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Hybridization biosensor using diaquabis[N-(2-pyridinylmethyl)benzamide-κ2N,O]-cadmium(II) dinitrate as a new electroactive indicator for detection of human hepatitis B virus DNA
Sensors and Actuators B 124 (2007) 290–296
Hybridization biosensor using diaquabis[N-(2-pyridinylmethyl)benzamide-2N,O]-cadmium(II) dinitrate as a new electroactive indicator for detection of human hepatitis B virus DNA Shusheng Zhang ∗ , Qianqian Tan, Feng Li, Xiaoru Zhang College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042, PR China Received 4 December 2006; accepted 18 December 2006 Available online 28 December 2006
Abstract The complex diaquabis[N-(2-pyridinylmethyl) benzamide-2 N,O]-cadmium(II) dinitrate {[CdL2 (H2 O)2 ](NO3 )2 , where L = N-(2pyridinylmethyl) benzamide} was synthesized and characterized by X-ray diffraction analysis. Fluorescence spectroscopy and voltammetry were used to probe the interaction between [CdL2 ]2+ and salmon sperm DNA. Results showed that [CdL2 ]2+ had excellent electrochemical activity on glassy carbon electrode (GCE) and could intercalate into the double helix of double-stranded DNA (dsDNA). In 0.2 mol L−1 NaAc-HAc media (pH 7.02), the binding ratio between [CdL2 ]2+ and salmon sperm DNA was calculated to be 2:1 and the binding constant was 25.56 L1/2 mol−1/2 . An electrochemical DNA biosensor for the detection of human hepatitis B virus (HBV) DNA fragment was developed. The biosensor relied on the covalent immobilization of the 21-mer single-stranded DNA (ssDNA) related to HBV gene on the modified glassy carbon electrode (GCE). Using [CdL2 ]2+ as novel electroactive indicator, the hybridization between the probe and its complementary ssDNA, as the target, was investigated by differential pulse voltammetry (DPV). Experiment with non-complementary oligonucleotides was carried out to assess the selectivity of the developed electrochemical DNA biosensor. The target HBV DNA could be quantified ranged from 1.01 × 10−8 to 1.62 × 10−6 mol L−1 with good linearity (γ = 0.9962). The detection limit was 7.19 × 10−9 mol L−1 (3σ, n = 11). © 2007 Elsevier B.V. All rights reserved. Keywords: Electrochemical DNA biosensor; Hepatitis B virus; Hybridization; Voltammetry; [CdL2 ]2+
1. Introduction Deoxyribonucleic acid (DNA) is an important genetic material in organism and it is the basis of gene expression. The detection of DNA is currently an area of tremendous interest as it plays a major role in clinical, forensic, and pharmaceutical applications [1–6]. Electrochemical transducers offer attractive advantages, e.g., good sensitivity, accurate specificity, simplicity and low-cost, for converting nucleic acid hybridization events into useful analytical signals [7–13]. Nowadays, electrochemical DNA biosensors have become popular in DNA sequence analysis. These biosensors could be developed by immobilizing
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single-stranded DNA (ssDNA) probes on different electrodes. In general, protocols for detection hybridization events between DNA probes and their complementary DNA fragments are classified into major two, direct and indirect strategies. In direct detection protocol, oxidation of guanine moiety in DNA strands eliminates external labels (label-free detection). While indirect detection protocol is based on the incorporation of an electroactive indicator [14,15]. Amongst them, the later fashion, using an electroactive indicator to convert the hybridization/recognition event into measurable electrochemical signals, offers a very attractive route and is recently preferred. Development of more sensitive electrochemical indicators, therefore, is of particular significance. Owing to the potential of interaction with the nitrogenous bases of DNA, the interaction between transition metal complexes and DNA currently receive considerable attention. It has
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been reported that transition metal complexes have activity of chemistry nuclease, such as specificity splitting DNA. Additionally, transition metal complexes could be helpful in designing and synthesis of anticancer and antivirotic drugs. Since 1980s, interactions between DNA and transition metal complexes, including Co(III), Fe(II), and Ru(II) complexes, have been well documented [16–22]. In general, transition metal complexes usually consist of one or several transition metal ion(s) as center ion(s) and several organic heterocycles as ligands. The different interaction with single-stranded DNA and doublestranded DNA (dsDNA) coupled with good electrochemical properties of the center ion(s) make DNA hybridization events easily transformable into electrochemical signals [23]. Nowadays, transition metal complexes show potential and have been successfully applied as electroactive indicators for detecting DNA hybridization events. Most indicator-based electrochemical DNA biosensors use cationic metal complexes that interact in a different way with ssDNA and dsDNA [24,25]. In this study, the complex diaquabis[N-(2-pyridinylmethyl) benzamide-2 N,O]-cadmium(II) dinitrate {[CdL2 (H2 O)2 ] (NO3 )2 , where L = N-(2-pyridinylmethyl) benzamide} was synthesized and characterized by X-ray diffraction analysis. The interaction between [CdL2 ]2+ and DNA was studied by using cyclic voltammetry and fluorescence spectroscopy. A new electrochemical DNA biosensor for the detection of human hepatitis B virus (HBV) DNA fragment was developed. The biosensor was based on the covalent immobilization of ssDNA related to HBV gene on the modified glassy carbon electrode (GCE). Using [CdL2 ]2+ as novel electroactive indicator, surface hybridization between immobilized ssDNA and its complementary ssDNA was evidenced by differential pulse voltammetry (DPV). Experiment with non-complementary oligonucleotide showed the selectivity of the developed electrochemical DNA biosensor. The new electroactive indicator and electrochemical DNA biosensor might have potential application in DNA detection, drug designing and diagnosis of disease.
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tary to S1 ): 5 -AAT GTG CTC CCC CAA CTC CTC-3 ; S3 (non-complementary to S1 ): 5 -AAA AGG TGT AAG CGT TTG CCG-3 . All stock solutions of the 21-base oligomers (100 g mL−1 ) were prepared with 10 mmol L−1 Tris–HCl plus 1 mmol L−1 EDTA at pH 8.0 (TE solution). More dilute solutions of salmon sperm DNA and three ssDNA were also prepared with TE solution. The final concentrations of three ssDNA were all controlled at 3.19 × 10−5 mol L−1 (CS1 = CS2 = CS3 = 3.19 × 10−5 mol L−1 ). N-hydroxysuccinimide (NHS) and 1ethyl-3-(3-dimethyllaminopropyl) carbodiimide-hydrochloride (EDC) were used without further purification. Other chemicals employed were all of analytical grade and double distilled water (DDW) was used throughout. 2.2. Fluorescence spectroscopic study on the interaction between [CdL2 ]2+ and DNA Fluorescence spectroscopy was used to investigate the interaction between [CdL2 ]2+ and salmon sperm DNA. Appropriate amount of [CdL2 ]2+ and salmon sperm DNA solution was mixed in 0.2 mol L−1 NaAc-HAc media (pH 7.02) and reacted for 8 min at room temperature. Afterward, the fluorescence spectroscopy was recorded on fluorescence spectrophotometer. 2.3. Electrochemical study on the interaction between [CdL2 ]2+ and DNA Appropriate amount of [CdL2 ]2+ was added to 2 mL of 0.2 mol L−1 NaAc-HAc media at pH 7.02. Then different amounts of DNA were added, respectively. The changes on characteristic of cyclic voltammograms (CVs) were recorded. The potential scanning range was from −0.6 to −1.1 V. The scanning rate was 0.10 V s−1 . The sample interval was 0.001 V and the quiet time 2 s. 2.4. Preparation of the electrochemical DNA biosensor
2. Experimental 2.1. Apparatus and reagents The electrochemical measurement was carried out with Model CHI 832B electrochemical analyzer (ChenHua Instruments, China). A three-electrode system was employed using Pt wire as counter electrode, Ag/AgCl/KCl (sat) as reference electrode, and glassy carbon electrode (GCE) as working electrode. Spectroscopic experiments were conducted on Hitachi F-4500 fluorospectrometer (Hitachi, Japan). Double-stranded salmon sperm DNA (10 mg mL−1 , A260 / A280 > 1.8) was purchased from Huashun Biological Engineering Company (Shanghai, China). The complex of [CdL2 ]2+ was synthesized according to previous report [26]. The base sequences of three 21-base oligonucleotides (SBS Genetech Company, China) were as follows: ssDNA related to HBV gene (probe ssDNA, S1 ): 5 -GAG GAG TTG GGG GAG CAC ATT-3 ; HBV DNA fragment (S2 , target ssDNA, complemen-
The electrochemical DNA biosensor developed was based on the covalent immobilization of ssDNA on the modified electrode. Before the immobilization of probe DNA, the GCE was firstly polished using 1.0, 0.3 and 0.05 m ␣-Al2 O3 suspension, respectively, followed with extensively rinse in DDW with ultrasonic. Then, the electrode was oxidized at +0.50 V for 1 min in 50 mmol L−1 phosphate buffer solution (PBS, pH 7.4). After being rinsed repeatedly, the electrode was activated by evaporation to dryness of 20 L of a solution containing 5 mmol L−1 EDC plus 8 mmol L−1 NHS in PBS at pH 7.4. Salmon sperm DNA (dsDNA) or ssDNA (S1 ) was subsequently immobilized on modified-GCE. In detail, 20 L ssDNA solution (CS1 = 3.19 × 10−5 mol L−1 ) or salmon sperm DNA solution (CdsDNA = 4.68 × 10−2 mol L−1 ) was dropped on the modified GCE surface and then air-dried to dryness under an infrared lamp. Then the electrode was washed with DDW and Tris–HCl buffer solution (pH 7.0) orderly to eliminate the adsorbed DNA.
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2.5. Hybridization on the DNA-immobilized electrode
3. Results and discussion
To achieve DNA interaction, the S1 -immobilized electrode was immersed in 20 mmol L−1 Tris–HCl buffer (pH 7.0) containing complementary ssDNA segment (S2 ) or non-complementary ssDNA segment (S3 ), respectively. The hybridization was allowed for 1 h at 40 ◦ C with stirring. Afterwards, the electrode was dried at room temperature and successively rinsed with the same Tris–HCl buffer and DDW to remove S2 or S3 adsorbed on the electrode surface.
3.1. Strucrture characterization of the complex
2.6. Intercalation of hybridization indicator [CdL2 ]2+ was accumulated at the electrode by immersion in 0.2 mol L−1 NaAc-HAc media (pH 7.02) containing 6.7 × 10−5 mol L−1 [CdL2 ]2+ for 10 min at 40 ◦ C with stirring. Four types of electrodes, S1 -immobilized GCE, dsDNAimmobilized GCE and two electrodes obtained after DNA hybridization, were used. After the accumulation, the electrode was rinsed with DDW in order to eliminate the [CdL2 ]2+ adsorbed on the electrode surface. 2.7. Electrochemical detection The electrochemical investigation of hybridization was carried out by differential pulse voltammetry in a 10 mL electrochemical cell. The DPV measurements were performed in 0.2 mol L−1 NaAc-HAc media (pH 7.02) using [CdL2 ]2+ accumulated electrode as working electrode, an Ag/AgCl as the reference electrode and a platinum wire as the auxiliary electrode. The scan ranges were from −0.4 to −1.1 V. The peak related to the reduction of [CdL2 ]2+ at approximately −0.82 V was taken as the electrochemical detection signal.
The complex of diaquabis[N-(2-pyridinylmethyl) benzamide-2 N,O]-cadmium(II)dinitrate {[CdL2 (H2 O)2 ](NO3 )2 , where L = N-(2-pyridinylmethyl) benzamide} was synthesized and the crystal structure was determined [26]. It crystallized in the monoclinic system. The Cd(II) atom, which was located on a twofold rotation axis, was six-coordinated by two O atoms and two N atoms from two benzamide ligands, and two O atoms from two water molecules also located on the two-fold axis, as shown in Fig. 1. The geometry around the Cd(II) atom was octahedral, and two nitrate anions lied outside the coordination sphere, balancing the charge. The amide C–N bond was known to possess a partial double-bond character due to donation of the non-bonding electron pair on the nitrogen [27]. As expected, the benzamide chelate was not planar and the two aromatic rings made a dihedral angle of 60.6 (1)◦ . In the crystal structure, the water molecules acted as donors to form O–H· · ·O hydrogen bonds. These hydrogen bonds involving the nitrate anions linked the ions and molecules into chains along the b-axis. The packing was further stabilized into a three-dimensional framework by N–H· · ·O interactions. 3.2. Fluorescence spectroscopic study of the interaction between [CdL2 ]2+ and dsDNA To investigate the interaction between [CdL2 ]2+ and doublestranded salmon sperm DNA, fluorescence spectroscopic study was designed. Fig. 2 displayed the fluorescence features of [CdL2 ]2+ in the absence and presence of DNA. It was observed that [CdL2 ]2+ had the maximal emission wavelength at 523 nm with strong fluorescence intensity (curve 1). When DNA was added, fluorescence intensity of [CdL2 ]2+ was depressed (curve
Fig. 1. The molecular structure for [CdL2 (H2 O)2 ](NO3 )2 with the atomic numbering scheme.
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Fig. 2. Fluorescence spectrum of [CdL2 (H2 O)2 ](NO3 )2 in the absence (1) and presence of 2.48 × 10−3 mol L−1 double-stranded salmon sperm DNA (2) (C[CdL2 ]2+ = 1.00 × 10−6 mol L−1 ).
2), indicating the interaction between the complex and DNA helix. 3.3. Electrochemical interaction between [CdL2 ]2+ and dsDNA Electrochemical study on [CdL2 ]2+ and its interaction with double-stranded salmon sperm DNA were performed at 25 ◦ C. Fig. 3 shows the cyclic voltammograms of [CdL2 ]2+ in the absence and presence of DNA. The buffer solution used was 0.2 mol L−1 NaAc-HAc media (pH 7.02). Curve 1 demonstrated the typical voltammogram of 3 × 10−4 mol L−1 [CdL2 ]2+ , in which a cathodic peak for [CdL2 ]2+ was observed. The potential for such cathodic peak (Epc ) was −0.85 V. Curve 2 was the cyclic voltammogram obtained when [CdL2 ]2+ interacted with dsDNA. We could see that peak current decreased and Epc shifted
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to positive potentials. Both Eø shift and decrease of peak current implied the formation of a new association complex. Bard and co-workers [28] have reported the discrimination of binding modes between small molecules and DNA. If Eø shifted to more negative value, the interaction mode was electrostatic binding. On the contrary, if Eø shifted to more positive value, the interaction mode was intercalative binding. Therefore, we attributed the interaction between [CdL2 ]2+ and DNA to be the intercalation into the base pairs of DNA. Experimental conditions for the interacting between [CdL2 ]2+ and DNA, e.g., pH, reaction time, the concentrations of DNA, were optimized. It was found that the value of peak current changed distinctly when pH of the NaAc-HAc media changed. The maximum peak current was found at 0.2 mol L−1 NaAc-HAc media at pH 7.02. Consequently, pH 7.02 was set as the reactive pH. Experiments to optimize the reaction time were also performed. As the peak current reached a constant value after reaction for 8 min, therefore, 8 min was chosen as the reaction time. The influence of scan rate was also investigated. Results showed that the peak current of cathodic peak (ipc ) was not directly proportional to both the scan rate and the square root of scan rate in 0.01–0.5 V s−1 range. The phenomenon indicated that the electro-deoxidation process of [CdL2 ]2+ was controlled not only by the diffusion of [CdL2 ]2+ , but also the adsorption effect on the electrode surface. 3.4. The binding ratio and binding constant between [CdL2 ]2+ and dsDNA The binding ratio and binding constant between [CdL2 ]2+ and double-stranded salmon sperm DNA were studied based on the assumption that DNA and [CdL2 ]2+ only produced one single complex [29], which was DNA − n[CdL2 ]2+ , according to the following equation. DNA + n[CdL2 ]2+ DNA − n[CdL2 ]2+ (n = 1, 2, 3, . . . or1, 1/2, 1/3, . . .)
(1)
The equilibrium constant (β) was given as below: β=
[DNA − n[CdL2]2+ ] [DNA][[CdL2]2+ ]
n
(2)
And the following equation could be deduced. 1 1 1 = + −n ipc ipc,max βipc,max [[CdL2 ]2+ ]
Fig. 3. The cyclic voltammograms of [CdL2 (H2 O)2 ](NO3 )2 in the absence (1) and presence of 4.68 × 10−4 mol L−1 double-stranded salmon sperm DNA (2) (C[CdL2 ]2+ = 3 × 10−4 mol L−1 ).
(3)
where [DNA] was the equilibrium concentration of doublestranded salmon sperm DNA (dsDNA), while ipc represented the difference of reduction peak current, ipc , in the absence and presence of dsDNA. The maximum value of ipc was designated as ipc,max. [[CdL2 ]2+ ] represented the equilibrium concentration of the metal complex. We could see that different n gave different relationship of 2+ −n −1 i−1 pc versus [[CdL2 ] ] . With the suitable n, curve of ipc 2+ −n versus [[CdL2 ] ] could be a straight line. In such case, the binding constant β could be calculated from the slope and inter-
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down distinctly for n = 1 and 2. It indicated that [CdL2 ]2+ bound to DNA to form a 2:1 association complex. From the slope and intercept of the straight line, the binding constant β was calculated to be 25.56 L1/2 mol−1/2 , which correlated to the equation DNA + 1/2[CdL2 ]2+ DNA − 1/2[CdL2 ]2+ . 3.5. The selectivity of the prepared electrochemical DNA biosensor
Fig. 4. Curves of reduction peak current of [CdL2 (H2 O)2 ](NO3 )2 (ipc ), and ipc versus analytical concentration of [CdL2 ]2+ . (1)CdsDNA : 0; (2) CdsDNA : 4.68 × 10−5 mol L−1 ; (3) ipc = ipc l − ipc 2.
cept of the straight line, and the resulting n could be regarded as the binding ratio. In the absence and presence of dsDNA, the dependence of the reduction peak current (ipc ) of [CdL2 ]2+ on the analytical concentration of [CdL2 ]2+ was shown in Fig. 4. Curve 1 demonstrated the circumstance in the absence of dsDNA, whereas, curve 2 displayed the relationship between ipc and analytical concentration of [CdL2 ]2+ when 4.68 × 10−5 mol L−1 DNA was added. The relationship between the difference of ipc in curve1 and curve 2 (ipc , ipc = ipc1 − ipc2 ) and the analytical concentration of [CdL2 ]2+ is also shown in Fig. 4 (curve 3). We could also obtained i−1 pc based on calculating difference of ipc (ipc ) between curve1 and curve 2 (ipc = ipc1 − ipc2 ). Curves of i−1 pc versus the equilibrium concentration of 2+ −0.5 , [[CdL ]2+ ]−1 and [CdL2 ]2+ , i−1 2 pc versus [[CdL2 ] ] 2+ −2 [[CdL2 ] ] , were listed in Fig. 5. It could be seen that a straight line (γ = 0.9989) was obtained for n = 0.5, while the curves bent
Electrochemical DNA biosensor usually involved the immobilization of a single-stranded probe nucleic acid to a solid substrate and the production of electrochemical signal using redox groups. The DPV was used to study the selectivity of the prepared electrochemical DNA sensor. Fig. 6 shows the representative DPV curves obtained in NaAc-HAc media using S1 -modified GCE as working electrode (curve 1). S1 –S2 hybridized GCE (curve 2), dsDNA-modified GCE (curve 3) and S1 –S3 hybridized GCE (curve 4), respectively. Each measurement was performed after [CdL2 ]2+ was accumulated. It was found that no voltammetric response was detected in curve 1 for S1 -modified electrode. As shown in curve 2, a reduction peak which showed the same potential as bare GCE in the [CdL2 ]2+ appeared for S1 –S2 hybridized GCE. As we know, S2 was complementary to the immobilized S1 . Consequently, S1 –S2 hybridization resulted in double-stranded DNA. The reduction peak at about −0.82 V confirmed the intercalation of [CdL2 ]2+ . The peak current was 3.079 × 10−7 A. When dsDNA was immobilized on the electrode (curve 3), the reduction peak with the same potential as bare GCE in the [CdL2 ]2+ also appeared, indicating the intercalation of [CdL2 ]2+ . However, the peak current was 2.645 × 10−7 A, which was lower than S1 –S2 hybridized electrode did. We attributed this phenomenon to the steric hindrance effect caused by the immobilization of dsDNA, which greatly influenced the spread at the electrode surface. Additionally, no voltammetric response was detected in curve 4 for S1 –S3 hybridized GCE. As we know, no base pair of the dsDNA was formed because S3 was non-complementary to S1 . As a result, no [CdL2 ]2+ was accumulated on the surface of electrode. In other words, only the target ssDNA, that was complementary to the immobilized probe ssDNA, could be detected. The results indicated the selectivity of the new electrochemical DNA biosensor. 3.6. Quantitative analysis of target ssDNA
Fig. 5. Curves of i−1 pc versus C[[CdL
2]
2+ ]−n
,
n = 1/2, 1, and 2.
The sensitivity of the prepared DNA electrochemical sensor was investigated by varying the concentration of target ssDNA, which was complementary to the probe ssDNA immobilized on GCE. Results showed that differential current obtained in DPV mearsurement increased when the concentration of target ssDNA increased. The increase of peak current was linear with the concentration of target ssDNA ranging from 1.01 × 10−8 to 1.62 × 10−6 mol L−1 (Fig. 7). The regression equation was ip = 0.1116C + 3.0365 × 10−8 (C, mol L−1 ; ip , A) and the correlation coefficient was 0.9962. Thus, a detection limit of 7.19 × 10−9 mol L−1 of target ssDNA (3σ, n = 11) was achieved.
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Fig. 6. The DPV curves of S1 -modified (1), S1 –S2 hybridized (2), dsDNA-modified (3), S1 –S3 hybridized (4) GCE.
indicator and developed electrochemical DNA sensor might have promising in transducing DNA hybridization, drug designing and diagnosis of diseases. Acknowledgements The work was supported by the Natural Science Foundation of Shandong Province (No. Z2006B01) and the Program for New Century Excellent Talents in Universities (No. NCET-04-0649). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.snb.2006.12.040. References Fig. 7. The increase of peak current (ip ) versus concentration of target ssDNA (S2 ).
4. Conclusions Interaction between [CdL2 ]2+ and double-stranded salmon sperm DNA was studied using fluorescence spectroscopy and voltammetry. Results showed that [CdL2 ]2+ could intercalate into the base pairs of the dsDNA. The binding ratio between [CdL2 ]2+ and salmon sperm DNA was calculated to be 2:1 and the binding constant was 25.56 L1/2 mol−1/2 . Using [CdL2 ]2+ as a new electroactive indicator, the new electrochemical DNA biosensor could selectively detect human hepatitis B virus DNA fragment. The utility of the new electrochemical hybridization
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Biographies Shusheng Zhang was born in 1966. He received his PhD in chemistry from Nanjing University, China, in 1999. He has been working as a professor and dean at the College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology since 1999. He has published 30 papers and 2 scientific books. His research interests cover analytical chemistry, bioelectrochemistry and chemical biosensors. Qianqian Tan is currently a postgraduate student of Qingdao University of Science and Technology. Majoring in Analytical Chemistry. Feng Li is a professor of chemistry, College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao, China. He obtained his PhD from Nankai University in 2005. His research interests cover analytical chemistry and chip-based capillary electrophoresis. Xiaoru Zhang is an associate professor of chemistry, College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao, China. She obtained her PhD from Ocean University of China in 2003. Her research interests cover organic synthesis.
Hybridization biosensor using diaquabis[N-(2-pyridinylmethyl)benzamide-2N,O]-cadmium(II) dinitrate as a new electroactive indicator for detection of human hepatitis B virus DNA Shusheng Zhang ∗ , Qianqian Tan, Feng Li, Xiaoru Zhang College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042, PR China Received 4 December 2006; accepted 18 December 2006 Available online 28 December 2006
Abstract The complex diaquabis[N-(2-pyridinylmethyl) benzamide-2 N,O]-cadmium(II) dinitrate {[CdL2 (H2 O)2 ](NO3 )2 , where L = N-(2pyridinylmethyl) benzamide} was synthesized and characterized by X-ray diffraction analysis. Fluorescence spectroscopy and voltammetry were used to probe the interaction between [CdL2 ]2+ and salmon sperm DNA. Results showed that [CdL2 ]2+ had excellent electrochemical activity on glassy carbon electrode (GCE) and could intercalate into the double helix of double-stranded DNA (dsDNA). In 0.2 mol L−1 NaAc-HAc media (pH 7.02), the binding ratio between [CdL2 ]2+ and salmon sperm DNA was calculated to be 2:1 and the binding constant was 25.56 L1/2 mol−1/2 . An electrochemical DNA biosensor for the detection of human hepatitis B virus (HBV) DNA fragment was developed. The biosensor relied on the covalent immobilization of the 21-mer single-stranded DNA (ssDNA) related to HBV gene on the modified glassy carbon electrode (GCE). Using [CdL2 ]2+ as novel electroactive indicator, the hybridization between the probe and its complementary ssDNA, as the target, was investigated by differential pulse voltammetry (DPV). Experiment with non-complementary oligonucleotides was carried out to assess the selectivity of the developed electrochemical DNA biosensor. The target HBV DNA could be quantified ranged from 1.01 × 10−8 to 1.62 × 10−6 mol L−1 with good linearity (γ = 0.9962). The detection limit was 7.19 × 10−9 mol L−1 (3σ, n = 11). © 2007 Elsevier B.V. All rights reserved. Keywords: Electrochemical DNA biosensor; Hepatitis B virus; Hybridization; Voltammetry; [CdL2 ]2+
1. Introduction Deoxyribonucleic acid (DNA) is an important genetic material in organism and it is the basis of gene expression. The detection of DNA is currently an area of tremendous interest as it plays a major role in clinical, forensic, and pharmaceutical applications [1–6]. Electrochemical transducers offer attractive advantages, e.g., good sensitivity, accurate specificity, simplicity and low-cost, for converting nucleic acid hybridization events into useful analytical signals [7–13]. Nowadays, electrochemical DNA biosensors have become popular in DNA sequence analysis. These biosensors could be developed by immobilizing
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single-stranded DNA (ssDNA) probes on different electrodes. In general, protocols for detection hybridization events between DNA probes and their complementary DNA fragments are classified into major two, direct and indirect strategies. In direct detection protocol, oxidation of guanine moiety in DNA strands eliminates external labels (label-free detection). While indirect detection protocol is based on the incorporation of an electroactive indicator [14,15]. Amongst them, the later fashion, using an electroactive indicator to convert the hybridization/recognition event into measurable electrochemical signals, offers a very attractive route and is recently preferred. Development of more sensitive electrochemical indicators, therefore, is of particular significance. Owing to the potential of interaction with the nitrogenous bases of DNA, the interaction between transition metal complexes and DNA currently receive considerable attention. It has
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been reported that transition metal complexes have activity of chemistry nuclease, such as specificity splitting DNA. Additionally, transition metal complexes could be helpful in designing and synthesis of anticancer and antivirotic drugs. Since 1980s, interactions between DNA and transition metal complexes, including Co(III), Fe(II), and Ru(II) complexes, have been well documented [16–22]. In general, transition metal complexes usually consist of one or several transition metal ion(s) as center ion(s) and several organic heterocycles as ligands. The different interaction with single-stranded DNA and doublestranded DNA (dsDNA) coupled with good electrochemical properties of the center ion(s) make DNA hybridization events easily transformable into electrochemical signals [23]. Nowadays, transition metal complexes show potential and have been successfully applied as electroactive indicators for detecting DNA hybridization events. Most indicator-based electrochemical DNA biosensors use cationic metal complexes that interact in a different way with ssDNA and dsDNA [24,25]. In this study, the complex diaquabis[N-(2-pyridinylmethyl) benzamide-2 N,O]-cadmium(II) dinitrate {[CdL2 (H2 O)2 ] (NO3 )2 , where L = N-(2-pyridinylmethyl) benzamide} was synthesized and characterized by X-ray diffraction analysis. The interaction between [CdL2 ]2+ and DNA was studied by using cyclic voltammetry and fluorescence spectroscopy. A new electrochemical DNA biosensor for the detection of human hepatitis B virus (HBV) DNA fragment was developed. The biosensor was based on the covalent immobilization of ssDNA related to HBV gene on the modified glassy carbon electrode (GCE). Using [CdL2 ]2+ as novel electroactive indicator, surface hybridization between immobilized ssDNA and its complementary ssDNA was evidenced by differential pulse voltammetry (DPV). Experiment with non-complementary oligonucleotide showed the selectivity of the developed electrochemical DNA biosensor. The new electroactive indicator and electrochemical DNA biosensor might have potential application in DNA detection, drug designing and diagnosis of disease.
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tary to S1 ): 5 -AAT GTG CTC CCC CAA CTC CTC-3 ; S3 (non-complementary to S1 ): 5 -AAA AGG TGT AAG CGT TTG CCG-3 . All stock solutions of the 21-base oligomers (100 g mL−1 ) were prepared with 10 mmol L−1 Tris–HCl plus 1 mmol L−1 EDTA at pH 8.0 (TE solution). More dilute solutions of salmon sperm DNA and three ssDNA were also prepared with TE solution. The final concentrations of three ssDNA were all controlled at 3.19 × 10−5 mol L−1 (CS1 = CS2 = CS3 = 3.19 × 10−5 mol L−1 ). N-hydroxysuccinimide (NHS) and 1ethyl-3-(3-dimethyllaminopropyl) carbodiimide-hydrochloride (EDC) were used without further purification. Other chemicals employed were all of analytical grade and double distilled water (DDW) was used throughout. 2.2. Fluorescence spectroscopic study on the interaction between [CdL2 ]2+ and DNA Fluorescence spectroscopy was used to investigate the interaction between [CdL2 ]2+ and salmon sperm DNA. Appropriate amount of [CdL2 ]2+ and salmon sperm DNA solution was mixed in 0.2 mol L−1 NaAc-HAc media (pH 7.02) and reacted for 8 min at room temperature. Afterward, the fluorescence spectroscopy was recorded on fluorescence spectrophotometer. 2.3. Electrochemical study on the interaction between [CdL2 ]2+ and DNA Appropriate amount of [CdL2 ]2+ was added to 2 mL of 0.2 mol L−1 NaAc-HAc media at pH 7.02. Then different amounts of DNA were added, respectively. The changes on characteristic of cyclic voltammograms (CVs) were recorded. The potential scanning range was from −0.6 to −1.1 V. The scanning rate was 0.10 V s−1 . The sample interval was 0.001 V and the quiet time 2 s. 2.4. Preparation of the electrochemical DNA biosensor
2. Experimental 2.1. Apparatus and reagents The electrochemical measurement was carried out with Model CHI 832B electrochemical analyzer (ChenHua Instruments, China). A three-electrode system was employed using Pt wire as counter electrode, Ag/AgCl/KCl (sat) as reference electrode, and glassy carbon electrode (GCE) as working electrode. Spectroscopic experiments were conducted on Hitachi F-4500 fluorospectrometer (Hitachi, Japan). Double-stranded salmon sperm DNA (10 mg mL−1 , A260 / A280 > 1.8) was purchased from Huashun Biological Engineering Company (Shanghai, China). The complex of [CdL2 ]2+ was synthesized according to previous report [26]. The base sequences of three 21-base oligonucleotides (SBS Genetech Company, China) were as follows: ssDNA related to HBV gene (probe ssDNA, S1 ): 5 -GAG GAG TTG GGG GAG CAC ATT-3 ; HBV DNA fragment (S2 , target ssDNA, complemen-
The electrochemical DNA biosensor developed was based on the covalent immobilization of ssDNA on the modified electrode. Before the immobilization of probe DNA, the GCE was firstly polished using 1.0, 0.3 and 0.05 m ␣-Al2 O3 suspension, respectively, followed with extensively rinse in DDW with ultrasonic. Then, the electrode was oxidized at +0.50 V for 1 min in 50 mmol L−1 phosphate buffer solution (PBS, pH 7.4). After being rinsed repeatedly, the electrode was activated by evaporation to dryness of 20 L of a solution containing 5 mmol L−1 EDC plus 8 mmol L−1 NHS in PBS at pH 7.4. Salmon sperm DNA (dsDNA) or ssDNA (S1 ) was subsequently immobilized on modified-GCE. In detail, 20 L ssDNA solution (CS1 = 3.19 × 10−5 mol L−1 ) or salmon sperm DNA solution (CdsDNA = 4.68 × 10−2 mol L−1 ) was dropped on the modified GCE surface and then air-dried to dryness under an infrared lamp. Then the electrode was washed with DDW and Tris–HCl buffer solution (pH 7.0) orderly to eliminate the adsorbed DNA.
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2.5. Hybridization on the DNA-immobilized electrode
3. Results and discussion
To achieve DNA interaction, the S1 -immobilized electrode was immersed in 20 mmol L−1 Tris–HCl buffer (pH 7.0) containing complementary ssDNA segment (S2 ) or non-complementary ssDNA segment (S3 ), respectively. The hybridization was allowed for 1 h at 40 ◦ C with stirring. Afterwards, the electrode was dried at room temperature and successively rinsed with the same Tris–HCl buffer and DDW to remove S2 or S3 adsorbed on the electrode surface.
3.1. Strucrture characterization of the complex
2.6. Intercalation of hybridization indicator [CdL2 ]2+ was accumulated at the electrode by immersion in 0.2 mol L−1 NaAc-HAc media (pH 7.02) containing 6.7 × 10−5 mol L−1 [CdL2 ]2+ for 10 min at 40 ◦ C with stirring. Four types of electrodes, S1 -immobilized GCE, dsDNAimmobilized GCE and two electrodes obtained after DNA hybridization, were used. After the accumulation, the electrode was rinsed with DDW in order to eliminate the [CdL2 ]2+ adsorbed on the electrode surface. 2.7. Electrochemical detection The electrochemical investigation of hybridization was carried out by differential pulse voltammetry in a 10 mL electrochemical cell. The DPV measurements were performed in 0.2 mol L−1 NaAc-HAc media (pH 7.02) using [CdL2 ]2+ accumulated electrode as working electrode, an Ag/AgCl as the reference electrode and a platinum wire as the auxiliary electrode. The scan ranges were from −0.4 to −1.1 V. The peak related to the reduction of [CdL2 ]2+ at approximately −0.82 V was taken as the electrochemical detection signal.
The complex of diaquabis[N-(2-pyridinylmethyl) benzamide-2 N,O]-cadmium(II)dinitrate {[CdL2 (H2 O)2 ](NO3 )2 , where L = N-(2-pyridinylmethyl) benzamide} was synthesized and the crystal structure was determined [26]. It crystallized in the monoclinic system. The Cd(II) atom, which was located on a twofold rotation axis, was six-coordinated by two O atoms and two N atoms from two benzamide ligands, and two O atoms from two water molecules also located on the two-fold axis, as shown in Fig. 1. The geometry around the Cd(II) atom was octahedral, and two nitrate anions lied outside the coordination sphere, balancing the charge. The amide C–N bond was known to possess a partial double-bond character due to donation of the non-bonding electron pair on the nitrogen [27]. As expected, the benzamide chelate was not planar and the two aromatic rings made a dihedral angle of 60.6 (1)◦ . In the crystal structure, the water molecules acted as donors to form O–H· · ·O hydrogen bonds. These hydrogen bonds involving the nitrate anions linked the ions and molecules into chains along the b-axis. The packing was further stabilized into a three-dimensional framework by N–H· · ·O interactions. 3.2. Fluorescence spectroscopic study of the interaction between [CdL2 ]2+ and dsDNA To investigate the interaction between [CdL2 ]2+ and doublestranded salmon sperm DNA, fluorescence spectroscopic study was designed. Fig. 2 displayed the fluorescence features of [CdL2 ]2+ in the absence and presence of DNA. It was observed that [CdL2 ]2+ had the maximal emission wavelength at 523 nm with strong fluorescence intensity (curve 1). When DNA was added, fluorescence intensity of [CdL2 ]2+ was depressed (curve
Fig. 1. The molecular structure for [CdL2 (H2 O)2 ](NO3 )2 with the atomic numbering scheme.
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Fig. 2. Fluorescence spectrum of [CdL2 (H2 O)2 ](NO3 )2 in the absence (1) and presence of 2.48 × 10−3 mol L−1 double-stranded salmon sperm DNA (2) (C[CdL2 ]2+ = 1.00 × 10−6 mol L−1 ).
2), indicating the interaction between the complex and DNA helix. 3.3. Electrochemical interaction between [CdL2 ]2+ and dsDNA Electrochemical study on [CdL2 ]2+ and its interaction with double-stranded salmon sperm DNA were performed at 25 ◦ C. Fig. 3 shows the cyclic voltammograms of [CdL2 ]2+ in the absence and presence of DNA. The buffer solution used was 0.2 mol L−1 NaAc-HAc media (pH 7.02). Curve 1 demonstrated the typical voltammogram of 3 × 10−4 mol L−1 [CdL2 ]2+ , in which a cathodic peak for [CdL2 ]2+ was observed. The potential for such cathodic peak (Epc ) was −0.85 V. Curve 2 was the cyclic voltammogram obtained when [CdL2 ]2+ interacted with dsDNA. We could see that peak current decreased and Epc shifted
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to positive potentials. Both Eø shift and decrease of peak current implied the formation of a new association complex. Bard and co-workers [28] have reported the discrimination of binding modes between small molecules and DNA. If Eø shifted to more negative value, the interaction mode was electrostatic binding. On the contrary, if Eø shifted to more positive value, the interaction mode was intercalative binding. Therefore, we attributed the interaction between [CdL2 ]2+ and DNA to be the intercalation into the base pairs of DNA. Experimental conditions for the interacting between [CdL2 ]2+ and DNA, e.g., pH, reaction time, the concentrations of DNA, were optimized. It was found that the value of peak current changed distinctly when pH of the NaAc-HAc media changed. The maximum peak current was found at 0.2 mol L−1 NaAc-HAc media at pH 7.02. Consequently, pH 7.02 was set as the reactive pH. Experiments to optimize the reaction time were also performed. As the peak current reached a constant value after reaction for 8 min, therefore, 8 min was chosen as the reaction time. The influence of scan rate was also investigated. Results showed that the peak current of cathodic peak (ipc ) was not directly proportional to both the scan rate and the square root of scan rate in 0.01–0.5 V s−1 range. The phenomenon indicated that the electro-deoxidation process of [CdL2 ]2+ was controlled not only by the diffusion of [CdL2 ]2+ , but also the adsorption effect on the electrode surface. 3.4. The binding ratio and binding constant between [CdL2 ]2+ and dsDNA The binding ratio and binding constant between [CdL2 ]2+ and double-stranded salmon sperm DNA were studied based on the assumption that DNA and [CdL2 ]2+ only produced one single complex [29], which was DNA − n[CdL2 ]2+ , according to the following equation. DNA + n[CdL2 ]2+ DNA − n[CdL2 ]2+ (n = 1, 2, 3, . . . or1, 1/2, 1/3, . . .)
(1)
The equilibrium constant (β) was given as below: β=
[DNA − n[CdL2]2+ ] [DNA][[CdL2]2+ ]
n
(2)
And the following equation could be deduced. 1 1 1 = + −n ipc ipc,max βipc,max [[CdL2 ]2+ ]
Fig. 3. The cyclic voltammograms of [CdL2 (H2 O)2 ](NO3 )2 in the absence (1) and presence of 4.68 × 10−4 mol L−1 double-stranded salmon sperm DNA (2) (C[CdL2 ]2+ = 3 × 10−4 mol L−1 ).
(3)
where [DNA] was the equilibrium concentration of doublestranded salmon sperm DNA (dsDNA), while ipc represented the difference of reduction peak current, ipc , in the absence and presence of dsDNA. The maximum value of ipc was designated as ipc,max. [[CdL2 ]2+ ] represented the equilibrium concentration of the metal complex. We could see that different n gave different relationship of 2+ −n −1 i−1 pc versus [[CdL2 ] ] . With the suitable n, curve of ipc 2+ −n versus [[CdL2 ] ] could be a straight line. In such case, the binding constant β could be calculated from the slope and inter-
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down distinctly for n = 1 and 2. It indicated that [CdL2 ]2+ bound to DNA to form a 2:1 association complex. From the slope and intercept of the straight line, the binding constant β was calculated to be 25.56 L1/2 mol−1/2 , which correlated to the equation DNA + 1/2[CdL2 ]2+ DNA − 1/2[CdL2 ]2+ . 3.5. The selectivity of the prepared electrochemical DNA biosensor
Fig. 4. Curves of reduction peak current of [CdL2 (H2 O)2 ](NO3 )2 (ipc ), and ipc versus analytical concentration of [CdL2 ]2+ . (1)CdsDNA : 0; (2) CdsDNA : 4.68 × 10−5 mol L−1 ; (3) ipc = ipc l − ipc 2.
cept of the straight line, and the resulting n could be regarded as the binding ratio. In the absence and presence of dsDNA, the dependence of the reduction peak current (ipc ) of [CdL2 ]2+ on the analytical concentration of [CdL2 ]2+ was shown in Fig. 4. Curve 1 demonstrated the circumstance in the absence of dsDNA, whereas, curve 2 displayed the relationship between ipc and analytical concentration of [CdL2 ]2+ when 4.68 × 10−5 mol L−1 DNA was added. The relationship between the difference of ipc in curve1 and curve 2 (ipc , ipc = ipc1 − ipc2 ) and the analytical concentration of [CdL2 ]2+ is also shown in Fig. 4 (curve 3). We could also obtained i−1 pc based on calculating difference of ipc (ipc ) between curve1 and curve 2 (ipc = ipc1 − ipc2 ). Curves of i−1 pc versus the equilibrium concentration of 2+ −0.5 , [[CdL ]2+ ]−1 and [CdL2 ]2+ , i−1 2 pc versus [[CdL2 ] ] 2+ −2 [[CdL2 ] ] , were listed in Fig. 5. It could be seen that a straight line (γ = 0.9989) was obtained for n = 0.5, while the curves bent
Electrochemical DNA biosensor usually involved the immobilization of a single-stranded probe nucleic acid to a solid substrate and the production of electrochemical signal using redox groups. The DPV was used to study the selectivity of the prepared electrochemical DNA sensor. Fig. 6 shows the representative DPV curves obtained in NaAc-HAc media using S1 -modified GCE as working electrode (curve 1). S1 –S2 hybridized GCE (curve 2), dsDNA-modified GCE (curve 3) and S1 –S3 hybridized GCE (curve 4), respectively. Each measurement was performed after [CdL2 ]2+ was accumulated. It was found that no voltammetric response was detected in curve 1 for S1 -modified electrode. As shown in curve 2, a reduction peak which showed the same potential as bare GCE in the [CdL2 ]2+ appeared for S1 –S2 hybridized GCE. As we know, S2 was complementary to the immobilized S1 . Consequently, S1 –S2 hybridization resulted in double-stranded DNA. The reduction peak at about −0.82 V confirmed the intercalation of [CdL2 ]2+ . The peak current was 3.079 × 10−7 A. When dsDNA was immobilized on the electrode (curve 3), the reduction peak with the same potential as bare GCE in the [CdL2 ]2+ also appeared, indicating the intercalation of [CdL2 ]2+ . However, the peak current was 2.645 × 10−7 A, which was lower than S1 –S2 hybridized electrode did. We attributed this phenomenon to the steric hindrance effect caused by the immobilization of dsDNA, which greatly influenced the spread at the electrode surface. Additionally, no voltammetric response was detected in curve 4 for S1 –S3 hybridized GCE. As we know, no base pair of the dsDNA was formed because S3 was non-complementary to S1 . As a result, no [CdL2 ]2+ was accumulated on the surface of electrode. In other words, only the target ssDNA, that was complementary to the immobilized probe ssDNA, could be detected. The results indicated the selectivity of the new electrochemical DNA biosensor. 3.6. Quantitative analysis of target ssDNA
Fig. 5. Curves of i−1 pc versus C[[CdL
2]
2+ ]−n
,
n = 1/2, 1, and 2.
The sensitivity of the prepared DNA electrochemical sensor was investigated by varying the concentration of target ssDNA, which was complementary to the probe ssDNA immobilized on GCE. Results showed that differential current obtained in DPV mearsurement increased when the concentration of target ssDNA increased. The increase of peak current was linear with the concentration of target ssDNA ranging from 1.01 × 10−8 to 1.62 × 10−6 mol L−1 (Fig. 7). The regression equation was ip = 0.1116C + 3.0365 × 10−8 (C, mol L−1 ; ip , A) and the correlation coefficient was 0.9962. Thus, a detection limit of 7.19 × 10−9 mol L−1 of target ssDNA (3σ, n = 11) was achieved.
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Fig. 6. The DPV curves of S1 -modified (1), S1 –S2 hybridized (2), dsDNA-modified (3), S1 –S3 hybridized (4) GCE.
indicator and developed electrochemical DNA sensor might have promising in transducing DNA hybridization, drug designing and diagnosis of diseases. Acknowledgements The work was supported by the Natural Science Foundation of Shandong Province (No. Z2006B01) and the Program for New Century Excellent Talents in Universities (No. NCET-04-0649). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.snb.2006.12.040. References Fig. 7. The increase of peak current (ip ) versus concentration of target ssDNA (S2 ).
4. Conclusions Interaction between [CdL2 ]2+ and double-stranded salmon sperm DNA was studied using fluorescence spectroscopy and voltammetry. Results showed that [CdL2 ]2+ could intercalate into the base pairs of the dsDNA. The binding ratio between [CdL2 ]2+ and salmon sperm DNA was calculated to be 2:1 and the binding constant was 25.56 L1/2 mol−1/2 . Using [CdL2 ]2+ as a new electroactive indicator, the new electrochemical DNA biosensor could selectively detect human hepatitis B virus DNA fragment. The utility of the new electrochemical hybridization
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Biographies Shusheng Zhang was born in 1966. He received his PhD in chemistry from Nanjing University, China, in 1999. He has been working as a professor and dean at the College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology since 1999. He has published 30 papers and 2 scientific books. His research interests cover analytical chemistry, bioelectrochemistry and chemical biosensors. Qianqian Tan is currently a postgraduate student of Qingdao University of Science and Technology. Majoring in Analytical Chemistry. Feng Li is a professor of chemistry, College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao, China. He obtained his PhD from Nankai University in 2005. His research interests cover analytical chemistry and chip-based capillary electrophoresis. Xiaoru Zhang is an associate professor of chemistry, College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao, China. She obtained her PhD from Ocean University of China in 2003. Her research interests cover organic synthesis.