- Email: [email protected]
Electrochemical detection of nonlabeled oligonucleotide DNA using biotin-modified DNA(ss) on a streptavidin-modified gold electrode
JOURNAL OF BIOSCIENCE AND BIOENGINEERING Vol. 97, No. 1, 29–32. 2004
Electrochemical Detection of Nonlabeled Oligonucleotide DNA Using Biotin-Modified DNA(ss) on a Streptavidin-Modified Gold Electrode JONG WAN PARK,1 HEA-YEON LEE,1* JONG MIN KIM,1 RYUJIRO YAMASAKI,1 TAKASHI KANNO,1 HIROYUKI TANAKA,1 HIDEKAZU TANAKA,1 AND TOMOJI KAWAI1 The Institute of Scientific and Industrial Research, Osaka University, 8-1 Mihogaoka, Ibaraki, Osaka 567-0047, Japan1 Received 12 August 2003/Accepted 14 October 2003
In the electrochemical detection of nonlabeled DNA, it is important to control the bonding at the interface between the DNA and the electrode. Atomic force microscope (AFM) was taken for the commonly used thiol-modified DNA on a gold surface. It was found that the coverage of the DNA was very low. On the other hand, a streptavidin-modified gold electrode provided a much better alternative where DNA hybridization resulted in large changes in the electrochemical reaction responses. This work demonstrates that streptavidin-modified gold electrodes could be used in the development of a new electrochemical protocol for the detection of nonlabeled DNA. [Key words: bioassay, streptavidin-modified electrode, nonlabeled DNA, electrochemical detection]
use of nonlabeled DNA makes these techniques very cost effective, they are seldom used since their sensitivities are extremely low. It is therefore highly desirable to develop a new type of nonlabeled biosensor for the rapid detection of DNA hybridization with high sensitivity. To meet this task, we set out to develop a new protocol to detect the hybridization of nonlabeled DNA using an electrochemical method. When attaching nonlabeled DNA probe molecules to an electrode for electrochemical analysis, it is important to control the bonding between the DNA and the electrode. Although bonding between thiol-treated DNA and gold surfaces is easily achieved, requiring a rather simple treatment process, Yang et al. reported that stability of thiolnucleic acids is poor due to the electrostatic interaction between the large hydrophilic nucleic acid groups (2). Recently, Sosnowski et al. used a bio-functionalized gold surface containing streptavidin and showed that biotinylated DNA could be easily immobilized to the streptavidin-modified gold surface by an electric field (5). Yu et al. employed this surface treatment in the development of an electrochemical protocol to probe the hybridization of DNA with a ferrocene-modified indicator (6). In this paper, we demonstrate that it is feasible to utilize a streptavidin-modified gold surface in the electrochemical detection of nonlabeled DNA. Additionally, we will show what is to the best of our knowledge, the first atomic force microscope (AFM) picture of a gold surface with thiol-treated DNA. This picture explains why the latter surface is unable to provide satisfactory results in the detection of DNA.
DNA chip technology has developed very rapidly in the biosensor field and will continue to do so in the near future. From academic and industrial standpoints, the DNA array chip can be seen as representing a more commonly used biochip approach, in which the extreme miniaturization of analytical sensors makes it possible to place millions of detection elements performing different assays into a single chip. In an effort to make use of these miniaturized detection elements, it is necessary to adapt existing protocols that have very high sensitivities. Until now, the most commonly used protocol was to use a fluorescently labeled DNA probe and to measure the change in fluorescence when the probe hybridized to its target DNA sequence. Although this technique is highly sensitive, it is expensive, requires a great deal of effort to prepare the labeled probe, and is dependent on rather large-sized and expensive equipment for the detection process. Since the development of electrochemical blood-glucose monitoring, there has been considerable enthusiasm in applying electrochemical techniques as detection methods in the biotechnology industry (1). In general, electrochemical strategies for the detection of DNA use intercalating redox-active indicators. Although these labeling strategies require simple detection techniques based on electric signals, the preparation of the DNA-indicator samples remains difficult and costly. On the other hand, protocols employing the use of nonlabeled DNA molecules have also been developed. These types of biosensors can detect DNA hybridization through the use of optical methods (2), a quartz-crystal microbalance (3), or surface plasmon resonance (4). Although the
MATERIALS AND METHODS * Corresponding author. e-mail: [email protected] phone: +81-(0)6-6879-8447 fax: +81-(0)6-6875-2440
Thiol-modified 21-mer single-strand DNAs (ssDNAs), 5¢-HS29
30
PARK ET AL.
GAGGAGTTGGGGGAGCACATT-3¢, in a buffer solution consisting of 5 mM phosphate and 50 mM NaCl (pH 7) were used in the AFM studies. Fifty ml of 50 mM thiol-modified oligonucleotide was immobilized on an Au(111) electrode for 12 h. AFM imaging was performed by an intermittent contact mode (SPI3800; Seiko Instruments, Tokyo) at room temperature. The scanning conditions were a scan speed of 1 Hz and 512 pixels ´ 256 lines without any filtering. Streptavidin was attached to the thiol-treated gold electrodes according to previously published procedures (5). A biotinylated 21-mer ssDNA of 5¢-biotin-GAGGAGTTGGGGGAGCACATT-3¢ and 3¢-AATGTGCTCCCCCAACTCCTC-5¢ were used as probeand target-DNA, respectively. All DNA synthesis reagents were obtained from Nishimbo Co. (Kyoto). The ssDNA was diluted to 50 mM with Millipore milli-Q (18 MW ×cm) water. The ssDNA solution was dropped onto the streptavidin-modified gold electrode and immobilization of the probe DNA was performed in an electric field of 300 mV for 3 min. Following immobilization, the electrode was rinsed three times for 3 min each with double-distilled water at room temperature. To check the feasibility of the DNA chip using the streptavidinmodified gold surface by the electrochemical detection of nonlabeled DNA, we have investigated the electrochemical signals of immobilization and hybridization. The hybridization of nonlabeled target DNA was carried out by applying an electric field of 300 mV for 3 min after dropping target DNA on the modified electrode of the immobilized probe DNA. Electrochemical experiments were performed using a CHI1030 multi-potentiostat (Austin, USA) at room temperature. Cyclic voltammetry (CV) was performed in 10 mM K3Fe(CN)6 solution at a scan rate of 100 mV/s. Also, differential pulse voltammetry (DPV) was used with a pulse amplitude of 50 mV and pulse width of 50 ms. The reference and counter electrodes used were an Ag/AgCl solid bar (with 3 M KCl) and platinum wire (1 mm), respectively.
RESULTS AND DISCUSSION Figure 1a shows an AFM image of the thiol-modified ssDNA on the surface of the gold electrode. The hybridization efficiency in a DNA chip is known to be dependent on how much of the electrode surface is covered with the probe DNA and how the immobilized DNA is attached to the electrode. Quantitative analysis on the AFM image showed that only approximately 10% of the gold surface was covered with immobilized oligonucleotide. This low coverage can be accounted for in terms of the electrostatic repulsive forces that exist between the hydrophilic DNA molecules (2). Furthermore, the AFM data shows that the height of the immobilized oligonucleotides varied from 2 to 7 nm, indicating that some ssDNA molecules lay flat while others stood vertically on the surface. Since the probe DNA lying flat on the surface is unable to hybridize to target DNA, the proportion of active immobilized probe DNA on the gold surface would be quite small. The avidin-biotin system has been frequently used in an effort to generate bio-functional surfaces (7, 8). Avidinbiotin technology is a universal molecular system utilized in biological science research. However, the use of avidin (Av) from egg white is often restricted due to its high isoelectric point and the presence of sugar moieties that could attach a biotin molecule at an unspecific region (1). The molecular structure of streptavidin is remarkably similar to that of Av and has the ability to specifically bind biotin. Moreover, it is
J. BIOSCI. BIOENG.,
FIG. 1. (a) AFM image of the thiol-DNA modified gold surface (Au(111)) with 21-mer ssDNA in buffer solution (5 mM phosphate, 50 mM NaCl buffer, pH 7). The height contrast is about from 2 to 7 nm. (b) AFM image of the streptavidin-modified surface on mica. All image sizes are 2 ´2 mm and scans were performed at a speed of 1 Hz.
slightly anionic and contains no sugar groups, so the high affinity constant and the stability of the streptavidin-biotin complex allow for reliable bio-functionalization. Additionally, the streptavidin protein molecules can be attached to a thiol-treated gold electrode without significant electrostatic repulsive forces being present, resulting in large surface coverage. Figure 1b shows an AFM image of the streptavidinmodified mica surface. The streptavidin is filled closely in the surface by high density with a uniform grain size. The biotinylated probe DNA can be immobilized by high density on the streptavidin-modified electrode caused by the streptavidin-biotin interaction that is in contrast with the thiol-modified ssDNA on the gold electrode. Figure 2a shows CV data which were taken (i) on a bare gold electrode, (ii) on a streptavidin-modified gold electrode, (iii) after immobilization of the probe DNA, and (iv) after hybridization with target DNA. As the peaks for both the reduction and the oxidation curves were clearly observed, a redox reaction of the K3Fe(CN)6 solution clearly occurred in all four cases. The redox current peaks increased or decreased compared to the previous process and the position of the redox peak is slightly shifted. At the streptavidin-modified gold electrode, the oxidation peak decreased by about 20% compared with a bare gold electrode.
VOL. 97, 2004
FIG. 2. Cyclic voltammetries (a) and differential pulse voltammetries (b) in 10 mM K3Fe(CN)6 solutions with a scan rate of 100 mV/s. Curves: i, bare gold electrode; ii, electrode with chemically modifiedstreptavidin; iii, electrode with immobilized probe DNA; and iv, electrode with hybridized target DNA. The concentrations of biotinylated probe-DNA and target-DNA were both 50 mM.
This result suggested that streptavidin inhibits the electrochemical reactions in the ferricyanide solution due to the low conductivity of the protein monolayer. However, the oxidation current peak after immobilization of the probe DNA showed an increase similar to that for the bare gold electrode. This pronounced increase in the peak current was observed with an increasing density of biotinylated probe DNA on the streptavidin-modified surface. The measured redox peak intensities were significantly higher when the Fe3+ ion of the ferricyanide solution was coupled with the biotinylated oligonucleotide DNA immobilized to the streptavidin modified electrode. Although the origin of the increasing current in the redox peak signal is not yet fully understood, this increasing peak current offers advantages when using the streptavidin-modified electrode for the electrochemical detection of target DNA. After hybridization of the nonlabeled target DNA, the current shows a decrease (curve iv in Fig. 2a). Though more consideration is necessary, it is assumed this may be caused by blocking of the charge transfer within the ferricyanide solution by the hybridization. The DPV signal can provide a larger electrochemical change than the CV signal because the former can suppress the capacitive current in the latter, which is due to charging effects in the protein layer. Figure 2b presents DPV data obtained for the same cases as those of Fig. 2a. Hybridization of target DNA to the probe DNA resulted in a significant
ELECTROCHEMICAL DETECTION OF NONLABELED DNA
31
decrease in the redox current. The variation of the peak current between immobilization and hybridization was typically decreased by approximately 50%. Additionally, another target DNA, the sequence of which is 3¢-TTACACGAGGG GGTTGAGGAG-5¢ (negative DNA), was also investigated for the current change after hybridization. However, the peak current was observed to decrease within 20% (data not shown). This result indicates that the variation of the current is directly related to the sequence of target DNA. Though not all steps are confirmed by the AFM technique, Fig. 1b suggests that it is possible to use the streptavidin-modified gold electrode in a nonlabeled DNA detection chip. Additionally, the streptavidin-modified electrode exhibited reasonably good stability, as reflected by the fact the electrochemical signal was only slightly decreased by approximately 10% after 7 d. An AFM picture was obtained for thiol-modified ssDNA on a gold electrode surface. It was found that the coverage of ssDNA was only approximately 10% and that a significant proportion of the DNA lay flat on the surfaces making it inaccessible for the hybridization process. The AFM picture clearly demonstrated that the ssDNA chemisorbed with thiol molecules could not be used as an effective binding medium for the DNA detection. Additionally, the feasibility of the streptavidin-modified gold electrodes was tested for the electrochemical detection of hybridization of nonlabeled nucleic acids to protein-bound nucleic acids using soluble mediators with K3Fe(CN)6 solutions. We found that the stability of the streptavidin-modified gold electrodes was reasonably good and that hybridization could result in large changes in the electrochemical reaction responses. This work demonstrates that this electrochemical technique based on the use of a streptavidin-modified gold electrode could lead to the development of a new protocol for the fast and costeffective detection of nonlabeled DNA. ACKNOWLEDGMENTS One of the authors (Jong Wan Park) gratefully acknowledges a JSPS Researcher Fellowship. This work was supported by the Center of Excellence (COE) program under the Japanese Ministry of Education, Culture, Sports, Science and Technology.
REFERENCES 1. Millelsen, S. R.: Electrochemical biosensors for DNA sequence detection. Electroanalysis, 8, 15–19 (1996). 2. Yang, M., McGovern, M. E., and Thompson, M.: Genosensor technology and the detection of interfacial nucleic acid chemistry. Anal. Chim. Acta, 346, 259–275 (1997). 3. Etchenique, R. A. and Calvo, E. J.: Electrochemical quartz crystal impedance study of redox hydrogel mediators for amperometric enzyme electrodes. Anal. Chem., 69, 4833–4841 (1997). 4. Lee, H. J., Goodrich, T. T., and Corn, R. M.: SPR imaging measurements of 1-D and 2-D DNA microarrays created from microfluidic channels on gold thin films. Anal. Chem., 73, 5525–5531 (2001). 5. Sosnowski, R. G., Tu, E., Butler, W. F., O’Connell, J. P., and Heller, M. J.: Rapid determination of single base mismatch mutations in DNA hybrids by direct electric field control. Proc. Natl. Acad. Sci., 94, 1119–1123 (1997). 6. Yu, C. J., Wan, Y., Yowanto, H., Li, J., Tao, C., James,
32
PARK ET AL.
M. D., Tan, C. L., Blackburn, G. F., and Meade, T. J.: Electronic detection of single-base mismatches in DNA with ferrocene-modified probes. J. Am. Chem. Soc., 123, 11155– 11161 (2001). 7. Dontha, N., Nowall, W. B., and Kuhr, W. G.: Generation of biotin/avidin/enzyme nanostructures with maskless photolithography. Anal. Chem., 69, 2619–2625 (1997). 8. Gorton, L., Lindgren, A., Larsson, T., Munteanu, F. D.,
J. BIOSCI. BIOENG.,
Ruzgas, T., and Gazaryan, I.: Direct electron transfer between heme-containing enzymes and electrodes as basis for third generation biosensors. Anal. Chim. Acta, 400, 91–108 (1999). 9. Padeste, C., Grubelnik, A., and Tiefenauer, L.: Ferrocene– avidin conjugates for bioelectrochemical applications. Biosens. Bioelectron., 15, 431–438 (2000).
Electrochemical Detection of Nonlabeled Oligonucleotide DNA Using Biotin-Modified DNA(ss) on a Streptavidin-Modified Gold Electrode JONG WAN PARK,1 HEA-YEON LEE,1* JONG MIN KIM,1 RYUJIRO YAMASAKI,1 TAKASHI KANNO,1 HIROYUKI TANAKA,1 HIDEKAZU TANAKA,1 AND TOMOJI KAWAI1 The Institute of Scientific and Industrial Research, Osaka University, 8-1 Mihogaoka, Ibaraki, Osaka 567-0047, Japan1 Received 12 August 2003/Accepted 14 October 2003
In the electrochemical detection of nonlabeled DNA, it is important to control the bonding at the interface between the DNA and the electrode. Atomic force microscope (AFM) was taken for the commonly used thiol-modified DNA on a gold surface. It was found that the coverage of the DNA was very low. On the other hand, a streptavidin-modified gold electrode provided a much better alternative where DNA hybridization resulted in large changes in the electrochemical reaction responses. This work demonstrates that streptavidin-modified gold electrodes could be used in the development of a new electrochemical protocol for the detection of nonlabeled DNA. [Key words: bioassay, streptavidin-modified electrode, nonlabeled DNA, electrochemical detection]
use of nonlabeled DNA makes these techniques very cost effective, they are seldom used since their sensitivities are extremely low. It is therefore highly desirable to develop a new type of nonlabeled biosensor for the rapid detection of DNA hybridization with high sensitivity. To meet this task, we set out to develop a new protocol to detect the hybridization of nonlabeled DNA using an electrochemical method. When attaching nonlabeled DNA probe molecules to an electrode for electrochemical analysis, it is important to control the bonding between the DNA and the electrode. Although bonding between thiol-treated DNA and gold surfaces is easily achieved, requiring a rather simple treatment process, Yang et al. reported that stability of thiolnucleic acids is poor due to the electrostatic interaction between the large hydrophilic nucleic acid groups (2). Recently, Sosnowski et al. used a bio-functionalized gold surface containing streptavidin and showed that biotinylated DNA could be easily immobilized to the streptavidin-modified gold surface by an electric field (5). Yu et al. employed this surface treatment in the development of an electrochemical protocol to probe the hybridization of DNA with a ferrocene-modified indicator (6). In this paper, we demonstrate that it is feasible to utilize a streptavidin-modified gold surface in the electrochemical detection of nonlabeled DNA. Additionally, we will show what is to the best of our knowledge, the first atomic force microscope (AFM) picture of a gold surface with thiol-treated DNA. This picture explains why the latter surface is unable to provide satisfactory results in the detection of DNA.
DNA chip technology has developed very rapidly in the biosensor field and will continue to do so in the near future. From academic and industrial standpoints, the DNA array chip can be seen as representing a more commonly used biochip approach, in which the extreme miniaturization of analytical sensors makes it possible to place millions of detection elements performing different assays into a single chip. In an effort to make use of these miniaturized detection elements, it is necessary to adapt existing protocols that have very high sensitivities. Until now, the most commonly used protocol was to use a fluorescently labeled DNA probe and to measure the change in fluorescence when the probe hybridized to its target DNA sequence. Although this technique is highly sensitive, it is expensive, requires a great deal of effort to prepare the labeled probe, and is dependent on rather large-sized and expensive equipment for the detection process. Since the development of electrochemical blood-glucose monitoring, there has been considerable enthusiasm in applying electrochemical techniques as detection methods in the biotechnology industry (1). In general, electrochemical strategies for the detection of DNA use intercalating redox-active indicators. Although these labeling strategies require simple detection techniques based on electric signals, the preparation of the DNA-indicator samples remains difficult and costly. On the other hand, protocols employing the use of nonlabeled DNA molecules have also been developed. These types of biosensors can detect DNA hybridization through the use of optical methods (2), a quartz-crystal microbalance (3), or surface plasmon resonance (4). Although the
MATERIALS AND METHODS * Corresponding author. e-mail: [email protected] phone: +81-(0)6-6879-8447 fax: +81-(0)6-6875-2440
Thiol-modified 21-mer single-strand DNAs (ssDNAs), 5¢-HS29
30
PARK ET AL.
GAGGAGTTGGGGGAGCACATT-3¢, in a buffer solution consisting of 5 mM phosphate and 50 mM NaCl (pH 7) were used in the AFM studies. Fifty ml of 50 mM thiol-modified oligonucleotide was immobilized on an Au(111) electrode for 12 h. AFM imaging was performed by an intermittent contact mode (SPI3800; Seiko Instruments, Tokyo) at room temperature. The scanning conditions were a scan speed of 1 Hz and 512 pixels ´ 256 lines without any filtering. Streptavidin was attached to the thiol-treated gold electrodes according to previously published procedures (5). A biotinylated 21-mer ssDNA of 5¢-biotin-GAGGAGTTGGGGGAGCACATT-3¢ and 3¢-AATGTGCTCCCCCAACTCCTC-5¢ were used as probeand target-DNA, respectively. All DNA synthesis reagents were obtained from Nishimbo Co. (Kyoto). The ssDNA was diluted to 50 mM with Millipore milli-Q (18 MW ×cm) water. The ssDNA solution was dropped onto the streptavidin-modified gold electrode and immobilization of the probe DNA was performed in an electric field of 300 mV for 3 min. Following immobilization, the electrode was rinsed three times for 3 min each with double-distilled water at room temperature. To check the feasibility of the DNA chip using the streptavidinmodified gold surface by the electrochemical detection of nonlabeled DNA, we have investigated the electrochemical signals of immobilization and hybridization. The hybridization of nonlabeled target DNA was carried out by applying an electric field of 300 mV for 3 min after dropping target DNA on the modified electrode of the immobilized probe DNA. Electrochemical experiments were performed using a CHI1030 multi-potentiostat (Austin, USA) at room temperature. Cyclic voltammetry (CV) was performed in 10 mM K3Fe(CN)6 solution at a scan rate of 100 mV/s. Also, differential pulse voltammetry (DPV) was used with a pulse amplitude of 50 mV and pulse width of 50 ms. The reference and counter electrodes used were an Ag/AgCl solid bar (with 3 M KCl) and platinum wire (1 mm), respectively.
RESULTS AND DISCUSSION Figure 1a shows an AFM image of the thiol-modified ssDNA on the surface of the gold electrode. The hybridization efficiency in a DNA chip is known to be dependent on how much of the electrode surface is covered with the probe DNA and how the immobilized DNA is attached to the electrode. Quantitative analysis on the AFM image showed that only approximately 10% of the gold surface was covered with immobilized oligonucleotide. This low coverage can be accounted for in terms of the electrostatic repulsive forces that exist between the hydrophilic DNA molecules (2). Furthermore, the AFM data shows that the height of the immobilized oligonucleotides varied from 2 to 7 nm, indicating that some ssDNA molecules lay flat while others stood vertically on the surface. Since the probe DNA lying flat on the surface is unable to hybridize to target DNA, the proportion of active immobilized probe DNA on the gold surface would be quite small. The avidin-biotin system has been frequently used in an effort to generate bio-functional surfaces (7, 8). Avidinbiotin technology is a universal molecular system utilized in biological science research. However, the use of avidin (Av) from egg white is often restricted due to its high isoelectric point and the presence of sugar moieties that could attach a biotin molecule at an unspecific region (1). The molecular structure of streptavidin is remarkably similar to that of Av and has the ability to specifically bind biotin. Moreover, it is
J. BIOSCI. BIOENG.,
FIG. 1. (a) AFM image of the thiol-DNA modified gold surface (Au(111)) with 21-mer ssDNA in buffer solution (5 mM phosphate, 50 mM NaCl buffer, pH 7). The height contrast is about from 2 to 7 nm. (b) AFM image of the streptavidin-modified surface on mica. All image sizes are 2 ´2 mm and scans were performed at a speed of 1 Hz.
slightly anionic and contains no sugar groups, so the high affinity constant and the stability of the streptavidin-biotin complex allow for reliable bio-functionalization. Additionally, the streptavidin protein molecules can be attached to a thiol-treated gold electrode without significant electrostatic repulsive forces being present, resulting in large surface coverage. Figure 1b shows an AFM image of the streptavidinmodified mica surface. The streptavidin is filled closely in the surface by high density with a uniform grain size. The biotinylated probe DNA can be immobilized by high density on the streptavidin-modified electrode caused by the streptavidin-biotin interaction that is in contrast with the thiol-modified ssDNA on the gold electrode. Figure 2a shows CV data which were taken (i) on a bare gold electrode, (ii) on a streptavidin-modified gold electrode, (iii) after immobilization of the probe DNA, and (iv) after hybridization with target DNA. As the peaks for both the reduction and the oxidation curves were clearly observed, a redox reaction of the K3Fe(CN)6 solution clearly occurred in all four cases. The redox current peaks increased or decreased compared to the previous process and the position of the redox peak is slightly shifted. At the streptavidin-modified gold electrode, the oxidation peak decreased by about 20% compared with a bare gold electrode.
VOL. 97, 2004
FIG. 2. Cyclic voltammetries (a) and differential pulse voltammetries (b) in 10 mM K3Fe(CN)6 solutions with a scan rate of 100 mV/s. Curves: i, bare gold electrode; ii, electrode with chemically modifiedstreptavidin; iii, electrode with immobilized probe DNA; and iv, electrode with hybridized target DNA. The concentrations of biotinylated probe-DNA and target-DNA were both 50 mM.
This result suggested that streptavidin inhibits the electrochemical reactions in the ferricyanide solution due to the low conductivity of the protein monolayer. However, the oxidation current peak after immobilization of the probe DNA showed an increase similar to that for the bare gold electrode. This pronounced increase in the peak current was observed with an increasing density of biotinylated probe DNA on the streptavidin-modified surface. The measured redox peak intensities were significantly higher when the Fe3+ ion of the ferricyanide solution was coupled with the biotinylated oligonucleotide DNA immobilized to the streptavidin modified electrode. Although the origin of the increasing current in the redox peak signal is not yet fully understood, this increasing peak current offers advantages when using the streptavidin-modified electrode for the electrochemical detection of target DNA. After hybridization of the nonlabeled target DNA, the current shows a decrease (curve iv in Fig. 2a). Though more consideration is necessary, it is assumed this may be caused by blocking of the charge transfer within the ferricyanide solution by the hybridization. The DPV signal can provide a larger electrochemical change than the CV signal because the former can suppress the capacitive current in the latter, which is due to charging effects in the protein layer. Figure 2b presents DPV data obtained for the same cases as those of Fig. 2a. Hybridization of target DNA to the probe DNA resulted in a significant
ELECTROCHEMICAL DETECTION OF NONLABELED DNA
31
decrease in the redox current. The variation of the peak current between immobilization and hybridization was typically decreased by approximately 50%. Additionally, another target DNA, the sequence of which is 3¢-TTACACGAGGG GGTTGAGGAG-5¢ (negative DNA), was also investigated for the current change after hybridization. However, the peak current was observed to decrease within 20% (data not shown). This result indicates that the variation of the current is directly related to the sequence of target DNA. Though not all steps are confirmed by the AFM technique, Fig. 1b suggests that it is possible to use the streptavidin-modified gold electrode in a nonlabeled DNA detection chip. Additionally, the streptavidin-modified electrode exhibited reasonably good stability, as reflected by the fact the electrochemical signal was only slightly decreased by approximately 10% after 7 d. An AFM picture was obtained for thiol-modified ssDNA on a gold electrode surface. It was found that the coverage of ssDNA was only approximately 10% and that a significant proportion of the DNA lay flat on the surfaces making it inaccessible for the hybridization process. The AFM picture clearly demonstrated that the ssDNA chemisorbed with thiol molecules could not be used as an effective binding medium for the DNA detection. Additionally, the feasibility of the streptavidin-modified gold electrodes was tested for the electrochemical detection of hybridization of nonlabeled nucleic acids to protein-bound nucleic acids using soluble mediators with K3Fe(CN)6 solutions. We found that the stability of the streptavidin-modified gold electrodes was reasonably good and that hybridization could result in large changes in the electrochemical reaction responses. This work demonstrates that this electrochemical technique based on the use of a streptavidin-modified gold electrode could lead to the development of a new protocol for the fast and costeffective detection of nonlabeled DNA. ACKNOWLEDGMENTS One of the authors (Jong Wan Park) gratefully acknowledges a JSPS Researcher Fellowship. This work was supported by the Center of Excellence (COE) program under the Japanese Ministry of Education, Culture, Sports, Science and Technology.
REFERENCES 1. Millelsen, S. R.: Electrochemical biosensors for DNA sequence detection. Electroanalysis, 8, 15–19 (1996). 2. Yang, M., McGovern, M. E., and Thompson, M.: Genosensor technology and the detection of interfacial nucleic acid chemistry. Anal. Chim. Acta, 346, 259–275 (1997). 3. Etchenique, R. A. and Calvo, E. J.: Electrochemical quartz crystal impedance study of redox hydrogel mediators for amperometric enzyme electrodes. Anal. Chem., 69, 4833–4841 (1997). 4. Lee, H. J., Goodrich, T. T., and Corn, R. M.: SPR imaging measurements of 1-D and 2-D DNA microarrays created from microfluidic channels on gold thin films. Anal. Chem., 73, 5525–5531 (2001). 5. Sosnowski, R. G., Tu, E., Butler, W. F., O’Connell, J. P., and Heller, M. J.: Rapid determination of single base mismatch mutations in DNA hybrids by direct electric field control. Proc. Natl. Acad. Sci., 94, 1119–1123 (1997). 6. Yu, C. J., Wan, Y., Yowanto, H., Li, J., Tao, C., James,
32
PARK ET AL.
M. D., Tan, C. L., Blackburn, G. F., and Meade, T. J.: Electronic detection of single-base mismatches in DNA with ferrocene-modified probes. J. Am. Chem. Soc., 123, 11155– 11161 (2001). 7. Dontha, N., Nowall, W. B., and Kuhr, W. G.: Generation of biotin/avidin/enzyme nanostructures with maskless photolithography. Anal. Chem., 69, 2619–2625 (1997). 8. Gorton, L., Lindgren, A., Larsson, T., Munteanu, F. D.,
J. BIOSCI. BIOENG.,
Ruzgas, T., and Gazaryan, I.: Direct electron transfer between heme-containing enzymes and electrodes as basis for third generation biosensors. Anal. Chim. Acta, 400, 91–108 (1999). 9. Padeste, C., Grubelnik, A., and Tiefenauer, L.: Ferrocene– avidin conjugates for bioelectrochemical applications. Biosens. Bioelectron., 15, 431–438 (2000).