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J. Phys. Chem. B 2009, 113, 894–896
Conformational Transitions of Immobilized DNA Chains Driven by pH with Electrochemical Output Fanben Meng,† Yuexing Liu,‡ Lei Liu,† and Genxi Li*,† Department of Biochemistry and National Key Laboratory of Pharmaceutical Biotechnology, Nanjing UniVersity, Nanjing 210093, People’s Republic of China, and Laboratory of Biosensing Technology, School of Life Science, Shanghai UniVersity, Shanghai 200444, People’s Republic of China ReceiVed: March 3, 2008; ReVised Manuscript ReceiVed: NoVember 26, 2008
A pH-driven DNA sway rod is prepared by immobilizing thiolated DNA, mercaptohexanol, and cysteine on a gold electrode surface. As pH changes around the pI of cysteine, contrary electrostatic effect is produced between the negative DNA and amphoteric cysteine, which actuates reversible conformational transitions, such as sway of the DNA molecules, rodlike chain-to-globule, and so forth. The nanoscale motion can be detected by commonly used electrochemical technique and reversible electrochemical signal may be observed. Introduction The design and exploiture of nanodevices has attracted more and more research interest,1-4 and DNA has been proven to be a versatile and feasible building material with steady chemical structure and specific biological function.5-11 Meanwhile, electrochemistry has furnished a good means to detect the motion of the nanodevices. For instance, some electrochemical ON-OFF nanoswitches12,13 and AND-OR logic gates14,15 have been reported. In this paper, we present a DNA nanodevice, which may be driven by pH, so a pH-driven DNA sway rod is reported. Experimental Methods Thiolated ssDNA (HS-ssDNA) was manufactured by Invitrogen Biotechnology Co., Ltd. The sequence is 5′-HS-(CH2)6-CAC GAC GTT GTA AAA CGA CGG CCA G-3′. 6-Mercapto-1-hexanol (MCH), cysteine, and tris(2-carboxyethyl)phosphine hydrochloride (TCEP) were from Sigma. All solutions were prepared with doubly distilled water, which was purified with a Milli-Q purification system (Branstead, U.S.A.) to a specific resistance of >18 MΩ cm. The substrate gold electrode was prepared by inserting a gold rod into a glass tube and fixing it with epoxy resin. Electrical contact was made by adhering a copper wire to the rod with the help of Woods Alloy. The gold electrode was first polished on fine sand paper and alumina (particle size of about 0.05 µm)/water slurry on silk. Then it was thoroughly washed by ultrasonicating in both doubly distilled water and ethanol for about 5 min. Finally, the electrode was electrochemically cleaned to remove any remaining impurities.16 After drying in nitrogen atmosphere, the gold electrode was immersed in a solution of 1 µM HS-ssDNA, 10 mM Tris-HCl, 1 mM EDTA, 1.0 M NaCl, and 1 mM TCEP (pH 8.0) for 16 h, followed by a 2 h treatment with an aqueous solution of 1 mM spacer thiol molecules. The spacer thiol molecules are the mixture of MCH and cysteine (1:1). After thoroughly rinsed with pure water and dried again in nitrogen atmosphere, the modified electrode can be then ready for use. The surface density of ssDNA could be known as 6.0 × 1012 molecules/cm2 by chronocoulometric method,17,18 and the rest of the electrode surface was all covered by MCH and cysteine. * To whom correspondence should be addressed. E-mail: genxili@ nju.edu.cn. Tel.: (+86-25) 8359-3596. Fax: (+86-25) 8359-2510. † Nanjing University. ‡ Shanghai University.
Cyclic voltammetry (CV) experiments were performed with a model 263A Potentiostat/Galvanostat (EG and G, PARC). Experiments with electrochemical impedance spectroscopy (EIS) were performed on a model 660C Eletrochemical Analyzer (CH Instruments). The CV and EIS experiments were separately carried out with 50 mM PBS and tris-HCl buffer both containing 5 mM [Fe(CN)6]3-/4-. A three-electrode system consisting of the modified gold electrode, saturated calomel reference electrode (SCE), and platinum counter electrode was used for all the electrochemical measurements. Experiments with atomic force microscopy (AFM) were performed on SPM-9600 (Shimadzu). They were carried out in the fluid cell with PBS solution using the tapping mode. The NSG 11S cantilevers (NT-MDT) were used with a nominal spring constant of 2.5 N/m. The self-assembled monolayer (SAM) of HSssDNA on the electrode surface was imaged alternately in PBS with the pH values of 6.0 and 3.9. After each manipulation, the remaining buffer was removed from the fluid cell, and the surface was thoroughly washed with pure water. Then, a new buffer with the other pH value was introduced. Results and Discussion Self-assembled HS-ssDNA chains on the surface of gold electrode with MCH as the dilution spacer molecule have been reported to be able to keep standing.18-21 Here, we introduce another spacer molecule also with thiol group, cysteine, which will be coadsorbed together with MCH onto a gold electrode surface. Because of the amphoteric character of cysteine, the electrostatic effect between DNA and cysteine will be changed with the pH of the test system around the isoelectric point (pI) of this amino acid, which may be employed to develop a nanodevice driven by pH. Figure 1 may illustrate the mechanism. The pI of cysteine is 5.02 (30 °C). Since it is immobilized on the surface of gold electrode via Au-S and the effect of -SH on the pI is eliminated, its pI will be changed to be about 6.0 in the condition of this system. Therefore, as is illustrated in Figure 1, reversibly conformational transitions of the DNA molecules can be achieved in the test solution with a pH around 6.0. At the beginning, when the pH of the test solution is higher than 6.0, the DNA molecules will stand on the surface of the gold electrode. As pH falls from 6.0, cysteine takes more and more positive charges, so electrostatic attraction between DNA and cysteine will become stronger and stronger. Consequently, the DNA molecules will swing toward the electrode
10.1021/jp806268z CCC: $40.75 2009 American Chemical Society Published on Web 01/07/2009
Immobilized DNA Chains Driven by pH
J. Phys. Chem. B, Vol. 113, No. 4, 2009 895
Figure 1. (A) Schematic drawing of the conformational transitions of HS-ssDNA molecules driven by pH, (B) for control.
surface until lying down, while some of the DNA molecules will be chain-to-globule changed. If pH of the test solution rises back little by little, the DNA molecules will go back to the “standing” state again gradually. Meanwhile, if the pH jumps back to 6.0 or higher, the DNA molecules will go back to “standing” immediately. Therefore, reversible operation of up-and-down swinging of the surfaceimmobilized DNA and the rodlike chain-to-globule change is achieved. Accordingly, reversible changes in electrochemical response may be observed. The surface topography images of the HS-ssDNA-MCH-cysteine modified electrode with two different pH values (6.0 and 3.9) have been provided by AFM with the reference of the previous work.19 When the pH is 6.0, the state of the ssDNA molecules is “standing” and the height of ssDNA SAM is about 10 nm (Figure 2A), which is the same as the length of the ssDNA. But as the pH falls to 3.9, the height is less than 4 nm (Figure 2B), which is consistent with the width of accumulated ssDNA molecules on the surface of electrode and demonstrates the state of “lying” or globulelike. As the surface is scanned alternately in the solutions of pH 6.0 and 3.9, the changes of the height phase are reversible. By contrast, the AFM experiments of the electrode without cysteine attached have also been carried out, and no obvious change is observed in the solutions with different pH values (data not shown).
Figure 3. Cyclic voltammograms of 5 mM [Fe(CN)6]3-/4- obtained at an (A) HS-ssDNA-MCH-cysteine (B) HS-ssDNA-MCH (C) MCHcysteine modified electrode for a 50 mM PBS buffer solution with pH from 6.0 to 3.9 in the potential scan range between 0 and 400 mV. Scan rate: 100 mV s-1.
We have used CV, the most popular electrochemical technique22 to characterize the conformational transitions of the DNA molecules. Figure 3A shows the cyclic voltammograms obtained at the HS-ssDNA-MCH-cysteine modified electrode corresponding to Figure 1A with 5 mM [Fe(CN)6]3-/4- as the redox species. It can be observed that no electrochemical wave can be observed at first when the pH of the system is 6.0. However, with pH decreasing, apparent redox waves appear gradually, indicative of the reduction and oxidation of [Fe(CN)6]3-/4-. The peaks currents will reach maximum and keep unchanged, as pH falls to 3.9. Further studies reveal that the well-defined peaks will disappear little by little, if the pH of the test solution is changed to be 6.0 again gradually. And, the redox peaks may exhibit a
Figure 2. AFM images of HS-ssDNA-MCH-cysteine modified electrode surface with (A) pH 6.0 and (B) pH 3.9.
896 J. Phys. Chem. B, Vol. 113, No. 4, 2009 switchable change reversibly, as the pH of the system jumps between 6.0 and 3.9. By contrast, we have also studied the electrochemical behavior of [Fe(CN)6]3-/4- by using an HS-ssDNA-MCH modified electrode corresponding to Figure 1B. It can be observed from Figure 3B that no redox peak can be obtained, and the cyclic voltammogram keeps unchanged no matter how the pH of the system changes between 3.9 and 6.0. On the other hand, as shown in Figure 3C if MCH-cysteine alone are modified on the substrate electrode surface with no ssDNA attached, although [Fe(CN)6]3-/4- may exhibit electroactive, the peaks currents only show slight changes as the pH of the system falls from 6.0 to 3.9. Therefore, the obvious changes of cyclic voltammograms in Figure 3A should be ascribed to the interaction of ssDNA and cysteine caused by the changes of pH. The above results are obviously reasonable. As is well known, the SH-ssDNA molecules at the electrode surface take abundant negative charges because of the anionic phosphate backbones. These negative charges of the single stranded nucleic acid will repel [Fe(CN)6]3-/4- molecules in the solution, which are also negatively charged, from getting close to the electrode surface to achieve redox reactions. Nevertheless, since cysteine molecules take more and more charges to become more and more positive with the pH falling gradually, the negatively charged ssDNA will get closer to the electrode surface due to the electrostatic effect, as shown in Figure 1A. So, the repellence of the ssDNA to [Fe(CN)6]3-/4- becomes weaker, and the redox species can be closer to the electrode surface to achieve electron transfer reactions. Consequently, redox peaks can be observed. As pH falls to 3.9, the ssDNA will lie on the electrode surface, and the peaks currents will ultimately reach maximum. On the other hand, if pH jumps back to be higher than 6.0, the electrostatic effect between cysteine and ssDNA is repellent again, so the DNA molecules get back to the original state. The repellence of the ssDNA to the electrochemical probes occurs again, so the CV waves disappear. Therefore, pH is the motivity and the key factor to the conformational transitions of the ssDNA molecules. EIS has also been employed to demonstrate and characterize the nanoscale motion of the SH-ssDNA molecules immobilized on the electrode surface. Figure 4 shows the electrochemical impedance spectra (Nyquist plots) of HS-ssDNA-MCH-cysteine modified electrode for the test solution with different pH value between 6.0 and 3.9. As is shown in Figure 4, because of the increase of the pH value, the interfacial electron resistance will be enlarged, which is consistent with the experimental results obtained with CV method to reveal the conformational transitions of ssDNA chains. Conclusions In summary, we report in this paper a pH-driven nanodevice, which is based upon the self-assembled ssDNA strands on a gold electrode surface. The ssDNA strands can reversibly changed with pH. The conformational transitions of the ssDNA chains has been detected and demonstrated by electrochemical method. This nanodevice may have some useful applications, such as sensitive sensor for pH or motorial cantilever, and so forth. Furthermore, since the controllable DNA microarray is a basic step for the design of more complex and functional DNAbased nanomachine, this work might be followed by more researches in the future. On the other hand, since the confor-
Meng et al.
Figure 4. Electrochemical impedance spectroscopy (Nyquist plots) of an HS-ssDNA-MCH-cysteine modified electrode for a 50 mM trisHCl buffer solution containing 5 mM [Fe(CN)6]3-/4- with different pH value from 3.9 to 6.0, in the frequency range of 1 Hz to 100 kHz. Alternative voltage: 10 mV. Inset: theoretical equivalent circuits where Rs is solution resistance, Cml is capacitance of monolayer, and Rct is charge-transfer resistance.
mational transitions of DNA molecules is driven by pH, this work may also have some very important biological significance. Acknowledgment. This work is supported by the National Natural Science Foundation of China (Grants 90406005 and 20575028) and the Program for New Century Excellent Talents in University, the Chinese Ministry of Education (NCET-040452). References and Notes (1) Barth, J. V.; Costantini, G.; Kern, K. Nature 2005, 437, 671. (2) Lu, W.; Lieber, C. M. Nat. Mater. 2007, 6, 841. (3) Masmanidis, S. C.; Karabalin, R. B.; De Vlaminck, I.; Borghs, G.; Freeman, M. R.; Roukes, M. L. Science 2007, 317, 780. (4) Dalgleish, H.; Kirczenow, G. Nano Lett. 2006, 6, 1274. (5) Ding, B.; Seeman, N. C. Science 2006, 314, 1583. (6) Bishop, J. D.; Klavins, E. Nano Lett. 2007, 7, 2574. (7) Simmel, F. C.; Dittmer, W. U. Small 2005, 1, 284. (8) Seeman, N. C. Nature 2003, 421, 427. (9) Liu, D.; Bruckbauer, A.; Abell, C.; Balasubramanian, S.; Kang, D.; Klenerman, D.; Zhou, D. J. Am. Chem. Soc. 2006, 128, 2067. (10) Gill, R.; Patolsky, F.; Katz, E.; Willner, I. Angew. Chem., Int. Ed. 2005, 44, 4554. (11) Rant, U.; Arinaga, K.; Fujita, S.; Yokoyama, N.; Abstreiter, G.; Tornow, M. Nano Lett. 2004, 4, 2241. (12) Zuo, X.; Song, S.; Zhang, J.; Pan, D.; Wang, L.; Fan, C. J. Am. Chem. Soc. 2007, 129, 1042. (13) Katz, E.; Baron, R.; Willner, I. J. Am. Chem. Soc. 2005, 127, 4060. (14) Tokarev, H.; Orlov, M.; Katz, E.; Minko, S. J. Phys. Chem. B 2007, 111, 12141. (15) Weizmann, Y.; Elnathan, R.; Lioubashevski, O.; Willner, I. J. Am. Chem. Soc. 2005, 127, 12666. (16) Fan, C.; Zhong, J.; Guan, R.; Li, G. Biochim. Biophys. Acta 2003, 1649, 123. (17) Steel, A. B.; Herne, T. M.; Tarlov, M. J. J. Am. Chem. Soc. 1998, 70, 4670. (18) Lao, R.; Song, S.; Wu, H.; Wang, L.; Zhang, Z.; He, L.; Fan, C. Anal. Chem. 2005, 77, 6475. (19) Rant, U.; Arinaga, K.; Fujita, S.; Yokoyama, N.; Abstreiter, G.; Tornow, M. Langmuir 2004, 20, 10086. (20) Herne, T. M.; Tarlov, M. J. J. Am. Chem. Soc. 1997, 119, 8916. (21) Levicky, R.; Herne, T. M.; Tarlov, M. J.; Satija, S. K. J. Am. Chem. Soc. 1998, 120, 9787. (22) Bard, A. J.; Fauikner, L. R. Electrochemical Methods, 2nd ed.; Wiley: New York, 2000.
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