resulting DNAmodified electrode was characterized with the cyclic voltammetry. Despite of some advantages of sol–gel immobilization matrix, the brittleness of the sol–gel immobilization matrix confined their wide application in biomolecules immobilization. Zhu et al. have presented a simple and practical DNA hybridization detection based on Methylene Blue (MB) and zirconia (ZrO2 ) thin films modified gold electrode for literature [23]. However, the integration of MWNTs, ZrO2 and CHIT film for DNA hybridization detection has not been explored to enlarge the electrochemical signal of the DNA indicator and increase sensitivity for DNA detection previously. In this study, the MWNTs/ZrO2 /CHIT film modified glassy carbon electrode (GCE) provides a synergistic augmentation of the response current toward indicator of DNA hybridization (daunomycin) compared to MWNTs/CHIT or ZrO2 /CHIT modified GCE. The large amplification of the current could be due to the MWNTs/ZrO2 /CHIT synergy effect leading to improve the loading amount of DNA. The high sensitivity of the modified electrode toward daunomycin was used to immobilize DNA for the fabrication of DNA biosensor.
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Table 1 Oligonucleotide sequences used in this work Target DNA Mismatch DNA Probe DNA
5 -AAAACTTGTGGTAGTTGGAGCTGATGGCGTAGGCAAGAGTGCCC-3 5 -AAATCTTGTGGTAGTTGTAGCTGATGGCGCAGGCAAGAGTGCGC-3 5 -GGGCACTCTTGCCTACGCCATCAGCTCCAACTACCACAAGTTTT-3
otech. Port Co., Ltd. (Shenzhen, China). Daunomycin (DNR) was purchased from Shenzhen Main Luck Pharmaceutical Inc. (Shenzhen, China). Hybridization buffer solution was prepared with 0.05 mol L−1 NaCl, 0.01 mol L−1 Tris, 0.001 mol L−1 EDTA (pH 8.8, STE) in asepsis water and stored in a refrigerator. The supporting electrolyte for the determination of target DNA was 0.01 mol L−1 phosphate buffer solution containing 0.1 mol L−1 NaCl (pH 7.0, PBS). 1.0 mol L−1 DNR solution was prepared freshly with 0.01 mol L−1 phosphate buffer solution containing 0.3 mol L−1 NaCl. Other chemicals were of analytical reagent grade. All solutions were prepared with doubly distilled water. 2.2. Apparatus Cyclic voltammetry (CV) and differential pulse voltammetry (DVP) experiments were carried out using a CHI 760b Electrochemical Analyzer (Chen Hua Instrument Inc., Shanghai, China). A three-electrode cell (10 mL) with the modified glassy carbon (GC) electrode as the working electrode, a saturated calomel electrode (SCE) as reference electrode and a platinum foil electrode as counter electrode was used. All potentials were measured and reported versus the SCE. Scanning electron microscopy (SEM) image of the carbon nanotube/ZrO2 /chitosan film was obtained by using XL30ESEM-TMP (Philips, Holland). 2.3. Treatment of nanoparticles zirconia Nanoparticles zirconia can be treated by adding 0.5 g of nanoparticles zirconia into 100 mL 0.7% sodium dodecylbenzene sulfonate and adjusting pH to 4. After stirred for 6 h, the resulting solution was filtered. Washed with water and dried, the surface-treated nanoparticles zirconia was obtained [24].
2. Experimental 2.4. Treatment of MWNTs 2.1. Reagents and materials Oligonucleotides were received from Dalian Biotechnology Co., Ltd. (Dalian, China). The oligonucleotides sequences were shown in Table 1. The target DNA sequence is the gene sequence associated with colorectal cancer. The probe DNA is the sequence completely complementary with the target DNA. The mismatch DNA is shown with four-mismatched bases underlined. Nanoparticles ZrO2 was produced by Nano Material Application Engineering Technology Center (Zhejiang, China). Chitosan (CHIT, MW ∼ 1 × 106 ; ∼80% deacetylation) were purchased from Sigma (St. Louis, MO, USA). Multi-walled carbon nanotubes (MWNTs) were obtained from Shenzhen Nan-
The MWNTs were first treated with a 3:1 (v/v) mixture of concentrated sulfuric acid and nitric acid with the aid of ultrasonication for 6 h to introduce carboxylic acid groups on the nanotubes surface in order to improve its dispersibility in CHIT. Then, the oxidized carbon were filtrated and washed with distilled water until the pH of the solution was neutral, then dried at 100 ◦ C. 2.5. Preparation of ZrO2 /MWNTs/CHIT solution A 0.2 wt.% CHIT solution was prepared by dissolving appropriate amount of CHIT flakes into 0.05 mol L−1 acetic acid
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and stirring for 3 h at room temperature until complete dissolution. Appropriate amount of surface-treated nanoparticles zirconia and MWNTs dispersed in 0.1% of chitosan. The mass ratio of ZrO2 :MWNTs:chitosan was 1:2.5:100.The mixture was sonicated for 15 min after stirring 1 h. Finally, a high dispersed colloidal solution was formed. 2.6. Immobilization of ssDNA probe on MWNTs–ZrO2 -modified GC electrode A glass carbon (0.4 mm diameter) electrode was polished before each experiment with 0.05 m ␣-alumina powder, successively rinsed thoroughly with absolute alcohol and distilled water in ultrasonic bath and dried in air. Firstly, 10 l of MWNTs/ZrO2 /CHIT casting solution was coated on the glass carbon electrode surface and dried in air. Then, 10 L of probe DNA at 130 nmol L−1 was pipetted onto the surface of above modified electrode. The casting solution was allowed to absorb at room temperature for 1 h. Finally, the DNA electrode was rinsed vigorously with 0.01 mol L−1 PBS (pH 7.0) in order to wash out the unimmobilized probe DNA from the electrode surface before hybridization. 2.7. Determination of target DNA The ssDNA-modified electrode was immersed in hybridization buffer solution (STE, pH 8.8) containing the desired amount of target DNA for 30 min at 60 ◦ C. Subsequently, the electrode was soaked in a stirred PBS (pH 7.0) for removing nonspecifically absorbed target DNA. Similar procedures were also repeated by using the four-base-mismatched DNA sequence. Then, the electrode was immersed into the stirred solution of DNR (1.0 mol L−1 ) and allowed to react for 15 min at room temperature. After accumulation of DNR, the electrode was rinsed in a stirred PBS (pH 7.0) for 10 s. The response signal of DNR was measured by using differential pulse voltammetry and cyclic voltammetry in 0.01 mol L−1 PBS (pH 7.0) with three-electrode system consisted of a dsDNA-modified electrode with accumulated DNR as working electrode, a SCE as reference electrode and a Pt foil as counter electrode. The scan range was from −1.2 to 0.90 V with 50 mV s−1 of scan rate. DNR has an oxidation peak at 0.47 V under this experimental condition.
Fig. 1. SEM of MWNTs/ZrO2 /CHIT.
1.0 × 10−6 mol L−1 DNR of glass carbon electrodes modified with different films of ZrO2 /CHIT; MWNTs/CHIT and MWNTs/ZrO2 /CHIT were studied. Fig. 2 shows cyclic voltammetric recorded at film electrodes in 1.0 mol L−1 DNR solution at the potential scan rate of 50 mV s−1 . It can be seen that the CV response current increased gradually from ZrO2 /CHIT (a) and MWNTs/CHIT (b) to MWNTs/ZrO2 /CHIT (c) film. For the ZrO2 /CHIT film (a), no obvious voltammetric peak was observed in the solution, which indicated that ZrO2 has no catalysis to the oxidation of DNR. For the MWNTs/CHIT film (b), there is an increase of the peak current. The peak potentials of redox waves were at −650 and −710 mV. This is due to the unique catalysis feature and excellent electron-transfer ability of MWNTs. In particular, the introduction of MWNTs and nano ZrO2 in the biopolymer amplified the peak current of DNR by 1.36 times compared with MWNTs/CHIT film which revealed the presence of synergistic effects in MWNTs/ZrO2 /CHIT film leading to improved redox of DNR. The peak potentials of one couple of redox waves were at 474 and 260 mV, respectively, and the peak potentials of the other couple of redox waves
3. Results and discussion 3.1. Characterization of the MWNTS/ZrO2 /CHIT film The SEM image in Fig. 1 confirmed the co-existence of MWNTs and ZrO2 . The nanoparticle ZrO2 were dispersed among carbon nanotube. The diameter of the particles is around 100 nm, while the diameter of MWNTs is between 80 and 120 nm. 3.2. Electrochemical characterization of different film To discern the role of individual components and possible synergy between them, the cyclic voltammetry response to
Fig. 2. Cyclic voltammograms for different electrodes in 1.0 mol L−1 daunomycin solution. (a) ZrO2 /CHIT modified GC electrode; (b) MWNTs/CHIT modified GC electrode; (c) MWNTs/ZrO2 /CHIT modified GC electrode; scan rate was 50 mV s−1 .
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Fig. 3. Cyclic voltammograms of: (a) ZrO2 /CHIT film modified electrode, (b) MWNTs/CHIT film modified electrode and (c) MWNTs/ZrO2 /CHIT film modified electrode in 20 mM K3 Fe(CN)6 containing 0.2 M KCl. Scan rate, 100 mV s−1 .
were at −650 and −710 mV. The synergistic effects with the introduction of MWNTs and nano ZrO2 can be ascribed to the excellent electron-transfer ability of MWNTs and the increased effect surface area of electrode caused by nano ZrO2 . To further verify that peak current increase was really due to the synergistic effects, Fig. 3 shows the CVs of the ZrO2 /CHIT (a), MWNTs/CHIT (b) and ZrO2 /MWNTs/CHIT (c) films modified electrodes in 20 mM K3 Fe(CN)6 containing 0.2 M KCl at 100 mV s−1 . The well-defined oxidation and reduction peaks due to the Fe3+ /Fe2+ redox couple were noticeable at +0.243 and +0.169 V, respectively. The electroactive surface areas of the modified electrodes were determined according to the Randles–Sevcik equation [25]: Ip = 2.69 × 105 AD1/2 n3/2 γ 1/2 C where A represents the area of the electrode (cm2 ), n is the number of electrons participating in the reaction, D is the diffusion coefficient of the molecule in solution which is (6.70 ± 0.02) × 10−6 cm2 s−1 , C represents the concentration of the probe molecule in the solution and γ is the scan rate (0.1 V s−1 ). The average value of the electroactive surface area for ZrO2 /MWNTS /CHIT modified electrode was 0.487 ± 0.03 cm2 compared to 0.29 ± 0.03 cm2 for the MWNTs/CHIT modified electrode and 0.015 ± 0.001 cm2 for the ZrO2 /CHIT electrode. The electroactive surface area for ZrO2 /MWNTs/CHIT modified electrode was about 1.68 times larger than the MWNTs/CHIT modified electrode. DNA biosensor with DNR as hybridization indicator was ever reported in previous literatures [26,27]. Thus, the increased loading of ssDNA probe can enhance the signal response of DNR. Fig. 4 displays the DPV response of DNR in PBS (pH 7.0) obtained with above three different electrodes immobilized with ssDNA probe after incubated in STE containing 3.73 × 10−9 mol L−1 target DNA. The three electrodes were ssDNA/ZrO2 / CHIT/GC electrode (a), ssDNA/MWNTs/CHIT/GC electrode (b) and ssDNA/MWNTs/ZrO2 /CHIT/GC electrode (c). As can be seen, compared with the signal obtained with the
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Fig. 4. The DPV response of daunomycin as indicator in PBS (pH 7.0) recorded for: (a) ZrO2 /CHIT modified GC electrode; (b) MWNTs/CHIT modified GC electrode; (c) MWNTs/ZrO2 /CHIT modified GC electrode; after incubation in STE buffer solution containing 3.73 × 10−9 mol L−1 target DNA. Stirred in PBS (pH 7.0) containing 1.0 mol L−1 daunomycin solution for 5 min. Scan rate was 50 mV s−1 .
ssDNA/ZrO2 /CHIT/GC electrode and ssDNA/MWNTs/CHIT/ GC electrode, the ssDNA/MWNTs/ZrO2 /CHIT/GC electrode offered a maximal signal. This confirmed the synergistic effects of MWNTs/ZrO2 /CHIT composite film which can effectively increased the loading of the ssDNA probe, ascribed to the excellent electron-transfer ability of MWNTs and the high surface area of nano ZrO2 dispersed in CHIT film. 3.3. Optimization of experimental parameters 3.3.1. Hybridization time When the targets DNA in the hybridization buffer solution reach the probe DNA at the surface of the DNA sensor, it takes time for the contacting species to form dsDNA. The effect of hybridization time on DPV signals was also investigated. Fig. 5
Fig. 5. Effect of incubation time. The DNA biosensor was incubated in STE buffer solution containing 7.46 × 10−10 mol L−1 target DNA at 65 ◦ C.
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Fig. 6. The effect of daunomycin accumulation time. The hybridized-electrode was incubated in PBS (pH 7.0) containing 1.0 mol L−1 daunomycin.
demonstrates with the prolonging hybridization time from 0.25 to 1.5 h, the current increases dramatically and then tends to change only slightly. So an incubation time of 30 min was adopted in experiments. One would expect that most of the surface-exposed probes DNA are binding with the target DNA in the hybridization solution, forming compact complexes on the surface of the DNA sensor. 3.3.2. Hybridization temperature An additional parameter that affected the assay was the hybridization temperature. The effect of hybridization temperature on response current was examined from 20 to 65 ◦ C. It was found that the signal increases with an increase of temperature up to 60 ◦ C. The linear regression equation for the linear part is I = 0.0458 + 0.1108x, where I is the current (A) and x is the temperature (◦ C). At higher temperature, the dsDNA would untie. To obtain the maximum current, 60 ◦ C was selected in the subsequent work.
Fig. 7. The DPV response of the DNA biosensor was incubated in SET buffer solution of pH 8.8 containing: (a) 1.49 × 10−10 mol L−1 , (b) 3.72 × 10−10 mol L−1 , (c) 7.45 × 10−10 mol L−1 , (d) 1.86 × 10−8 mol L−1 and (e) 9.32 × 10−8 mol L−1 target DNA. The DPV response of daunomycin as indicator was measured in PBS (pH 7.0).
of the logarithm of the target DNA concentration in the range of 1.49 × 10−10 to 9.32 × 10−8 mol L−1 with a detection limit of 7.5 × 10−11 mol L−1 (S/N = 3). The linear regression equation is I = 32.62 + 3.037 log CDNA (mol L−1 ) with a correlation coefficient value of 0.9842. The detection limit was lower than the value using MWNTs or ZrO2 solely reported by Li et al. [10], Cai et al. [26] and Zhu et al. [23] due to the synergistic effects of MWNTs and nano ZrO2 . 3.5. The selectivity of the DNA sensor The selectivity of this assay was explored by using the ssDNA/MWNTs/ZrO2 /CHIT to hybridize with different kinds of DNA sequences. Fig. 9 shows DPV response of
3.3.3. Accumulation time The accumulation time of DNR was also investigated. The hybridized-electrode was incubated in PBS (pH 7.0) containing 1.0 mol L−1 DNR. With the increasing accumulation time from 5 to 15 min, the peak current of DNR increased significantly (Fig. 6). The response signal tended to stabilize when the accumulation time reached 15 min or above. Therefore, the optimum accumulation time was 15 min. 3.4. Calibration curve To obtain the calibration curve, the peak current values of DNR were measured by DPV under the optimum conditions after ssDNA probe hybridized with the target DNA sequences of different concentrations according to the procedure described. As can be seen from Fig. 7, the DPV peak current increased as target DNA concentration increased. Fig. 8 shows the calibration curve. The response signal increases linearly with the increase
Fig. 8. Calibration curve of the DNA biosensor. The DNA biosensor was incubated in SET buffer solution of pH 8.8 containing different amount of target DNA. The DPV response of daunomycin as indicator was measured in PBS (pH 7.0). Other conditions are optimal experimental conditions.
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Fig. 9. The DPV response of daunomycin as indicator in PBS (pH 7.0) using ssDNA probe-modified MWNTs/ZrO2 /CHIT/GC electrode (a), after exposure to 1.32 × 10−9 mol L−1 four-base-mismatched DNA sequence (b); the same after hybridization with the complementary target DNA sequence (7.45 × 10−10 mol L−1 ) (c).
the ssDNA-modified electrode (a), ssDNA-modified electrode hybridized with 1.32 × 10−9 mol L−1 of four-base-mismatched oligonucleotide (b), ssDNA-modified electrode hybridized with 7.45 × 10−10 mol L−1 of perfectly matched oligonucleotide (c). The sensor have much smaller responses when hybridization with four-base-mismatched oligonucleotide. The current ratio is only 0.66 when four-base mismatch sequence and complete complementary oligonucleotide concentration ratio is 1.77. As expected, only the complementary target sequence gave a significant increasing response, which shows the high selectivity of this hybridization assay. The high sequence-selectivity of this sensor is double-guaranteed by the complementary hybridization of target DNA with capture and detection probes. However, in Cai’s article [26], DPV response obtained with the colitoxin probe hybridized with same amount (1.25 × 10−8 mol L−1 ) of three-base mismatch sequence and complete complementary oligonucleotide are about 17.4 and 20.7 A. The current ratio is 0.84 while three-base mismatch sequence and complete complementary oligonucleotide concentration ratio is only 1.0. This comparison means that specificity of this DNA sensor is better than previous report [26]. 3.6. Reproducibility of the DNA sensor Reproducibility of the DNA sensor was tested by measuring 3.72 × 10−10 mol L−1 target DNA solution. Three DNA sensors, made independently, showed the response current values of 5.02, 4.94 and 5.39 A with an acceptable variation coefficient of 4.69% (n = 3). It indicated that a satisfactory reproducibility could be obtained by this system.
4. Conclusions In this article, an efficient DNA immobilization matrix based on a MWNTs/nano ZrO2 /chitosan composite film was firstly developed for the fabrication of DNA biosensor. The simple manipulation procedure, high selectivity, low-cost, fast response and broad linear range are the main features of proposed DNA sensor. Nano-porous ZrO2 and carbon nanotubes have synergistic effects on the redox of DNR. Compared with previous DNA sensors with oligonucleotides solely immobilized on MWNTs or ZrO2 film, this carbon nanotubes and nano ZrO2 based assay with its large surface area and good charge-transport characteristics increased ssDNA loading quantity and improved detection sensitivity for DNA hybridization. This is the first application of carbon nanotubes combined with nano ZrO2 to fabricate an electrochemical DNA biosensor with a favorable performance for the rapid detection of specific hybridization. Acknowledgements This work was supported by Foundation of Science Commission of Yunnan Province (No. 2006B0028M) and the National Natural Science Foundation of China (Grant Nos. 20435010, 20375012 and 20205005). References [1] R.H. Baughman, A. Zakhidov, W.A. De Heer, Science 297 (2002) 787. [2] C.N. Rao, B.C. Satishkumar, A. Govindaraj, M. Nath, Chem. Phys. Chem. 2 (2001) 78. [3] S. Iijima, Nature 354 (1991) 56.
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