Investigation of DNA Orientation on Gold by EC-STM - Bioconjugate

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Bioconjugate Chem. 2002, 13, 104−109

Investigation of DNA Orientation on Gold by EC-STM Zhi-Ling Zhang, Dai-Wen Pang,* and Rong-Ying Zhang Department of Chemistry, Wuhan University, Wuhan 430072, P. R. China

Jia-Wei Yan and Bing-Wei Mao State Key Laboratory for Physical Chemistry of Solid Surfaces, Xiamen University, Xiamen 361005

Yi-Peng Qi Department of Virology, Wuhan University, Wuhan 430072, P. R. China . Received July 8, 2001; Revised Manuscript Received October 18, 2001

The immobilization of thiol-derivatized DNA on a Au (111) single crystal surface by self-assembly has been investigated by electrochemical scanning tunneling microscopy (EC-STM). Continuous potential-dependent orientation changes of double-stranded oligodeoxynucleotides (ODN) have been observed in a certain potential range from 200 to 600 mV (versus SCE). It is suggested that the DNA duplexes stand straight on the gold surface at potentials negative of the potential of zero charge (pzc) and then lay down on the surface when the potential shifts positively. These results are in agreement with the expectation based on the Coulombic interaction consideration between negatively charged DNA helices and gold surface. As the applied potential shifts positively, the surface charge changes from negative to positive, that is, the Coulombic force between negatively charged DNA helices and gold surfaces changes from repulsion to attraction. However, for the single-stranded oligodeoxynucleotides, no distinct changes in the surface structure were observed with the applied potential.

INTRODUCTION

The immobilization of DNA is of great importance for the investigation of interaction between DNA and other molecules by surface-based methods, as well as for DNA biosensors (including genochips) with potential use in disease diagnosis and genome sequencing. Many researchers have highlighted the immobilization and characterization of DNA on surfaces. Numerous methods, including X-ray photoelectron spectroscopy (XPS) (1, 2), ellipsometry (1), electrochemical method (3-5), neutron reflectivity (6), surface plasmon resonance (SPR) (5, 7), Raman spectroscopy (8), and STM (8) have been used to study this highlighted topic. However various fundamental and underlying problems, such as the relationship between structure and orientation of surface-anchored DNA and their biological affinity, the relation between the microenvironment on the electrode surface, and the behavior of DNA-modified electrodes, still remain as open questions. The imaging of DNA immobilized on the surface, which could provide detailed and direct recognition of the structure and orientation of the immobilized DNA, has explored very little. By using scanning probe microscopy (SPM), this goal can be achieved. Scanning tunneling microscopy (STM) (9-16) and atom force microscopy (AFM) (17-21) have been used for investigation of the DNA structure. These techniques can, in principle, reach atomic resolution and can be operated under the natural conditions, for instance, in aqueous solution and at ambient temperature and pressure, under which conditions the biomolecules behave naturally. * Corresponding author Tel: +86-027-87686380; Fax: +86027-87882661; e-mail: [email protected].

Barton and co-workers investigated the applied potential effect on the orientation of self-assembled DNA helices on gold by electrochemical in-situ atom force microscopy (EC-AFM) (21). They found that the monolayer height varied with the applied potential, but no clear and detailed image of the DNA monolayer was acquired. We report herein the immobilization of thiol-derivatized oligodeoxynucleotides (HS-ODN) containing 15 base pairs on gold via self-assembly and the direct EC-STM observation of ODN monolayer on Au (111) surface. It was found that the STM images of double-stranded oligodeoxynucleotides changed with the applied potential, and the results seemed to agree with electrostatic interaction between negatively charged ODN helices and gold surface. However, perhaps due to their poor rigidity, the images of single-stranded oligodeoxynucleotides were not very clear and seemed not to change with applied potential. EXPERIMENTAL PROCEDURES

Materials. All solutions were prepared with deionized water (g18MΩ‚cm) from a Millipore MilliQ system. Oligodeoxynucleotides were purchased from Shanghai Sangon Biotechnical Co. Ltd. and used without further purification. A sequence of 5′-TGT ACA GTC ATC GGG3′ had a 6-mercaptohexyl linker at the 5′-terminus, the control sample had the same sequence but no 6-mercaptohexyl linker contained, and the complementary strand was 5′-CCC GAT GAC TGT ACA-3′. All oligodeoxynucleotides were dissolved in 10 mM pH 7.4 Tris-HCl buffer containing 1 mM EDTA (TE buffer). Double-stranded oligodeoxynucleotides were prepared through hybridization before immobilized on gold surfaces. Hybridization

10.1021/bc0155263 CCC: $22.00 © 2002 American Chemical Society Published on Web 12/13/2001

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Figure 1. Cyclic voltammograms of 20 µM Co(phen)33+/2+ on (A) HS-dsODN/Au and (B) HS-ssODN/Au electrodes in 5mM KNO3; scan rate, 100 mV/s.

Figure 2. XPS N1s and P2p spectra of (A) thiol-derivatized (HS-dsODN) and (B) non-thiol-derivatized (dsODN) dsODN.

Figure 3. XPS N1s and P2p spectra of (A) thiol-derivatized (HS-ssODN) and (B) non-thiol-derivatized (ssODN) ssODN.

was performed at 37 °C for 120 min in TE buffer (pH 7.4) containing 0.5 M NaCl. Preparation of the DNA-Modified Gold Electrodes. A flame-annealed Au (111) surface for EC-STM measurements prepared as reported previously (8) or a pretreated gold electrode for cyclic voltammetry (CV) had

been immersed in 8.1 µM thiol-derivatized oligodeoxynucleotide (pH 7.4 TE buffer, 0.5 M NaCl) (HS-ODN) for 12 h at 4 °C, followed by a thorough rinse with deionized water to remove unbound ODN; thus, an electrode modified with a self-assembled monolayer of ODN obtained. The concentration of oligodeoxynucleotides in XPS

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Figure 4. EC-STM 3D images (150 nm × 150 nm) of HS-dsODN on Au (111) recorded in the solution of 10 mM TE buffer containing 10 mM NaCl pH 7.4 at various potentials, (A) 200 mV, (B) -300 mV, (C) 300 mV, (D) 400 mV, (E) 500 mV, (F) 600 mV.

measurements was 2 µM. The preparation of the control electrode followed the same procedure described above except for the control sample of ODN used instead. Instrumentation. All STM images were acquired on a Naonscope IIIa (Digital Instruments) operated under the constant current mode with an electrochemically etched tungsten tip coated with polymethylstyrene. Monatomic height steps of bare gold surfaces in air were observed clearly under the conditions used. EC-STM images were recorded in the solution of TE buffer containing 10 mM NaCl with a tunneling current of 3 nA over the potential range of -300 to +600 mV (versus SCE). The potential was first decreased with an interval of 100 from 200 to -300 mV and then gradually back to 600 mV. XPS experiments were conducted on a VG ESCA-LAB MKII spectrometer with Mg radiation. The data were

processed with an XPS-AES sampling/processing program (Version 5.0, Tsinghua University). The radiation gun was operated at 11 KV and 20 mA; 25 scans were used, and the PE mode was used for the analyzer with 100 eV pass energy. The sensitivity factors of N and P are 0.42 and 0.39, respectively; all the XPS data discussed below are corrected with these sensitivity factors. Cyclic voltammograms (CV) were recorded on a CHI 660A electrochemical workstation using a Pt wire and a SCE as the counter and the reference electrode, respectively. Co(phen)33+/2+ (20 µM) was used as an electrochemical probe for ODN detection on gold. All solutions were deaerated with high purity nitrogen and kept under a nitrogen atmosphere throughout the electrohemical measurements. All reported potentials were against the SCE.

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Figure 5. Cross-sectional profiles of dsODN EC-STM images at various potentials (A) 200 mV, (B) -300 mV, (C) 300 mV, (D) 400 mV, (E) 500 mV, (F) 600 mV. RESULTS AND DISCUSSION

As demonstrated in our previous work (4), at the concentration used in the experiments, Co(phen)33+/2+ had little electrochemical response at the bare gold electrode, but a pair of redox peaks was generated at an ODNmodified gold electrode, which resulted from the enrichment of Co(phen)33+/2+ at the modified electrode because of the strong interaction between Co(phen)33+/2+ and ODN (22, 23). Therefore, the redox process can be used to verify whether the oligodeoxynucleotides were anchored on the gold surface or not. The cyclic voltammograms for Co(phen)33+/2+ at HS-dsODN and HS-ssODN modified electrodes (denoted as HS-dsODN/Au and HS-ssODN/Au, respectively) are given in Figure 1. Evident peaks with

a formal potential (E0′) of 126 mV at HS-dsODN/Au or 116 mV at HS-ssODN/Au, as shown in Figure 1, suggest that ODNs have been immobilized on the electrode surfaces. The peak current is directly proportional to the scan rate, which is a characteristic of the surface process. The value of peak potential separation, ∆Ep, of 46 mV at HS-dsODN/Au or 53 mV at HS-ssODN at a scan rate of 100 mV/s also characterizes the surface process. The N 1s (1) and P 2p (8) peaks in the XPS spectra can provide further reliable evidence for the immobilization of ODNs. The buffer solutions contained no phosphorus element, and there was no XPS-detectable nitrogen from bare gold surfaces exposed to buffer solutions. So we may conclude that any observed N 1s and P 2p

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Figure 6. EC-STM image (150 nm × 150 nm) of MCH at potential of 200 mV recorded in the solution of 10 mM TE buffer containing 10 mM NaCl pH 7.4, and the cross-sectional profile. The images at other potential just keep unchanged (see Supporting Information).

signals originate exclusively from the nitrogen-containing bases and phosphate groups of ODN, respectively. In this case, the peak area ratio of N 1s to P 2p should be equal to the molar ratio of nitrogen to phosphorus in DNA. Figure 2 shows the XPS N1s and P2p data of the HSdsODN/Au and the non-thiol-derivatized dsODN, which support that the HS-dsODN has been attached to the gold surface through the thiol-gold bond by self-assembly. There are very little XPS signals of the control samples, which indicates that nonspecific adsorption of ODN on gold surfaces can be ignored. The peak area ratio of N 1s to P 2p is 3.1, which is close to the value of 3.5 of molar nitrogen-to-phosphorus ratio of HS-dsODN. The XPS N1s and P2p spectra of the HS-ssODN and the nonthiol-derivatized ssODN are shown in Figure 3. The peak area ratio for HS-ssODN is 3.0, which is also close to its molar ratio of nitrogen to phosphorus. The effect of the applied potential on the surface orientation of ODN was investigated by EC-STM. The image of uniformly and ordered structure of HS-dsODN on Au (111) at 200 mV (Figure 4a) indicates that the HSdsODN stands straight up on the gold side by side with the thiol-end tethered on the gold and the other end floating in the buffer solution. This ordering may be a result of the electrostatic repulsion between the negatively charged deoxyribose-phosphate backbones of neighboring DNA helices. The cross-sectional profile indicated by a line on this image is shown in Figure 5a. The dsODN rods are measured to be 3 ( 1 nm in diameter, which is similar to that of B-form DNA helix reported by Watson and Crick (24). However, the height of dsODN rod measured is smaller than the expected height of 5.1 nm deduced from the 15 base-pair DNA helix structure. The apparent height difference between the measured and the expected is mainly due to the compact character of the dsODN monolayer. The HS-dsODN is perpendicular to the surface, which restricts both the precise measurement of the height of 15-base-pair dsODN and clear imaging of the dsODN helix structure. Moreover, the ends of dsODN helices tethering the surface seem to be interconnected as shown in the cross-sectional profile. This orientation maintained when potential was decreased from 200 mV to -300 mV as shown in Figures 4a and 4b. To avoid hydrogen evolution, which would destroy the DNA monolayer, further decrease of potential was not attempted. Distinct change of the DNA helix orientation was observed, when potential was reversed

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Figure 7. EC-STM image (100 nm × 100 nm) of HS-ssODN at potential of 200 mV recorded in the solution of 10 mMTE buffer containing 10 mMNaCl pH 7.4, and the cross-sectional profile.

to above 200 mV. The DNA helices start to tilt at 300 mV, and the tilted degree increases upon increasing the potential as shown in Figures 4c and 4d. As the applied potential was further increased to 500-600 mV, significant orientation changes were observed as can be seen in Figures 4e and 4f. The DNA helices no longer stand straight on but lie flat on the gold surface in this potential region. The cross-sectional profiles at different potentials are shown in Figure 5. The surface modified with the mercaptohexanol (MCH) exhibits the typical pinhole structure of thioalcohol self-assembly monolayer, instead of the ordered dsODN rod array on HS-dsODN modified gold surface. Moreover the image remained unchanged with varying potential as shown in Figure 6. This potential dependent orientation change is attributed to the requirement of minimum electrostatic repulsion between the DNA and the gold surface. Both the density and polarity of electric charges on the gold electrode surface will change with the potential if the electrode is driven away from the potential of zero charge (pzc). According to the reference (25), the value of pzc on Au (111) is 0.258 V versus SCE. Since the gold surface is negatively charged at the potentials negative of the pzc, as mentioned by Barton (20), the negatively charged DNA helices orient themselves vertically to minimize the electrostatic repulsion with the gold surface. Nevertheless, the electrostatic repulsion becomes weaker as the potential shifts positively. It is expected that as the potential is increased across the pzc, the Coulombic interaction between DNA helices and gold surfaces changes from repulsive to attractive, which is favorable for the DNA duplexes leaning toward gold surface. Eventually, the DNA helices tend to lie flat on the surface in virtue of Coulombic attractive forces. Figure 7 shows EC-STM images of HS-ssODN modified Au (111) at 200 mV. The gold surface exhibits an irregular configuration with the ssODN strands standing on the gold surface in a disorderly fashion. We believe that the difference in rigidity between dsODN and ssODN is mainly responsible for the distinctly different behaviors of the two DNA molecules on the surface. Both the rigidity of double-stranded ODN arising from Watson and Crick helix structure and the electrostatic repulsion between the DNA helices make the contribution in forming the regular arrangement. Since the singlestranded DNA cannot form stable and rigid helix structure, it is not surprising that ssODN does not form the ordered arrangement on the surface. Moreover, for its

DNA Orientation on Gold

poor rigidity, immobilized ssODN might dangle left and right along with the scrape of the tungsten tip. So the image of ssODN was blurred to some extent due to its irregular arrangement on gold surfaces, and no ssODN rod image as clear as dsODN could be obtained. No distinct changes in the surface structure of HS-ssODN were observed with the variation of applied potential, as shown in Supporting Information. CONCLUSIONS

In summary, EC-STM has been applied to study the behaviors of dsODN and ssODN containing 15 base pairs tethered on Au (111) surface through self-assembly. It is found that dsODN changes its orientation on gold with the applied potential while ssODN does not. The electrostatic interaction between HS-dsODN and gold surface plays an important role for the orientation change of dsODN on gold. The results presented in this paper show that a uniform and compact dsODN monolayer can be formed readily on gold surfaces through self-assembly. The orientation of dsDNA on metal substrates can be controlled by the applied potential. Thus, an appropriate method to acquire desired DNA surface orientation has been developed. ACKNOWLEDGMENT

This work was supported by the National Natural Science Foundation of China (Grant Nos. 29773034; 39770220), National Outstanding Young Investigator Fund (No. 20025311), and the State Key Laboratory for Physical Chemistry of Solid Surfaces (Xiamen University, Grant No. 9916). The authors also wish to thank Professor Zhong-Qun Tian, Professor Zhong-Hua Lin, Dr. Bin Ren, Professor Shui-Jiu Wang, Ms. Jing Tang, and Mr. Yi-An Chen for their kind help. Supporting Information Available: Plots of Ip-V for HS-ODN modified electrodes. EC-STM images of the bare gold surface and MCH on Au (111) surface. EC-STM images of HS-ssODN on Au (111) surface. This material is available free of charge via the Internet at http:// pubs.acs.org. LITERATURE CITED (1) Herne, T. M., and Tarlov, M. J. (1997) Characterization of DNA probes immobilized on gold surface. J. Am. Chem. Soc. 119, 8916-8920. (2) Strother, T., Cai, W., Zhao, X. S, Hamers, R. J., and Smith, L. M. (2000) Synthesis and characterization of DNA-modified cilicon (111) surface. J. Am. Chem. Soc. 122, 1205-1209. (3) Steel, A. B., Herne, T. M., and Tarlov, M. J. (1998) Electrochemical quantitation of DNA immobilized on gold. Anal. Chem. 70, 4670-4677. (4) Pang, D. W., and Abrun˜a, H. D. (1998) Micromethod for the investigation of the interactions between DNA and redoxactive molecules. Anal. Chem. 70, 3160-3169. (5) Yang, M. S., Yau, H. C. M., and Chan, H. L. (1998) Adsorption kinetics and ligand-binding properties of thiolmodified double-stranded DNA on a gold surface. Langmuir 14, 6121-6129. (6) Levicky, R., Herne, T. M., Tarlov, M. J., and Satija, S. K. (1998) Using self-assembly to control the structure of DNA monolayers on gold: a neutron reflectivity study. J. Am. Chem. Soc. 120, 9787-9792.

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