Electrical Switching of DNA Monolayers Investigated by Surface

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Electrical Switching of DNA Monolayers Investigated by Surface Plasmon Resonance Xiaohai Yang, Qing Wang, Kemin Wang,* Weihong Tan, Jing Yao, and Huimin Li State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Biomedical Engineering Center, Institute of Biological Technology, College of Material Science and Technology, Hunan UniVersity, Bio-Nano Technology Engineering Research Center of Hunan ProVince, Changsha 410082, People’s Republic of China ReceiVed October 28, 2005. In Final Form: April 15, 2006 The switching of DNA monolayers between a “lying” and a “standing” state initiated by applying electric field, and the subsequent DNA hybridization at different states were investigated in a contactless, label-free mode by surface plasmon resonance (SPR) technique. The results showed that the strength of the electric field and surface coverage could influence the switching of DNA monolayers. In addition, it was found that DNA hybridization efficiency could be enhanced or decreased when DNA probes stood straight up or lay flat on the gold surface, depending on the potential of the gold substrate. The enhancement of DNA hybridization efficiency reached the maximum when surface coverage reached 5.87 × 1012 molecules/cm2 and the potential of gold substrate was more negative than -0.7 V (versus ITO-coated glass). The research may be helpful for the construction of sensitive biosensors, biochips, and nanoscale electronic devices.

1. Introduction The controllable, reversible switching surfaces, which are initiated by either electric field,1-6 pH,7,8 or mechanical force,9 have been anticipated to be applicable in many fields such as biosensors, microfluidics, drug release systems, and nanoscale computers.1,2,7,10 These surfaces are usually formed by linear molecules such as 16-mercaptohexadecanoic acid,1,3 DNA,2,4 and bipyridinium.5 Among them, DNA monolayers have received a considerable amount of attention since DNA monolayers are the foundation of various biotechnology applications, including DNA microarrays,11 biosensors,12,13 biological ion channels,14,15 and so on. Meanwhile, DNA molecules have negative electric charges due to the phosphates in the sugar-phosphate backbone, so the molecules modified on the gold surface could be either * To whom correspondence should be addressed. Tel: +86 731 8821566. Fax: +86 731 8821566. E-mail: [email protected]. (1) Lahann, J.; Mitragotri, S.; Tran, T. N.; Kaido, H.; Sundaram, J.; Choi, I. S.; Hoffer, S.; Somorjai, G. A.; Langer, R. Science 2003, 299, 371-374. (2) Rant, U.; Arinaga, K.; Fujita, S.; Yokoyama, N.; Abstreiter, G.; Tornow, M. Nano Lett. 2004, 4, 2441-2445. (3) Liu, Y.; Mu, L.; Liu, B. H.; Zhang, S.; Yang, P. Y.; Kong, J. L. Chem. Commun. 2004, 1194-1195. (4) Kelley, S. O.; Barton, J. K.; Jackson, N. M.; McPherson, L. D.; Potter, A. B.; Spain, E. M.; Allen, M. J.; Hill, M. G. Langmuir 1998, 14, 6781-6784. (5) Wang, X. M.; Kharitonov, A. B.; Katz, E.; Willner, I. Chem. Commun. 2003, 1542-1543. (6) Pei Y.; Ma, J. J. Am. Chem. Soc. 2005, 127, 6802-6813. (7) Matthews, J. R.; Tuncel, D.; Jacobs, R. M. J.; Bain, C. D.; Anderson, H. L. J. Am. Chem. Soc. 2003, 125, 6428-6433. (8) Ionov, L.; Houbenov, N.; Sidorenko, A.; Stamm, M.; Luzinov, I.; Minko, S. Langmuir 2004, 20, 9916-9919. (9) Moresco, F.; Meyer, G.; Rieder, K. H.; Tang, H.; Gourdon, A.; Joachim, C. Phys. ReV. Lett. 2001, 86, 672-675. (10) Mao, Y. D.; Luo, C. X.; Deng, W.; Jin, G. Y.; Yu, X. M.; Zhang, Z. H.; Ouyang, Q.; Chen, R. S.; Yu, D. P. Nucleic Acids Res. 2004, 32, e144. (11) Riepl, M.; Enander, K.; Liedberg, B.; Schaferling, M.; Kruschina, M.; Ortigao, F. Langmuir 2002, 18, 7016-7023. (12) Boozer, C.; Ladd, J.; Chen, S.; Yu, Q.; Homola, J.; Jiang, S. Anal. Chem. 2004, 76, 6967-6972. (13) Nakamura, F.; Ito, E.; Sakao, Y.; Ueno, N.; Gatuna, I. N.; Ohuchi, F. S.; Hara, M. Nano Lett. 2003, 3, 1083-1086. (14) Harrell, C. C.; Kohli, P.; Siwy, Z.; Martin, C. R. J. Am. Chem. Soc. 2004, 126, 15646-15647. (15) Vlassiouk, I.; Takmakov, P.; Smirnov, S. Langmuir 2005, 21, 47764778.

driven away from, or pulled toward the gold substrate by applied electrochemical potential reversibly, which acts like a nanoscale “switch”.2,4 Usually, such switching of DNA monolayers on metal surface is studied by fluorescence spectroscopy,2 and atomic force microscopy (AFM),4 in which DNA molecules must be modified with fluorophores or be pressed by tips. Therefore, the obtained information is hard to reflect the electric-driven DNA monolayers purely and absolutely. Surface plasmon resonance (SPR) is a surface-sensitive analytical technique for chemical and biochemical sensing that is extremely sensitive to the refractive index of the medium next to the metal film.16-20 Since it is a label-free and contactless mode, SPR technology would be especially appropriate for detecting the conformational changes of the immobilized DNA monolayers. However, in the standard three-electrode cell, the applied electric field itself could alter SPR signals obviously.21 Herein, the switching of DNA monolayers between a “lying” and a “standing” state initiated by an electric field, along with the subsequent DNA hybridization at different states, was investigated by SPR technique. In this work, a capacitor-like cell consisting of Au film and indium-tin oxide (ITO) glass was designed to avoid the alternation of the SPR signal induced by the electric field. The results showed that the immobilized DNA monolayers could stand straight up or lie flat, depending on the charge of the Au surface. Such switching of DNA monolayers could be influenced by the strength of the electric field and surface coverage of the DNA probes. Meanwhile, the DNA hybridization efficiency can be enhanced or decreased following the standing or lying of DNA monolayers, respectively. (16) Englebienne, P.; Hoonacker, A. V.; Verhas, M. Spectroscopy 2003, 17, 255-273. (17) Homola, J. Anal. Bioanal. Chem. 2003, 377, 528-539. (18) Green, R. J.; Frazier, R. A.; Shakeshe, K. M.; Davies, M. C.; Roberts, C. J.; Tendler, S. J. B. Biomaterials 2000, 21, 1823-1835. (19) Shumaker-Parry, J. S.; Zareie, M. H.; Aebersold, R.; Campbell, C. T. Anal. Chem. 2004, 76, 918-929. (20) Chen, S.; Liu, L.; Zhou, J.; Jiang, S. Langmuir 2003, 19, 2859-2864. (21) Heaton, R. J.; Peterson, A. W.; Georgiadis, R. M. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 3701-3704.

10.1021/la052907m CCC: $33.50 © 2006 American Chemical Society Published on Web 05/16/2006

Electrical Switching of DNA Monolayers

Figure 1. Diagram of the electrostatic SPR apparatus. L: tungsten halogen lamp; C: collimator tube; P: polarizer; D: diaphragm; G: grating; E: DC electrical source; B: ITO-coated glass slide.

2. Experimental Section 2.1. Materials. 3-Mercaptopropionic acid (MPA, Fluka, Switzerland), N-hydroxysuccinimide (NHS, Nacalai Tesque, Inc., Kyoto, Japan) and 1-ethyl-3-(3-(dimethylamino)propyl) carbodiimide hydrochloride (EDC, Acros Organics, Morris Plains, NJ) were used for the gold surface modification. Hexaammineruthenium(III) chloride (98%, Aldrich, St. Louis, MO) was used for the measurement of the surface coverage of the DNA probes on Au films. All of the chemical reagents were of analytical grade or higher. Double-distilled water was used throughout. DNA oligonucleotides were purchased from TaKaRa Biotechnology Co., Ltd. (Dalian, China) and had the following sequences: (1) immobilized probe DNA, 5′-NH2 TCT AAA TCG CTA TGG TCG C-3′ and (2) cDNA, 3′-AGA TTT AGC GAT ACC AGC G-5′. All of them were dissolved into 0.01 M phosphate-buffered saline (PBS) (pH 7.3) before use. The ITO-coated glass slides (15 × 10 × 1.34 mm) were obtained from the Beijing Institute of Glass and Special Fiberglass (China). 2.2. Instrumentation. A homemade Kretschmann SPR instrument employing a tungsten halogen lamp was used. Details of the apparatus have been described previously. 22 The diagram of the device is shown in Figure 1. In this device, a 20 × 20 × 1 mm BK7 glass slide with a 1-2 nm adhesive layer of Cr followed by a 50 nm layer of gold deposited on top of the Cr was put in contact with a prism (BK7 glass; n ) 1.516) using a suitable index matching oil. The sensing mechanism of this SPR instrument utilized a fixed angle of incident light and modulated the wavelength. In this instrument, it was regarded as a signal when the shift of resonance wavelength (λr) was larger than 0.4 nm. A micro flow cell of about 20 µL was fabricated with the ITOcoated glass slide and gold substrate, which acted as two electrodes of a quasi parallel-plate capacitor, and the potential between the two electrodes was adjustable. In this cell, the gold surface and the glass side of the ITO-coated glass were directly in contact with the solutions. That is, the conductive layer of the ITO-coated glass did not contact the solution. Therefore, no oxidation/reduction reactions occurred at the Au film because there was no Faradaic current between the Au film and the ITO-coated glass. In this work, SPR spectra were obtained in the presence of an electric field when the switching of DNA monolayers was investigated, whereas SPR spectra were recorded at open circuit when the subsequent DNA hybridization at different states was studied. 2.3. Procedures. 2.3.1. Surface Modification of Gold Substrate. Gold substrate was modified as previously described.23 The gold substrate was first cleaned in piranha solution (H2SO4/H2O2, 3:1 (22) Chen, Z. Z.; Wang, K. M.; Yang, X. H.; Huang, S. S.; Huang, H. M.; Li, D.; Wang, Q. Acta Chim. Sin. 2003, 61, 137-140. (23) He, L.; Musick, M. D.; Nicewarner, S. R.; Salinas, F. G.; Benkovic, S. J.; Natan, M. J.; Keating, C. D. J. Am. Chem. Soc. 2000, 122, 9071-9077.

Langmuir, Vol. 22, No. 13, 2006 5655 (v/v)), followed by a thorough rinsing with water. Then, gold substrate was modified with a 10 mM MPA/ethanol solution over 70 min, followed by a thorough rinsing with 0.01 M PBS (pH 7.3), producing a carboxylate-modified surface on the gold substrate. After 60 min of incubation with 100 mM EDC and 100 mM NHS, the carboxylatemodified surface was thoroughly rinsed with PBS and then incubated with 0.57 µM DNA probes. A wide range of surface coverage of the immobilized DNA probes was obtained through varying the time (2 min to 2 h) of exposure to the DNA probes.24 Finally, the gold substrate was washed completely with 0.01 M PBS, and a DNA sensor chip was obtained. 2.3.2. Hybridization and Denaturation of DNA. Hybridization of DNA was conducted by exposing the immobilized DNA probes to a cDNA solution for 30 min at room temperature and then washing with 0.01 M PBS repeatedly. Hot water (90 °C) was used to denature the surface-immobilized DNA duplexes. For all hybridization experiments performed under electric field, the surface was first exposed to the target DNA solution at open circuit. After 10 s, the potential was applied for 30 min at room temperature, and then the gold surface was washed with 0.01 M PBS repeatedly at open circuit. 2.3.3. Surface CoVerage Measurements. The coverage of DNA probes on Au films was measured by electrochemical methods, which are described in the literature.25 Here, chronocoulometry was performed on CH660A (Shanghai Chenhua Instrument Co., Ltd., China). The gold substrate, an Ag/AgCl electrode, and a Pt wire served as the work, reference, and counter electrodes, respectively.

3. Results and Discussion 3.1. Switching of DNA Probes Modified on the Gold Surface by Applying Electric Field. 3.1.1. Influence of Electric Field on Bare Au Substrate. SPR is closely related to the oscillation of free electrons that propagates along with the metal surface.26 Since the application of an interfacial electric field changes the dielectric properties in the vicinity of the metal-liquid interface,27 the SPR signal of the metal can be altered by the electric field in the standard three-electrode cell (see Figure 2A).21 Therefore, to investigate the electrical switching of a DNA monolayer modified on Au film, it is necessary to avoid the effect of electric field on the Au film itself. In other words, when the electric field is applied, the SPR signal should only be related to the conformational change of the DNA strands. Here, a capacitorlike cell, which was composed of Au substrate and ITO-coated glass, was designed. As shown in Figure 2B, when the potential between Au substrate and ITO-coated glass ranged from 0 to 12 V, λr did not change regardless of whether the bare gold substrate had negative or positive charges. A possible reason was that the strength of the electric field at the metal/electrolyte interface of a standard three-electrode cell differs from that of a capacitorlike cell. The strength of the electric field in the capacitor-like cell was on the order of 107 V/m; 28 such electric field could not influence the SPR spectra of Au film itself according to Georgiadis’ work.21 Hence, the influence of electric field on bare Au was negligible (as shown in Figure 2B), and the capacitorlike cell was used to investigate the swing of DNA layer and hybridization. (24) Rant, U.; Arinaga, K.; Fujita, S.; Yokoyama, N.; Abstreiter, G.; Tornow, M. Langmuir 2004, 20, 10086-10092. (25) Steel, A. B.; Herne, T. M.; Tarlov, M. J. Anal. Chem. 1998, 70, 46704677. (26) Ikehata, A.; Itoh, T.; Ozaki, Y. Anal. Chem. 2004, 76, 6461-6469. (27) Lioubimov, V.; Kolomenskii, A.; Mershin, A.; Nanopoulos, D. V.; Schuessler, H. A. Appl. Opt. 2004, 43, 3426-3432. (28) When the voltage between ITO and Au (Au film was electronegative) was changed from 0 to 1.5 V, the potential of Au film changed from 23 to -40 mV (vs Ag/AgCl), accordingly. Since the thickness of the double layer was on the order of 1 nm (10-9 m), the strength of the electric field at the metal/electrolyte interface was estimated to be about 107 V/m according to the Gouy-Chapman theory.

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Figure 2. Effect of electric field at different modes on the SPR signal.

3.1.2. Influence of Electric Field on the Switching of DNA. First, the influence of electric field on the switching of DNA was investigated when the potential between the modified Au substrate and the ITO-coated glass was 1.5 V. It was observed that the SPR signals were affected under the applied electrostatic field (see Figure 3A). When the gold film was electronegative, λr blue shifted (as Figure 3A shows, the wavelength shift was negative). While the gold surface was electropositive, λr red shifted (that is, the wavelength shift was positive). Most importantly, these processes were reversible, which means that when the gold film was electropositively and electronegatively charged alternatively, λr red and blue shifted alternatively. When the electric field exerted on the microflow cell was removed, the SPR spectra could return to its original status. Since these changes were reversible, this system acted like a nanoscale switch. In addition, because of the low strength of the electric field and the inexistence of Faradaic current in the capacitor-like cell, such small negative voltages did not remove DNA probes, and thereby signal was not led to decrease after a few cycles of switching. After DNA probes were covalently coupled on the gold surface, the orientation of the DNA probes was somewhat random because of the interaction between the DNA bases and the gold surface (see Figure 3B1′).29,30 Depending on the charging state of the gold film, the intrinsically negatively charged DNA was either driven away from, or pulled toward the gold surface. Hence, a lying or standing state was adopted for negative and positive biases, respectively (Figure 3B). The orientation change of the DNA probes could affect the surface characteristic of the gold (29) Herne, T. M.; Tarlov, M. J. J. Am. Chem. Soc. 1997, 119, 8916-8920. (30) Han, S. B.; Lin, J. Q.; Zhou, F. M.; Vellanoweth, R. L. Biochem. Biophys. Res. Commun. 2000, 279, 265-269.

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substrate, and λr could shift accordingly. When the gold film was electronegative, the dielectric layer near the gold surface became rather uncompacted because of the vertical orientation of the DNA probes (Figure 3B2′), and λr blue shifted. While the gold surface was electropositive, owing to the increase in the dielectric constant caused by the horizontal orientation of the DNA probes (Figure 3B3′), λr red shifted. Then, the influence of the electric field on the switching of DNA was investigated at different electric intensities. It was surprising that not all intensities of electric field could cause the change of DNA probes. As shown in Figure 3C, no matter the gold film was electronegative or electropositive, a sudden change in the λr shift occurred when the potential between the Au film and the ITO-coated glass was about 0.6 V. A plateau of wavelength shift was observed when the potential was more negative than -0.7 V (when the Au surface was electronegative) or more positive than 0.7 V (when the Au surface was electropositive). It was reported that the orientation of dsDNA strands was determined by the balance between two factors: (1) statistical gyrations of the probes due to thermal agitation and (2) the total strength of the electrostatic interactions.2 Here, the switching behavior of the DNA probes may be similar. When the electric field was low, the electrostatic energy of the DNA probes was too small to compete with the thermal energy kBT (kB being the Boltzmann constant), and therefore no shift in λr was observed. As the electric field was progressively increased, the associated energy eventually exceeded kBT, and the probes were repelled from or attracted to the gold surface. Once the electrostatic energy sufficiently prevailed against kBT, the probes would stand straight up or lie down on the gold surface. Therefore, a further increase in the electric field (i.e., the potential between the gold film and the ITO glass) could not lead to an additional decrease or increase in the switching amplitude, and the shifts in λr did not change anymore. Similar to the orientation of DNA helices,4 the switching behavior occurred within narrow range of potentials (∼0.2 V). 3.1.3. Influence of Surface CoVerage on Switching of DNA. As shown in Figure 4, the mobility of DNA probes initiated by applying electric field was strongly affected by the surface coverage of immobilized DNA probes. For high surface coverage (> 1.0 × 1013 molecules/cm2) and low surface coverage (< 1 × 1012 molecules/cm2), regardless of whether the gold film had negative or positive charges, λr did not shift obviously. However, there was a maximum at ∼5.87 × 1012 molecules/cm2. This phenomenon can be understood in terms of the interplay of two factors: the quantity and the orientation change of the DNA probes tethered to the surface. The length of a 19-mer DNA probe was about 64 Å, and the distance between the DNA probes was about 17, 23, and 54 Å, when the surface coverage was 1.0 × 1013, 5.87 × 1012, and 1 × 1012 molecules/cm2, respectively.25 For high surface coverage (> 1.0 × 1013 molecules/ cm2), steric interactions prevented the free swing of DNA probes, and the DNA molecules were forced to take an upright orientation on the surface, so the shift of λr was very small. On the other hand, if the surface coverage was too low (< 1 × 1012 molecules/ cm2), the swing of DNA probes was free, but the induced SPR signal, that is, the shift of λr, was not high enough to be detectable because of the small quantity of DNA probes. In conclusion, the switching of DNA monolayers initiated by applying electric field could be monitored by SPR. Such DNA switching could be influenced by the strength of the electric field and the surface coverage of the DNA probes. The above results might also provide a convenient way to gain indirect information concerning the orientation of DNA on the gold surface.

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Figure 3. (A) Effects of electric field (1.5 V between Au and ITO) on DNA probes: (a) Au surface with positive charges; (b) open cycle; (c) Au surface with negative charges; (d) open cycle. (B) Orientation of the DNA probe on the Au surface: (1′) without charges, (2′) with negative charges, and (3′) with positive charges. (C) Effects of different electric field intensities on DNA probes: (1) Au surface with negative charges; (2) Au surface with positive charges.

3.2. Influence of the Switching of DNA on DNA Hybridization. DNA hybridization at a solid/solution interface relies on the monolayers of DNA immobilized on a solid substrate.31,32

As mentioned above, such monolayers could be switched by electric field. Hence, here, the SPR technique was used to investigate DNA hybridization when the immobilized DNA probes were at different orientations under the electric field. For the hybridization of DNA, all SPR spectra were recorded at open circuit. The extent of hybridization was evaluated with the shifts of λr, that is, the difference in λr before and after hybridization: the bigger the shift of λr, the higher the DNA hybridization efficiency. 3.2.1. Hybridization with cDNA of Different Concentrations. Hybridization with cDNA of different concentrations in the absence or presence of electric field was detected by SPR. First, a 1.5 V of the potential between the gold film and the ITO glass slide was applied. As shown in Figure 5, compared to opencircuit conditions, the shift of λr caused by DNA hybridization evidently increased when the gold film was electronegative, whereas the shift of λr decreased when the gold film had positive charges. The sensitivity of such a sensor was improved with the increase in the shift of λr. In comparison with the detection limit

(31) Wong, E. L. S.; Chow, E.; Gooding, J. J. Langmuir 2005, 21, 6957-6965.

(32) Su, X.; Wu, Y. J.; Robelek, R.; Knoll, W. Langmuir 2005, 21, 348-353.

Figure 4. The effect of electric field (1.5 V between Au and ITO) on the switching of DNA probes at different surface coverages.

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Figure 5. Effects of electric field (1.5 V between Au and ITO) on DNA hybridization under different concentrations of cDNA: (1) gold film with negative charges; (2) gold film in the absence of electric field; (3) gold film with positive charges.

Figure 6. Effects of different electric field intensities on hybridization with 2.83 nM cDNA. Here, the gold film was electronegative.

of 85.7 pM at open circuit, 17.1 pM cDNA could be detected when the gold film had negative charges. That is, it resulted in a 5-fold enhancement in sensitivity by applying a suitable electric field. 3.2.2. Hybridization with cDNA under Different Electric Field Intensities. DNA probes capturing cDNA (2.83 nM) under different electric field intensities were investigated when the gold film was electronegative. Similar to the case of DNA probes, a sudden change in wavelength shift was observed when the potential of the Au surface was equal to approximately -0.6 V (see Figure 6). Once the potential of the Au surface was more negative than -0.7 V, a plateau in the wavelength shift was observed. When the potential was more positive than -0.55 V, the wavelength shift could not be enhanced. Comparing Figure 3C with Figure 6, the effects of different electric field intensities on DNA hybridization and the switching of DNA were similar to some degree. Only when the orientation of the DNA probes was induced to change could the subsequent DNA hybridization efficiency be influenced. It reconfirmed that the orientation of the DNA monolayers is an important factor in DNA hybridization at a solid/solution interface or in the sensitivity of a DNA sensor. The results of SPR technique demonstrated that the application of the electric field could increase or decrease the hybridization efficiency. A possible explanation was that the switching of immobilized DNA probes under an electric field resulted in an alteration of the hybridization efficiency. When the gold film was electronegative, DNA probes stood straight up (Figure 3B2′) and were located farther away from the gold film, so DNA probes

Yang et al.

Figure 7. Effect of different surface coverages on DNA hybridization. Here, 5.65 nM cDNA was used, and the potential between Au and ITO was 1.5 V.

were closer to the solution and were more accessible for capturing the target DNA; the hybridization efficiency was enhanced, and the resonance wavelength shift was increased accordingly. Hence, the sensitivity of SPR DNA biosensors could be improved. However, DNA probes lay flat on the gold surface (Figure 3B3′) when the gold film was electropositive, so the gold surface acted as a shield for probes as a result of the associated steric factors. Hence, the ability of DNA probes to capture target DNA was reduced, and a decrease in wavelength shift was observed accordingly. These results seemed to differ from the work reported by Georgiadis and co-workers.21 However, they are not in contradiction to each other because the capacitor-like cell is different from the three-electrode cell used in Georgiadis’ work. The main difference between the two kinds of cells is the strength of their electric field. The strength of the electric field on a Au electrode surface may change two factors that are important for DNA hybridization: (1) the rate of adsorption of DNA targets onto a probe surface 21 and (2) the orientation of DNA probes.33 The observed results may be the balance between the two factors. In the work of Georgiadis et al., a three-electrode cell was used, and the strength of the electric field at the metal/electrolyte interface was on the order of 109 V/m. Therefore, the first factor was sufficiently dominant over the second one. Even if the DNA probes were in a good orientation for hybridization (straight up in negative charge), they could not capture enough targets in a certain time to show an increase in hybridization, and vice versa. Here, for the capacitor-like cell, when the applied potential was 1.5 V, the strength of the electric field at the metal/electrolyte interface was on the order of 107 V/m. It was low compared to the value reported by Georgiadis et al. (109 V/m). Therefore, for DNA hybridization, the influence of the applied potential on the rate of target DNA adsorption onto a probe surface may not be the primary factor, while the orientation of the DNA probes, that is, the steric hindrance effect, does control the interaction of targets and probes, and thus the contrary results to Georgiadis’ work are exhibited. 3.2.3. Influence of Surface CoVerage. Since the mobility of DNA probes was strongly affected by the surface coverage of DNA probes modified on the gold film, DNA hybridization efficiency was similarly influenced by the surface coverage of immobilized DNA probes. Figure 7 depicted the effect of surface coverage on DNA hybridization when the gold film had no or (33) Rant, U.; Arinaga, K.; Tornow, M.; Kim, Y. W.; Netz, R. R.; Fujita, S.; Yokoyama, N.; Abstreiteret, G. Biophys. J. BioFAST 2006, DOI: 10.1529/ biophysj.105.078857.

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negative charges. It showed that DNA hybridization could be enhanced when the gold film was electronegative within a certain range of surface coverage. However, at high or low surface coverage, DNA hybridization had not been enhanced obviously. This is in accordance with the effect of surface coverage on the switching of DNA (Figure 4). For high surface coverage, since the free swing of DNA probes was prevented by steric interactions, DNA hybridization could not be enhanced. On the other hand, for low surface coverage, the shift of λr was small because of the small quantity of DNA probes on the gold film.

4. Conclusion The electrically initiated, reversible switching of DNA monolayers was investigated by a homemade SPR device, in which a capacitor-like cell was used to avoid the influence of electric field on the SPR signal. As a label-free, contactless method, SPR can offer more information about such a reversibly switching surface. In addition, it was found that DNA hybridization could be enhanced by controlling the electric field. This

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work offers a novel alternative choice not only for the research of the controllable, reversible switching surface, but also for the purpose of developing sensitive DNA sensors. Since a number of biomolecules are electrosensitive, this work may provide a unique design feature for the construction of more sensitive biosensors, biochips, and nanoscale electronic devices. Acknowledgment. This work was partially supported by the National Key Basic Research Program (2002CB513110), the Key Project of the Natural Science Foundation of the People’s Republic of China (20135010), the Key Technologies Research and Development Program (2003BA310A16), the High Tech Research and Development Program (2003AA302250), the Key Project of International Technologies Collaboration Program of China (2003DF000039), the Natural Science Foundation of China (20475015), the China National Key Projects (05FK5029), and the Science Foundation of Hunan University of China. LA052907M