in Domain Boundaries in Monolayers of Single-Stranded DNA

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Langmuir 2007, 23, 1410-1413

Direct Imaging of Hexaamine-Ruthenium(III) in Domain Boundaries in Monolayers of Single-Stranded DNA Mikala Grubb,† Hainer Wackerbarth,*,† Jesper Wengel,‡ and Jens Ulstrup*,† Department of Chemistry, Nano DTU, Technical UniVersity of Denmark, Building 207, DK-2800 Kgs. Lyngby, Denmark, and Nucleic Acid Center, Department of Chemistry, UniVersity of Southern Denmark, DK-5230 Odense M, Denmark ReceiVed August 31, 2006. In Final Form: October 11, 2006 We describe adsorption and identification of the binding sites of [Ru(NH3)6]3+ (RuHex) molecules in a closely packed monolayer of a 13-base ss-DNA on Au(111) electrodes by electrochemical in situ scanning tunneling microscopy (STM), cyclic voltammetry and interfacial capacitance data. In situ STM at single-molecule resolution shows that RuHex adsorbs only at the domain borders and near defects. Together with the electrochemical data that show a negative redox potential shift for RuHex adsorbed to DNA strands, this strongly suggests that RuHex binds only to the exposed phosphate groups in the DNA backbone.

DNA-based sensor technology has developed broadly in recent years, with perspectives particularly for investigating DNA sequences at low cost.1 DNA sensors are based on single-stranded DNA (ss-DNA) immobilized on a solid surface. The broad application of DNA chip technology has also led to strong focus on the fundamental chemistry and physics of DNA and sensing at the nanoscale. Electrochemical sensing, for example, provides only indirect information about the surface, but morphological and dynamic surface properties at the molecular level must also be addressed directly. Scanning probe techniques here offer powerful tools. In this report we visualize structure and dynamics of a 13-base ss-DNA monolayer and its surface interaction with a redox probe by scanning tunneling microscopy in the in situ electrochemical mode (in situ STM) directly in an aqueous medium. Immobilization of DNA without loss of biological function has been a challenge. DNA strands are often immobilized on gold via a thiol linker that provides well-defined stable binding between gold and sulfur.2-8 The coverage of DNA on the surface is crucial for the hybridization efficiency,7 which can be controlled by the use of mixed monolayers3-7 of DNA molecules and a small spacer molecule, often mercaptohexanol (MCH). In addition to creating space for the DNA-based molecules to hybridize, the spacer molecules prevent DNA from adsorbing nonspecifically to the surface. Different redox probes have been used to identify and quantify DNA on the surface via an electrochemical signal, e.g., in cyclic voltammetry.4,6,8 Particular attention has been given to hexaammine-ruthenium(III)/(II), [Ru(NH3)6]3+/2+ (RuHex), * Authors to whom correspondence should be addressed. J. Ulstrup: e-mail, [email protected]; tel., +45 4525 2359; fax, +45 4588 3136. H. Wackerbarth: e-mail, [email protected]; tel., +45 4525 2353; fax, +45 4588 3136. † Technical University of Denmark. ‡ University of Southern Denmark. (1) Service, R. F. Science 1998, 282, 396. (2) Wackerbarth, H.; Grubb, M.; Zhang, J.; Hansen, A. G.; Ulstrup, J. Angew. Chem., Int. Ed. 2004, 43, 198. (3) Steel, A. B.; Herne, T. M.; Tarlov, M. J. Anal. Chem. 1998, 70, 4670. (4) Steel, A. B.; Herne, T. M.; Tarlov, M. J. Bioconjugate Chem. 1999, 10, 419. (5) Rant, U.; Arinaga, K.; Fujita, S.; Yokoyama, N.; Abstreiter, G.; Tornow, M. Nano Lett. 2004, 4, 2441. (6) Wong, E. L. S.; Gooding, J. J. Anal. Chem. 2003, 75, 3845. (7) Peterson, A. W.; Heaton, R. J.; Georgiadis, R. M. Nucl. Acid Res. 2001, 29, 5163. (8) Kelley, S. O.; Barton, J. K. Bioconjugate Chem. 1997, 8, 31.

with three phosphates of the DNA backbone required for stoichiometric electrostatic binding of one RuHex(III).3,4 We have previously studied full monolayers of ss-DNA molecules with 10 and 13 bases.2,9 The monolayers are disordered immediately after adsorption, but pack very densely with DNA in an upright position at negative sample potentials, leaving no space for hybridization. Pinholes are present but not a dominating surface feature.2 A recent theoretical study based on Monte Carlo simulations of the surface grafting of rodlike polyelectrolyte brushes supports a new nematic phase resembling the observed ordered thiol-modified oligonucelotide adlayers being formed at high densities, offering a rationale for both the upright orientation and the high surface density.10 Our results are also supported by a recent investigation of DNA on a mercury electrode, where a pinhole-free layer with very high coverage was disclosed.11 ss-DNA in a mixed monolayer cannot be identified directly by STM, presumably because the strands are too flexible and disordered but in a recent communication we showed that 13base ss-DNA molecules in a mixed monolayer with MCH could be identified by in situ STM after addition of RuHex.9 The lowlying RuHex redox level is likely to open new electron-transfer hopping channels in the tunneling gap via bound RuHex units. This leads to strongly enhanced tunneling current contrasts. Theoretical support for such a mechanism is available,12 and other redox systems have shown such behavior.13 In this way a new perspective is added to molecules as DNA-markers because RuHex can be observed only by in situ STM when bound to DNA. In this report we present a combined in situ STM and electrochemical study of RuHex binding to a densely packed pure monolayer of ss-DNA with a thiol linker, immobilized on a single-crystal gold surface. In situ STM discloses both the position and the conformation of molecules on the surface at the single-molecule level.14,15 The data show that RuHex first adsorbs (9) Grubb, M.; Wackerbarth, H.; Ulstrup, J. J. Am. Chem. Soc. 2006, 128, 7734. (10) Fazli, H.; Golestanian, R.; Hansen, P. L.; Kolahchi, M. R. Europhys. Lett. 2006, 73, 429. (11) Ostatna, V.; Palacek, E. Langmuir 2006, 22, 6481. (12) Kuznetsov, A. M.; Ulstrup, J. J. Phys. Chem. A 2000, 104, 11531. (13) Albrecht, T.; Guckian, A.; Ulstrup, J.; Vos, J. G. Nano Lett. 2005, 5, 1451. (14) Zhang, J.; Chi, Q.; Nielsen, J. U.; Friis, E. P.; Andersen, J. E. T.; Ulstrup, J. Langmuir 2000, 16, 7229. (15) Chi, Q.; Zhang, J.; Ulstrup, J. J. Phys. Chem. B 2005, 109, 15355.

10.1021/la062555z CCC: $37.00 © 2007 American Chemical Society Published on Web 12/09/2006

Direct Imaging of RuHex in a Monolayer of ss-DNA

Figure 1. 60 nm × 60 nm in situ STM image of HS-13-base monolayer after adsortion in 1 µM HS-13-base for 3 h and then setting the sample potential to -0.65 V (SCE) for 40 min. Tungsten tip. Isetpoint ) 0.35 nA, Vbias ) -0.15 V, Esample ) -0.25 V (SCE). Scan rate 9.8 lines/s. Buffer in the cell and during adsorption was 10 mM Tris + 50 mM NaCl (pH 7.6).

to the domain boundaries of the ordered DNA-based monolayers and then gradually penetrates the domains in parallel with the dissolution of the domain order. The ss-DNA 13-base sequence, HS-(CH2)6-5′- CGC ATT ATT ACG C, was from TAG Copenhagen (Denmark). The sample was diluted with Millipore water (18.2 mΩ cm) and used in 10 mM Tris + 50 mM NaCl (pH 7.6) buffer without further purification. All chemicals were reagent grade and from Sigma. The HS-13-base monolayer used in electrochemistry was prepared by soaking the Au(111) electrode in 1 µM HS-13-base solution for 3 h, in the presence or absence of 500 µM RuHex. The electrodes soaked in HS-13-base without RuHex were rinsed, soaked in 10 mM RuHex for 10 min, and then transferred to the electrochemical cell. The electrodes soaked for 3 h with both RuHex and DNA were rinsed with buffer and transferred to the electrochemical cell. The electrodes used for STM were soaked for 3 h in 1 µM ss-HS-13-base in 10 mM Tris + 50 mM NaCl (pH 7.6). After ordering and imaging of the ss-HS-13-base monolayer, 50 µL of 1 mM RuHex in 10 mM Tris + 50 mM NaCl (pH 7.6) was added to the STM cell and imaging was continued. Single-crystal Au(111) bead electrodes and the hanging meniscus method were used in voltammetric measurements, controlled by an Autolab system (Eco Chemie, The Netherlands) with a saturated calomel electrode (SCE) and a coiled platinum wire as reference and counter electrodes, respectively. The electrodes were prepared by the method of Hamelin and electropolished and annealed as described.16 An argon atmosphere was maintained throughout. Cells, glassware, and other utensils were boiled in 15% nitric acid, washed with Millipore water, and sonicated before use. A PicoSPM instrument (Molecular Imaging Co., USA), with a bipotentiostat for independent control of substrate and tip potential, and in-house-built three-electrode KEL-F cells were used. Au(111) discs purchased from Surface Preparation Labs (The Netherlands) were used for all STM measurements and prepared as the bead electrodes. Reference and counter electrodes (16) Hamelin, A. J. Electroanal. Chem. 1996, 407, 1.

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Figure 2. 60 nm × 60 nm in situ STM image after addition of 50 µM 1 mM RuHex in 10 mM Tris + 50 mM NaCl (pH 7.6) to the layer shown in Figure 1. Inset: 13 nm × 13 nm zoom-in on the layer. The image was recorded 30 min after RuHex addition. Tungsten tip. Isetpoint ) 0.35 nA, Vbias ) -0.15 V, Esample ) -0.25 V (SCE). Scan rate 9.8 lines/s. Buffer in the cell was 10 mM Tris + 50 mM NaCl (pH 7.6).

were platinum wires. Electrochemically etched W-tips coated with Apiezon wax and the constant current mode were used throughout. Pure HS-13-base monolayer formation was first imaged and showed no order. After the sample potential was set to -0.65 V (SCE) for 30 min, the monolayer was ordered (Figure 1). Similar behavior was also observed for a repetitive all-adenine 10-base sequence thiol-modified DNA strand2 except at -0.61 V (SCE) due to the more positive desorption potential of the 10-base adenine (-0.67 V (SCE)) compared with -0.71 V (SCE) for HS-13-base DNA. This shift is caused by the increased lateral interactions with increasing length of the DNA molecule. In situ STM of the same 13-base strand with the thiol linker attached to the 3′-end of the strand showed the same surface structures as the 5′-end modified strand (data not shown; see Supporting Information). Fifty microliters of 1 mM RuHex (in 10 mM Tris + 50 mM NaCl, pH 7.6) was then added to the electrolyte in the STM cell and imaging continued. After 10-15 min bright spots were observed at the domain edges, indicating by a change in contrast that RuHex had bound to the strands (Figure 2). After 2 h the monolayer showed a large number of bright spots in the domain boundaries, taking up about 6% of the total area. The domain edges have different properties compared to the domain interior. Particularly, the phosphates at the edges are exposed to RuHex binding. This is supported by the appearance of the bright spots (RuHex) in the initial binding phase only at the domain edges and at defects in the layer. Binding to the end of the DNA molecules would also expectedly show RuHex molecules inside the domains. This was not seen and confirms that RuHex binds to the backbone - not the end - of DNA. However, with time the RuHex molecules penetrate the domains that grow smaller at the same time. After about 8 h the domain sizes have decreased significantly with a large part of the previously ordered monolayer now disordered (data not shown). The behavior of RuHex in the 13-base DNA-based monolayers was also addressed by cyclic voltammetry and interfacial capacitance measurements. Figure 3 shows three cyclic voltammograms (CV) with differently prepared layers of DNA. The

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Figure 3. Cyclic voltammograms of HS-13-base ss-DNA on Au(111) in 10 mM Tris + 50 mM NaCl, pH 7.4. Scan rate 50 mV/s. No RuHex was present in the electrolyte in the cell and a freshly cleaned cell was used for every electrode. Red line: HS-13-base ss-DNA adsorbed on the Au(111), rinsed with buffer, dipped in a RuHex solution for 10 min, and transferred directly to the electrochemical cell. Blue line, 1 µM HS-13-base and 500 µM RuHex adsorbed together on the Au(111) electrode. Black line: HS-13base/MCH layer on Au(111), rinsed with buffer, dipped in 1 mM RuHex solution for ca. 10 min, and transferred directly to the electrochemical cell. Inset: Comparison of the peak positions of co-adsorbed HS-13-base and RuHex (blue) and the HS-13-base/ MCH layer after RuHex adsorption. The currents of the co-adsorbed HS-13-base and RuHex have been multiplied by a factor of 3.

Grubb et al.

Figure 4. Interfacial capacitances of HS-13-base adsorbed on a Au(111) electrode in 10 mM Tris + 50 mM NaCl, pH 7.5. Blue squares: Co-adsorption of HS-13-base ss-DNA and RuHex on the electrode. No RuHex in the electrolyte in the cell. Black squares: Mixed monolayer of HS-13-base and MCH with 500 µM RuHex present in the cell. Frequency 1 Hz, step potential 10 mV.

electrolyte was 10 mM Tris + 50 mM NaCl (pH 7.6) in all cases with no RuHex in the electrolyte. The freshly cleaned electrochemical cell was used for each measurement. The red curve shows a CV of a HS-13-base monolayer dipped in RuHex solution for 10 min after formation of the layer. The electrode was then rinsed and transferred to a cell with 10 mM Tris + 50 mM NaCl (pH 7.6) electrolyte. No voltammetric RuHex electrochemical signal is seen. The absence of a signal indicates that the amount of bound RuHex is very small. This is in accord with the in situ STM images of the HS-13-base where only small amounts of RuHex are found at the domain edges and at defects in the layer. The adsorption process of RuHex into the layer also takes much longer in a dense monolayer than the 10 min used. The blue curve is a CV of a HS-13-base ss-strand monolayer formed in the presence of RuHex. This CV has peaks at -254 and -217 mV (SCE) associated with RuHex but shifted negatively compared to RuHex in solution, supporting that the signal originates from RuHex bound in the monolayer.4 Ideally, there should be no cathodic and anodic peak separation for a surfaceconfined molecule. Peak separation can be induced by kinetic control or interfacial electron-transfer rates comparable to the scan rate. Other reasons for apparent “nonideality” could be dissociation and association of [Ru(NH3)6]3+/2+ that accompanies the electrochemical process due to different stochiometric PO43-/ RuHex ratios when RuHex is in the oxidized and reduced state. Other effects are discussed elsewhere.13 The peaks gradually decrease with time, probably due to slow RuHex desorption. A recent electrochemical study based on interfacial capacitance data suggests that the negatively charged backbone of the DNA strands in the layer keeps the counterions and maintains a high local ionic strength.17 Even though RuHex is bound strongly to the strands, the adsorption equilibrium would then be gradually shifted to the much more abundant sodium ions, when there is no RuHex in the bulk solution.

The black curve shows, finally, a CV for an electrode with a mixed monolayer of HS-13-base and MCH after soaking in RuHex solution for 10 min and transfer to RuHex-free buffer solution. The mixed monolayer has enough space for RuHex to adsorb to the strands (see, however, below). When the electrode is transferred to the electrochemical cell with no RuHex in the solution, most RuHex diffuses into bulk solution, but enough is left at the surface to give a notable RuHex signal. The peak positions (-146 and -193 mV (SCE)) here correspond closely to those for RuHex in solution.3 This is in accord with the observation that the negative potential shift of surface-confined RuHex increases with increasing DNA coverage.17 Various effects can cause the redox potential shift but the strongly negatively charged local environment around the RuHex molecules is a likely origin. Other possibilities could be changes in the dielectric environment, ion pairing, solvation, and image charge interactions. Desorption, reorientiation, protonation, or solvation of adsorbed species are reflected in capacitive currents or charging of the solution-electrode interface, also detectable by direct interfacial capacitance measurement.18 Capacitance data for the full HS13-base monolayer co-adsorbed with RuHex (blue curve) without RuHex in the electrolyte and for the mixed monolayer with RuHex present in the electrolyte (black curve) prepared like those for cyclic voltammetry were recorded, Figure 4. These data also show the peak shift. The capacitance shows a higher sensitivity in detecting the RuHex reduction and oxidation for the coadsorbed layer compared to cyclic voltammetry. As the potential is scanned toward negative values, a capacitance peak is detected, originating from Ru3+ f Ru2+ reduction and presumably corresponding cation migration to compensate for the missing positive charge in the layer.17 When the reduction of Ru3+ to Ru2+ is complete and the potential scanned to even more negative values, a new capacitance rise is observed. This is due to the increasing negative charge of the gold surface, leading to a charge imbalance close to the surface. This triggers further cation transport into the layer - leading again to the increase in capacitance. In conclusion, the STM data clearly show that RuHex adsorbs to the backbone of the DNA strand, as adsorbed RuHex is only observed at the domain edges. The time dependence of the domain

(17) Shen, G.; Tercero, N.; Gaspar, M. A.; Varughese, B.; Shepars, K.; Levicky, R. J. Am. Chem. Soc. 2006, 128, 8427.

(18) Wackerbarth, H.; Grubb, M.; Zhang, J.; Hansen, A. G.; Ulstrup, J. Langmuir 2004, 20, 1647.

Direct Imaging of RuHex in a Monolayer of ss-DNA

structure after adding RuHex to the solution also shows that the layer is far from static as RuHex seems to penetrate gradually into the layer. This may be because RuHex affects the structure of the ss-DNA. For example, the DNA strand may curl or bend over. If each DNA strand binds three or four RuHex molecules, the strand therefore - over time - occupies less vertical space, leaving space for RuHex to bind to the neighboring strand in the domain and gradually penetrate the domain. The electrochemical data show that the lack of space in a full DNA monolayer gives a weak signal when RuHex is co-adsorbed with DNA, while a mixed monolayer provides enough space for RuHex adsorption in larger amounts to give a stronger electrochemical signal. This observation supports the surface coverage of DNA being crucial for hybridization;5,7 i.e., the dense DNA monolayer would leave no space for hybridization when not even RuHex can be brought into the space between the strands. Recent results by Ostatna and Palacek11 together with our previous2 and present results thus show that the coverage of a ss-DNA monolayer on the surface is very high. The tightly packed DNA layer blocks RuHex from binding to the phosphates, although RuHex penetration can be observed over time. The packing density on a mercury electrode is even higher, probably due to lack of pinholes. Hence, no RuHex electrochemical signals were detected.11 Comparison of the number of RuHex molecules adsorbed in the full and mixed monolayers as detected by in situ STM discloses in fact a smaller difference than expected from a random distribution of “free” DNA strands in the MCH/DNA monolayer

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(based on the solution concentration ratio of the two components). Clusters, or disordered islands or small domains, in which the inner strands are prevented from RuHex binding, seem to remain even in the disordered mixed monolayer. If this is the case, RuHex molecules would be bound in smaller than stoichiometric amounts, with strands at the domain edges predominantly recorded in electrochemical signals. Coulometric quantification of surfaceimmobilized DNA strands based on RuHex binding, as commonly used, would then have to be viewed with caution except for very dilute layers. The data show that new DNA-based conductivity mechanisms in electrochemical environments induced by binding of redox molecules can be opened. The new conductivity channels can be exploited to disclose details of the surface organization of the DNA-based monolayer at the molecular level of resolution. Such observations may offer new concepts in chemical and biological sensing based on single-molecule phenomena. Acknowledgment. Financial support from NanoScience Center at the University of Copenhagen, the EU programme CIDNA (Contract No. NMP4-CT-2003-505669), and the Danish Research Council for Technology and Production Sciences (Contract No. 26-00-0034) is acknowledged. Supporting Information Available: In situ STM image of HS-3-13-base DNA for comparison with HS-5-13-base DNA. This material is available free of charge via the Internet at http://pubs.acs.org. LA062555Z