Electrochemical Interrogation of DNA Monolayers

mobilization at gold surfaces with electrochemical tech- ..... at least 1 nm (diameter of dsDNA is 2 nm) (Scheme 1A). Note .... We attempted to incuba...
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Anal. Chem. 2005, 77, 6475-6480

Electrochemical Interrogation of DNA Monolayers on Gold Surfaces Ruojun Lao,†,‡ Shiping Song,† Haiping Wu,† Lihua Wang,† Zhizhou Zhang,*,‡,§ Lin He,‡ and Chunhai Fan*,†

Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai 201800, China, Bio-X Life Science Research Center, Shanghai Jiao Tong University, Shanghai 200030, China, and Tianjin University of Science and Technology, Tianjin 300040, China

Electrochemical DNA sensors1-7 have attracted significant research interest because electrochemical tranducers offer a simple, sensitive, and inexpensive platform for applications including clinical genetic analysis,8,9 environmental monitoring,10-12 and forensic identification.13,14 A typical electrochemical DNA sensor consists of a solid electrode, an electrochemical analyzer, and

surface-confined DNA probes. Upon recognition of DNA sequences that are complementary to the probes, this hybridization event is transduced to electrochemical signals that manifest the presence of specific DNA target sequences. Immobilization of DNA probes is one of the key steps toward DNA sensor development.15-18 It has been well demonstrated that the DNA sensor performance (e.g., sensitivity, selectivity, and stability) is highly dependent on properties of immobilized DNA probes, such as orientation, conformation, and surface density.19 Patterning thiolated DNA probes on gold surfaces via selfassembly is among the most employed system.20,21 Tarlov and coworkers reported that the introduction of a dilution thiol, 6-mercapto1-hexanol (MCH), could well control surface properties of DNA probes.21-23 They proposed that MCH not only served as spacers that finely modulated surface density of DNA probes but also effectively prevented nonspecific DNA-gold interactions.1 Despite the progress, fundamental studies on characterization of DNA-gold surfaces as well as the effects of dilution thiol remain to be explored in order to fully understand this system and further improve sensor performance. A variety of technologies, such as electrochemistry, X-ray photoelectron spectroscopy (XPS),24,25 radiolabeling,1 surface plasmon resonance (SPR),26 and quartz

* Corresponding authors. Fax: (+86) 21-5955-6902. E-mail: [email protected]; [email protected]. † Chinese Academy of Sciences. ‡ Shanghai Jiao Tong University. § Tianjin University of Science and Technology. (1) Steel, A. B.; Herne, T. M.; Tarlov, M. J. Anal. Chem. 1998, 70 (22), 46707. (2) Boon, E. M.; Ceres, D. M.; Drummond, T. G.; Hill, M. G.; Barton, J. K. Nat. Biotechnol. 2000, 18, 1096-100. (3) Wang, J.; Xu, D.; Polsky, R. J. Am. Chem. Soc. 2002, 124, 4208-9. (4) Lapierre, M. A.; O’Keefe, M.; Taft, B. J.; Kelley, S. O. Anal. Chem. 2003, 75, 6327-6333. (5) Gore, M. R.; Szalai, V. A.; Ropp, P. A.; Yang, I. V.; Silverman, J. S.; Thorp, H. H. Anal. Chem. 2003, 75, 6586-92. (6) Fan, C.; Zhong, J.; Guan, R.; Li, G. Biochim. Biophys. Acta-Proteins Proteomes 2003, 1649, 123-6. (7) Fan, C.; Plaxco, K. W.; Heeger, A. J. Trends Biotechnol. 2005, 23, 186-92. (8) Staudt, L. M. Trends Immunol. 2001, 22, 35-40. (9) Debouck, C.; Goodfellow, P. N. Nat. Genet. 1999, 21, 48-50. (10) Nielsen, M.; Larsen, L. H.; Jetten, M. S.; Revsbech, N. P. Appl. Environ. Microbiol. 2004, 70, 6551-8. (11) Hanrahan, G.; Patil, D. G.; Wang, J. J. Environ. Monit. 2004, 6, 657-64. (12) Oliveira-Brett, A. M.; da Silva, L. A. Anal. Bioanal. Chem. 2002, 373, 71723. (13) Garcia-Repetto, R.; Gimenez, M. P.; Repetto, M. J. AOAC Int. 2001, 84, 342-9.

(14) Belosludtsev, Y. Y.; Bowerman, D.; Weil, R.; Marthandan, N.; Balog, R.; Luebke, K.; Lawson, J.; Johnston, S. A.; Lyons, C. R.; Obrien, K.; Garner, H. R.; Powdrill, T. F. Biotechniques 2004, 37 (4), 654-8, 660. (15) Bai, X. P.; Li, Z. M.; Jockusch, S.; Turro, N. J.; Ju, J. Y. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 409-13. (16) Lowe, L. B.; Brewer, S. H.; Kramer, S.; Fuierer, R. R.; Qian, G. G.; AgbasiPorter, C. O.; Moses, S.; Franzebm, S.; Feldheim, D. L. J. Am. Chem. Soc. 2003, 125, 14258-9. (17) Tatan, T. A.; Mirkin, C. A.; Letsinger, R. L. Science 2000, 289, 1757-60. (18) Moses, S.; Brewer, S. H.; Lowe, L. B.; Lappi, S. E.; Gilvey, L. B. G.; Sauthier, M.; Tenent, R. C.; Feldheim, D. L.; Franzen, S. Langmuir 2004, 20 (25), 11134-40. (19) Southern, E.; Mir, K.; Shchepinov, M. Nat. Genet. Suppl. 1999, 21, 5-9. (20) Okahata, Y.; Matsunobu, Y.; Ijiro, K.; Mukae, M.; Murakami, A.; Makino, K. J. Am. Chem. Soc. 1992, 114, 8299-300. (21) Herne, T. M.; Tarlov, M. J. J. Am. Chem. Soc. 1997, 119, 8916-20. (22) Levicky, R.; Herne, T. M.; Tarlov, M. J.; Satija, S. K. J. Am. Chem. Soc. 1998, 120, 9787-92. (23) Steel, A. B.; Herne, T. M.; Tarlov, M. J. Bioconjugate Chem. 1999, 10, 41923. (24) O’Donnell, M. J.; Tang, K.; Koster, H.; Smith, C. L.; Cantor, C. R. Anal. Chem. 1997, 69, 2438-43. (25) Kimura-Suda, H.; Petrovykh, D. Y.; Tarlov, M. J.; Whitman, L. J. J. Am. Chem. Soc. 2003, 125 (30), 9014-5. (26) Thiel, A. J.; Frutos, A. G.; Jordan, C. E.; Corn, R. M.; Smith, L. M. Anal. Chem. 1997, 69, 4948-56.

In this report, we systematically investigated DNA immobilization at gold surfaces with electrochemical techniques. Comparative cyclic voltammetric and chronocoulometric studies suggested that DNA monolayers immobilized at gold surfaces were not homogeneous. Nonspecific Au-DNA interactions existed even with the treatment of mercaptohexanol, which was known to competitively remove loosely bound DNA at gold surfaces. While both thiolated and nonthiolated DNA formed monolayers on gold surfaces, their hybridization abilities were distinctly different. In contrast to thiolated DNA probes, nonthiolated DNA probes immobilized at gold surfaces were essentially nonhybridizable. The experimental results presented here might be useful for the design of high-performance electrochemical DNA sensors.

10.1021/ac050911x CCC: $30.25 Published on Web 09/10/2005

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crystal microbalance (QCM),27 have been employed to investigate this two-component self-assembled monolayer (SAM). We are particularly interested in electrochemical interrogation of the system because (1) in contrast to often confronted interferences from measurements of mass-sensitive SPR28 and QCM,29 electrochemical measurements are usually sensitive and qualitative; (2) in contrast to radiolabeling analysis, electrochemical measurements are nontoxic; (3) in contrast to XPS, electrochemical measurements are nondestructive; thus electrochemically characterized electrode surfaces are ready for use as electrochemical DNA sensors. In this report, we studied the immobilization process of DNA probes on gold surfaces with electrochemical means, aiming at discrimination of specific against nonspecific adsorption of DNA probes at gold surfaces. EXPERIMENTAL SECTION Materials. DNA oligonucleotides were purchased from Sangon Inc. (Shanghai, China). The sequences are 5′-CACGA CGTTG TAAAA CGACG GCCAG-3′ (nonthiolated probe, non SH-ssDNA); 5′-SH-CACGA CGTTG TAAAA CGACG GCCAG-3′ (thiolated probe at 5′ terminus with a C6 spacer, SH-ssDNA); and 5′-CTGGC CGTCG TTTTA CAACG TCGTG-3′ (target DNA, c-DNA). Tris(hydroxymethyl)aminomethane was purchased from Cxbio Biotechnology Ltd. MCH, hexaammineruthenium(III) chloride ([Ru(NH3)6]3+, RuHex) and tris(2-carboxyethyl)phosphine hydrochloride (TCEP) were from Sigma. All solutions were prepared with Nanopure water (18MΩ‚cm resistivity) from a Millipore MilliQ system. The buffers involved in this work are as follows: DNA immobilization buffer (I-buffer), 10 mM Tris-HCl, 1 mM EDTA, 1.0 M NaCl, and 1 mM TCEP (pH 8.0). Note that TCEP was employed to cleave disulfides. DNA hybridization buffer (Hbuffer), 10 mM tris-HCl, 1 mM EDTA, and 1.0 M NaCl (pH 8.0). Buffer for electrochemistry (E-buffer), 10 mM Tris-HCl buffer, pH 7.0. Pretreatment of Electrodes and DNA Immobilization. Gold electrodes (2 mm in diameter, CH Instruments Inc.) were first polished on microcloth (Buehler) with Gamma micropolish deagglomerated alumina suspension (0.05 µm) for 5 min. These electrodes were then sonicated in ethanol and Milli-Q water for 5 min, respectively. Finally, the electrodes were then electrochemically cleaned to remove any remaining impurities.6,30,31 After drying with nitrogen, electrodes were subjected to oligonucleotides of appropriate concentrations, either thiolated or nonthiolated for 1416 h, followed by a 2-h deposition in 1 mM MCH in water. DNA duplex was prepared by hybridization of the two complementary sequences (0.5 µM each) in H-buffer for at least 1 h and was then ready for immobilization at surfaces. Electrochemical Measurements. Cyclic voltammetry (CV) and chronocoulometry (CC) were performed on a CH Instruments (27) Huang, E.; Satjapipat, M.; Han, S.; Zhou, F. Langmuir 2001, 17 (4), 121524. (28) Georgiadis, R.; Peterlinz, K. P.; Peterson, A. W. J. Am. Chem. Soc. 2000, 122, 3166-73. (29) Olofsson, L.; Rindzevicius, T.; Pfeiffer, I.; Kall, M.; Hook, F. Langmuir 2003, 19 (24), 10414-9. (30) Hoogvliet, J. C.; Dijksma, M.; Kamp, B.; van Bennekom, W. P. Anal. Chem. 2000, 72, 2016-21. (31) Janek, R. P.; Fawcett, W. R.; Ulman, A. J. Phys. Chem. B 1997, 101, 85508.

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model 430 eletrochemical analyzer. CV was carried out at a scan rate of 50 mV/s, and CC at a pulse period of 250 ms and pulse width of 700 mV. A three-electrode cell consisting of Ag/AgCl reference electrode and platinum counter electrode was used for all electrochemical measurements. The E-buffers were thoroughly purged with pure nitrogen before experiments. The surface density of ssDNA can be calculated using the assumption that one RuHex molecule stoichiometrically binds to the anionic phosphodiester backbone of DNA. When an electrode modified with DNA is placed in a low ionic strength electrolyte containing a mulitivalent redox cation, the redox cation exchanges with the native charge compensation cation and becomes electrostatically trapped at that interface. The surface densities of DNA probes can then be calculated from the redox charges of RuHex according to the chronocoulometric methods first proposed by Tarlov and co-workers.1 QCM-D Measurements. QCM-D is a novel QCM technique developed by Q-Sense Inc., which simultaneously monitors frequency (F) and dissipation (D) changes at surfaces.32 F indicates mass changes occurred at surfaces while D suggests conformational variations of surface-confined species.32,33 In our experiments, QCM gold electrodes were extensively cleaned as previously reported33 and modified with both DNA and MCH as described above. During hybridization, ∼2 mL aliquots of target DNA solutions (1 µM in 10 mM Tris-HCl/1 M NaCl, pH 8.0) were injected to the flow cell, and then F and D were simultaneously monitored. RESULTS AND DISCUSSION Cyclic Voltammetric Studies on DNA Surfaces. CV is the most popular electrochemical technique that provides important information of electron-transfer reactions at electrode surfaces.34 Previous studies have addressed that redox reactions of charged transition metal complexes (e.g., RuHex) are highly influenced by surface-confined polyelectrolyte.35 Thus, we initially employed CV to characterize the redox reaction of RuHex at gold surfaces. At 50 µM RuHex, we observed a pair of well-defined peaks corresponding to the reduction and oxidation of RuHex at a pure MCH-coated gold surface (Figure 1). The peak currents are linearly proportional to the square root of scan rates (v) (I ∝ v1/2, data not shown), suggesting that the electron-transfer reaction of RuHex is diffusion-controlled at the prepared MCH surface. This correlates well with the previous report that RuHex does not adsorb on MCH SAMs.10 Notably, two pairs of peaks appeared for a mixed SAM surface (SH-DNA/MCH, Figure 1). One pair of peaks were similar to that observed in the absence of SH-DNA, which were thus ascribed to the redox reaction of RuHex diffused to the MCH component in the mixed SAM. The other pair of peaks at ∼ -0.3 V were characteristic of a surface-confined redox process. This is because the peak separation is close to zero and peak currents are linearly proportional to scan rates (I ∝ v, data not shown). This pair of (32) Rodahl, M.; Hook, F.; Fredriksson, C.; Keller, C. A.; Krozer, A.; Brzezinski, P.; Voinova, M.; Kasemo, B. Faraday Discuss. 1997, (107), 229-46. (33) Hook, F.; Rodahl, M.; Kasemo, B.; Brzezinski, P. Proc. Natl. Acad. Sci., U.S.A. 1998, 95 (21), 12271-6. (34) Bard, A. J.; Faulkner, L. R. Electrochemical Methods, 2nd ed.; Wiley: New York, 2000. (35) Oyama, N.; Shimomura, T.; Shigehara, K.; Anson, F. C. J. Electroanal. Chem. 1980, 112, 271-80.

Figure 1. Cyclic voltammograms of gold electrodes modified with SH-ssDNA/MCH (thick line), non SH-ssDNA/MCH (solid line) and pure MCH (dashed line). The concentration of RuHex was 50 µM in 10 mM Tris buffer, pH 7.0. Scan rates, 50 mV/s.

peaks were ascribed to RuHex electrostatically bound to DNA backbones. This assignment was further confirmed by CV at 3.5 µM RuHex (Figure 1-S, Supporting Information). At this low concentration, the diffusion-controlled peak pair of RuHex disappeared, presumably because diffusion of RuHex to the surface was insufficient to produce signals. In contrast, the peaks corresponding to bound RuHex remained. Previous crystallographic studies have demonstrated that RuHex-DNA binding was completely based on electrostatic interactions.36 This is because RuHex lacks planar organic groups that can intercalatively insert into DNA base pairs. As a result, cationic RuHex stoichiometrically binds to the anionic phosphate backbones of DNA in solutions of low ionic strengths, which forms the basis of electrochemical quantification of DNA surface density.1 We also studied CV of RuHex at non SH-ssDNA/MCH surfaces. Similar to that obtained at SH-ssDNA/MCH surfaces, the CV had two pairs of peaks. Note, however, that the surfaceconfined peak pair is much smaller, suggesting that a large amount of non SH-DNA has been displaced during the MCH deposition process. In the absence of MCH, RuHex was still electroactive at both bare and DNA-coated gold surfaces. However, capacitive currents were significantly higher than those obtained in the presence of MCH (Figure 1-S). Chronocoulometric Studies on DNA Surfaces. CC was initially employed by Tarlov and co-workers to quantify DNA surface density via measuring redox charges of RuHex at surfaces (see Experimental Section for details).1 More recently, Yu et al. reported that they could quantify immobilized DNA with CV, which in principle was more direct and convenient.37 They observed that the RuHex binding reached equilibrium while the diffusion peak pair was negligible at an appropriate concentration of RuHex (∼3.5 µM). Therefore, by simply integrating CV peaks, they could obtain redox charges of surface-confined RuHex, which were linearly proportional to DNA surface densities.37 We then evaluated these two methods by comparing CV with CC results. We first compared the redox charge obtained from CV and from CC at 3.5 µM RuHex, with the former significantly lower (36) Ho, P. S.; Frederick, C. A.; Saal, D.; Wang, A.; Rich, A. J. Biomol. Struct. Dynam. 1987, 4, 521-34. (37) Yu, H. Z.; Luo, C. Y.; Sankar, C. G.; Sen, D. Anal. Chem. 2003, 75 (15), 3902-7.

than the latter. This means that some surface-bound RuHex is “electroinactive” in CV while “electroactive” in CC. This phenomenon may reflect the heterogeneity within DNA monolayers. In the ideal condition for a thiolated DNA monolayer, all DNA strands are well aligned and nearly perpendicular to the surface with a slight tilt angle (Scheme 1B). In this case, within a single DNA strand, individual RuHex molecules are separated by ∼0.34 nm (DNA base distance). Therefore, electron hopping occurs between these RuHex molecules, which efficiently transfer electrons between electrodes and involved RuHex molecules. Moreover, since DNA strands are well aligned, bound RuHex molecules should be uniformly distributed in this monolayer. As a result, the electron-transfer properties of RuHex should be homogeneous and equally fast. However, if DNA nonspecifically adsorbs to gold, for example, DNA strands contact gold via Au-base interactions (see Discussion below), RuHex is separated from electrodes by at least 1 nm (diameter of dsDNA is 2 nm) (Scheme 1A). Note that electron-transfer rates exponentially decay with increased donor-acceptor distances;38 thus, the increase in distance (from 0.34 to 1 nm) should make the electron transfer of these portions of RuHex molecules more sluggish than those bound to specifically adsorbed DNA strands. We thus could speculate that nonspecific DNA adsorption does exist even with MCH treatment. Under the experimental conditions employed in this work, we observed that the redox charge of RuHex obtained from CV (3.5 µM RuHex) was significantly lower than that from CC (50 µM RuHex). This result strongly suggests that CV performed at low concentrations of RuHex could not accurately reflect the surface density of DNA probes, at least under our experimental conditions (e.g., DNA probe density). This may arise due to several combined effects. First, as we have discussed, the distribution of RuHex at surfaces is not homogeneous; thus, the electron transfer of some RuHex molecules are always more sluggish than others. As a result, the contribution of these portions of surface-confined RuHex may not be reflected due to the “dynamic” nature of CV. Second, the adsorption rate of RuHex is much smaller at 3.5 µM than at 50 µM. As shown in Figure 2, it only took seconds for RuHex at 50 µM to reach equilibrium but tens of minutes for RuHex at 3.5 µM. Third, even at the equilibrated condition, a RuHex concentration of 3.5 µM may not be sufficient to saturate the surface. Indeed, the redox charge of RuHex obtained at 3.5 µM RuHex was smaller than that obtained at 50 µM RuHex (both with the CC method, Figure 3). This was confirmed by comparing the redox charges of RuHex obtained with CC and CV methods at the same concentration of RuHex (3.5 µM, Figure 3). Based on these observations, we concluded that it was more accurate to use CC to quantify surface density of DNA probes, which was then employed in the following quantification experiments. Quantification of DNA Surfaces. We employed CC to investigate different adsorption properties of thiolated DNA and nonthiolated DNA. Figure 4 demonstrates the surface densities of SH-ssDNA, non SH-ssDNA, and non SH-dsDNA on gold electrodes. Note that in all cases DNA-immobilized gold electrodes were exposed to MCH solution for 2 h, which effectively removed loosely bound DNA. The surface density of non SH-ssDNA was significantly lower than that of SH-ssDNA; however, it is apparent that MCH treatment did not displace all nonthiolated DNA. In (38) Marcus, R. A.; Sutin, N. Biochim. Biophys. Acta 1985, 811, 265-322.

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Scheme 1. Schematic Drawing of (A) Non SH-ssDNA and (B) SH-ssDNA Monolayers on Gold Surfaces (with MCH Treatment)a

a

The trivalent cation in the figure stands for RuHex.

Figure 2. Chronocoulometric curves of electrodes modified with SH-ssDNA/MCH in a RuHex solution with concentrations of 3.5 and 50 µM, respectively. In the case of 50 µM RuHex, the charge of the initial state is very close to that of the steady state, and the equilibrium time is only seconds. In contrast, in the case of 3.5 µM RuHex, the difference between two states is noticeable and the equilibrium time is tens of minutes. Also of note, the obtained steady-state charge at 3.5 µM is significantly smaller than that at 50 µM RuHex.

fact, prolonged treatment with MCH (overnight) did not further affect DNA surface density in both cases (data not shown). These results strongly suggest that some nonspecific Au-DNA interactions might even be comparable to the strong Au-S interaction. Consistently, recent studies also suggest that DNA bases interacts with gold surface via nitrogen atoms.25,39 In fact, poly(adenine)Au interaction was reported to be comparable to or even larger than Au-S interaction.25 While both thiolated and nonthiolated DNA can be assembled on gold surfaces, their hybridization properties differ significantly. We employed QCM-D to explore the hybridization ability of these two DNA monolayers (Figure 5). Noticeably, SH ssDNA monolayers demonstrated normal frequency decay in the presence of target DNA, indicating the occurrence of DNA hybridization.40 In contrast, non SH-ssDNA monolayers were essentially unhybridizable under the same conditions. Dissipation studies, which reflect conformational variations of surface-confined species,33 further confirmed that non SH-ssDNA monolayers were almost unperturbed in the presence of target DNA sequences. This result (39) Demers, L. M.; Ostblom, M.; Zhang, H.; Jang, N. H.; Liedberg, B.; Mirkin, C. A. J. Am. Chem. Soc. 2002, 124 (38), 11248-9. (40) Su, X.; Robelek, R.; Wu, Y.; Wang, G.; Knoll, W. Anal. Chem. 2004, 76 (2), 489-94.

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Figure 3. Surface density of a SH-ssDNA monolayer obtained with different techniques: cyclic voltammetry at 3.5 µM RuHex; chronocoulometry 3.5 µM RuHex; and chronocoulometry 50 µM RuHex. DNA immobilization was performed by incubation of electrodes at 2 µM SH-ssDNA probes in the I-buffer. CV, scan rates 50 mV/s; CC, pulse period 250 ms, and pulse width 700 mV.

Figure 4. Surface densities of SH-ssDNA, non SH-ssDNA, and non SH-dsDNA monolayers obtained with chronocoulometry at 50 µM RuHex. DNA immobilization was performed by incubation of electrodes at either 1 µM ssDNA probes or prehybridized dsDNA (0.5 µM probe + 0.5 µM target) in the I-buffer. CC, pulse period 250 ms, and pulse width 700 mV.

is not unexpected since DNA hybridization requires the approach and subsequent pairing via hydrogen bonds of bases on two complementary strands. SH-ssDNA monolayers offer such freedom since strands are nearly perpendicular to surfaces. In contrast, DNA bases are attached to gold during nonspecific adsorption, which prevents the pairing process with complementary base. Since nonspecifically adsorbed DNA still existed even when we employed thiolated DNA probes and MCH treatment,

Figure 6. Surface densities of non SH-ssDNA monolayers obtained with chronocoulometry at 50 µM RuHex. DNA immobilization was performed by incubation of electrodes at either 1 or 0.2 µM DNA probes in the I-buffer containing MCH (MCH:non SH-ssDNA ) 0:1, 1:1, 1:10, respectively). CC, pulse period 250 ms and pulse width 700 mV.

Figure 5. QCM-D sensorgrams for hybridization of SH-ssDNA and non SH-ssDNA monolayers at 1 µM target DNA in H-buffers. The upper and lower panels show the frequency and dissipation changes, respectively.

the results presented here strongly imply that these nonspecifically adsorbed DNA should reduce hybridization efficiencies of DNA sensors. Should we be able to completely prevent nonspecific DNA adsorption, we could then significantly improve the performance (both sensitivity and selectivity) of DNA hybridization-based sensors. Given that nonspecific Au-DNA interactions arise due to Aubase interactions, would the formation of a double helix prevent these nonspecific interactions? We then attempted to immobilize prehybridized DNA probes, in which case DNA bases are wrapped inside the helix. Indeed, we observed a significant decrease in surface density by immobilization of non SH-dsDNA as compared with immobilization of non SH-ssDNA. Nevertheless, the displacement was not 100%, either. At present, we are still not sure how dsDNA sticks to gold surfaces. Possible explanations include partial denaturation of dsDNA during the assembly process,25 interactions at the large groove region, or both. While the adsorption of both nonthiolated ssDNA and dsDNA is not negligible (approximately 1/3 and 1/10 of SH-ssDNA adsorption), we suggest that the contribution of the nonspecific adsorption during SH-ssDNA immobilization should be much less significant due to the competition between interactions of Au-S and Aubases. This could explain that one still obtains relatively high hybridization efficiency even in the presence of nonspecific AuDNA adsorption. Effects of MCH Treatment. It has been well known that MCH treatment is essential in well controlled DNA immobilization. We then investigated the effects of MCH treatment with two different protocols: (1) first with pure DNA and then with 1 mM MCH

and (2) codeposition of DNA and MCH at appropriate ratios and then with 1 mM MCH. We expect that the introduction of MCH in the initial DNA deposition process might largely prevent nonspecific interactions. However, in contrast, we did not observe a significant difference between DNA surface densities by varying MCH concentrations. As demonstrated in Figure 6, at both DNA probe concentration (0.2 and 1.0 µM), the surface densities of DNA are largely unaffected either in the absence of MCH or in the presence of MCH (with DNA:MCH ) 1:1 or 1:10). These results suggest that the protocol originally employed by Tarlov and co-workers works equally well as the codeposition protocol. Nevertheless, neither of these two protocols could remove all nonspecifically adsorbed DNA probes. In addition, we observed that MCH concentration was important for the quality of prepared DNA monolayers. As demonstrated in CVs, capacitive currents (background currents) are rather large without MCH treatment, which arises due to large uncovered areas (Figure 1-S). MCH treatment significantly reduces capacitive currents, indicating the coverage of small-molecule MCH at gold surfaces. Nevertheless, 2-h treatment of either 1 or 10 µM MCH is not sufficient to cover all bare gold surfaces (data not shown). Thus, 1 mM MCH deposition is important to both competitively remove nonspecific DNA adsorption and fill “pinholes” in DNA monolayers. Also of note, 1-2 h of MCH treatment is sufficient for covering gold surfaces. We attempted to incubate DNA monolayers in 1 mM MCH overnight; however, we did not observe significant change of capacitive currents. CONCLUSION In summary, we have electrochemically investigated DNA immobilization at gold surfaces. In particular, we attempted to distinguish specific adsorption via Au-S interaction and nonspecific adsorption via Au-base interactions. We have demonstrated that DNA probes adsorbed via Au-bases were nearly unhybridizable. However, nonspecific DNA adsorption did occur even when we employed thiolated DNA probes. We thus propose that this part of nonspecifically adsorbed DNA reduces hybridization efficiencies of DNA sensors. MCH has proven effective in Analytical Chemistry, Vol. 77, No. 19, October 1, 2005

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removing nonspecific DNA adsorption, which should improve the performance of DNA sensors. Despite this, we have provided evidence that neither MCH posttreatment nor MCH codeposition could remove all nonspecifically adsorbed DNA. Further efforts should be taken toward this direction in order to improve the DNA sensing performance. ACKNOWLEDGMENT This work was supported by National Natural Science Foundation (20404016), Shanghai Municipal Commission for Science and Technology (0452nm068, 03DZ14025, 02JC14029), Shanghai Rising-Star Program, Ministry of Science and Technology of China

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(2003AA226011), Ministry of Personnel and Chinese Academy of Sciences. SUPPORTING INFORMATION AVAILABLE Additional cyclic voltammetric and chronocoulometric curves for RuHex at DNA surfaces. This material is available free of charge via the Internet at http://pubs.acs.org.

Received for review May 25, 2005. Accepted August 15, 2005. AC050911X