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Langmuir 2006, 22, 5560-5562
The Role of Surface Charging during the Coadsorption of Mercaptohexanol to DNA Layers on Gold: Direct Observation of Desorption and Layer Reorientation K. Arinaga,*,†,‡ U. Rant,† M. Tornow,† S. Fujita,‡ G. Abstreiter,† and N. Yokoyama‡ Walter Schottky Institut, Technische UniVersitaet Muenchen, 85748 Garching, Germany, and Fujitsu Laboratories Ltd., Atsugi 243-0197, Japan ReceiVed February 2, 2006. In Final Form: May 7, 2006 We study the coadsorption of mercaptohexanol onto preimmobilized oligonucleotide layers on gold. Monitoring the position of the DNA relative to the surface by optical means directly shows the mercaptohexanol-induced desorption of DNA and the reorientation of surface-tethered strands in situ and in real time. By simultaneously recording the electrochemical electrode potential, we are able to demonstrate that changes in the layer conformation are predominantly of electrostatic origin and can be reversed by applying external bias to the substrate.
Introduction Oligonucleotide layers on solid substrates are receiving considerable attention for their applications in biosensing, in general, and DNA sensing in particular.1 Metal substrates are especially interesting because they provide the opportunity to actively manipulate the intrinsically negatively charged DNA layer2 or conduct electrochemical measurements.3 Usually, the immobilization of DNA layers onto solid supports occurs by self-assembly from solution; in the case of gold substrates, the oligonucleotides are commonly modified with a thiol linker, which serves as a specific anchor to covalently tether the strands to the surface. However, owing to the complex adsorption behavior of nucleic acids on Au surfaces (unspecific interactions via bases,4 electric image charge attraction of the charged backbone,5 etc.), generally, the layer structure is not well-defined, which in turn deteriorates the functionality of the biolayer. Herne and Tarlov introduced the coadsorption of mercaptohexanol (MCH) onto DNA-modified gold substrates6 and reported that MCH largely removes weakly, that is, unspecifically adsorbed nucleic acids by forming a dense MCH sublayer.7 Composite DNA/MCH layers of this kind were shown to exhibit excellent hybridization efficiencies with targets of complementary sequence as well as adequate stability.8 This two-step adsorption method has been adopted by numerous groups; however, details concerning the mutual interactions of MCH, DNA, and the gold surface during the process of film formation remain unrevealed. * Corresponding author. Tel:+81-46-250-8234; fax:+81-46-250-8844; e-mail:
[email protected]. † Technische Universitaet Muenchen. ‡ Fujitsu Laboratories Ltd. (1) Tarlov, M. J.; Steel, A. B. DNA-Based Sensors. In Biomolecular Films; Rusling, J. F., Ed.; Dekker, Inc: New York, 2003; Vol. 111. (2) Rant, U.; Arinaga, K.; Fujita, S.; Yokoyama, N.; Abstreiter, G.; Tornow, M. Nano Lett. 2004, 4 (12), 2441-2445. (3) Drummond, T. G.; Hill, M. G.; Barton, J. K. Nat. Biotechnol. 2003, 21, 1192-1199. (4) Kimura-Suda, H.; Petrovykh, D. Y.; Tarlov, M. J.; Whitman, L. J. J. Am. Chem. Soc. 2003, 125, 9014-9015. (5) Netz, R. R.; Andelman, D. Phys. Rep. 2003, 380, 1-95 and references therein. (6) Herne, T. M.; Tarlov, M. J. J. Am. Chem. Soc. 1997, 119, 8916-8920. (7) Levicky, R.; Herne, T. M.; Tarlov, M. J.; Satija, S. K. J. Am. Chem. Soc. 1998, 120, 9787-9792. (8) Peterlinz, K. A.; Georgiadis, R. M.; Herne, T. M.; Tarlov, M. J. J. Am. Chem. Soc. 1997, 119, 3401-3402.
Here, we directly evidence the desorption and reorientation of preimmobilized nucleic acids, induced by the coadsorption of MCH in situ and in real time. Most importantly, we show that changes in the structure of the DNA layer are directly correlated to the surface charging that accompanies the MCH adsorption, suggesting that the reorientation of DNA strands on the surface is predominantly of electrostatic nature. Materials and Methods All chemicals were purchased from general suppliers and used without further purification. DNA was obtained from IBA GmbH in Goettingen, Germany, and the sequences of the non-selfcomplementary 24 and 48-mer single-stranded (ss) oligonucleotides were 5′-HS-(CH2)6-TAG TCG GAA GCA TCG AAG GCT GATCy3-3′ and 5′-HS-(CH2)6-TAG TCG TAA GCT GAT ATG GCT GAT TAG TCG GAA GCA TCG AAG GCT GAT-Cy3-3′, respectively. For fluorescence detection, the ssDNA was labeled with a cyanine dye, Cy3, at the 3′ end, whereas the 5′ end was derivatized with a thiol linker to tether the DNA to Au surfaces. Au electrodes of 2.0 mm diameter were prepared on 3 in. singlecrystalline sapphire wafers by subsequently depositing Ti(10 nm)/ Pt(40 nm)/Au(200 nm) using standard optical lithography and e-beam metallization techniques. The average roughness of the prepared Au surfaces was measured by AFM and found to be less than 1 nm, that is, insignificant compared to the oligonucleotide length. The substrates were cleaned in Piranha solution (H2SO4/H2O2(30%) ) 7:3) for 15 min (Note that piranha solution must be handled with care: it is extremely oxidizing, reacts Violently with organics, and should only be stored in loosely sealed containers to aVoid pressure buildup.) and, prior to DNA adsorption, exposed to HNO3 (60%) for 15 min, followed by a final rinse with deionized (DI) water. Immobilization of ssDNA onto the Au surface was accomplished by exposing the electrodes to DNA-containing, buffered aqueous solution ([Tris] ) 10 mM, pH ) 7.3) using the following parameters: time of exposure to the solution containing the DNA was 1 h for ss24-mer DNA and 5 min for ss48-mer DNA; the DNA concentration was 10 µM for ss24-mer DNA and 1 µM for ss48-mer DNA; the concentration of added NaCl was 50 mM for ss24-mer DNA and 3 mM for ss48-mer DNA. After the adsorption process, the electrodes were thoroughly rinsed with buffer solution ([Tris] ) 10 mM, pH ) 7.3, [NaCl] ) 50 mM]). Following ssDNA adsorption, the modified Au surfaces were subjected to a second adsorption step by exposing them to MCH ([MCH] ) 1 mM, 1 h exposure time), which led to the formation of a mixed DNA/MCH monolayer. Here, MCH is used as a spacer molecule that specifically binds to Au by its thiol group. In the case
10.1021/la060324m CCC: $33.50 © 2006 American Chemical Society Published on Web 05/27/2006
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Figure 1. Desorption of 24-mer, ss nucleic acids from gold and the reorientation of the remaining DNA layer, induced by the coadsorption of MCH onto the surface (DNA surface coverage: 8 × 1011 cm-2; fluorescence background: ∼200 au). Bottom: schematic layer conformation and substrate charge. Dashed rays indicate the fluorescence emission of the attached dye label, which, because of nonradiative energy transfer, depends on the dye-metal distance. of 48-mer DNA samples, hybridization with complimentary DNA (c-DNA) was subsequently carried out in buffer solution ([c-DNA] ) 10 µM, [Tris] ) 10 mM, pH ) 7.3, [NaCl] ) 200 mM) for 1 h to prepare double-stranded DNA layers. After ssDNA adsorption onto Au electrodes, the wafers were installed in an unsealed electrochemical cell, which allowed for fluorescence measurements using optical fibers. A potentiostat (Autolab PGSTAT30, Eco Chemie, Utrecht, The Netherlands) was utilized for applying bias voltages to the Au work electrodes with respect to a Ag/AgCl reference electrode using a Pt-wire counter electrode in electrolyte solution. Fluorescence measurements of the immobilized Cy3-labeled DNA were conducted by positioning a special fiber mount over the electrode. Here, the green light from an Ar+ laser (λ ) 514 nm) is guided onto the electrode surface at an angle of 45°, whereas fluorescence from the Cy3 dyes is collected by a second fiber oriented normal to the surface plane. Note that the region of fluorescence detection included not only the electrode surface but also the electrolyte volume above, defined by the intersection of the excitation and detection beams. Cy3 fluorescence was measured by coupling light from the detection fiber into a monochromator (set to the Cy3-peak-emission wavelength, 565 nm) equipped with a cooled photomultiplier operating in single-photoncounting mode. According to reference measurements made on unmodified Au surfaces, the background signal was approximately 200 au.
Experimental Section For optical detection, oligonucleotides of mixed sequence are labeled with a fluorescent dye (Cy3) at their 3′ end, while the 5′ end was modified with a (CH2)6-SH linker. The oligonucleotides’ position (orientation) with respect to the surface can be inferred from the observed fluorescence of the marker, taking advantage of the distance-dependent, nonradiative energy transfer from the excited dye to the gold surface.9 Because of this proximity-quenching effect, almost no fluorescence is emitted when the DNA is lying on the substrate, whereas substantially enhanced emission can be observed from “standing” oligonucleotides. This effect has been described in (9) Barnes, W. L. J. Mod. Opt. 1998, 45 (4), 661-699.
detail in prior studies.2,10 Release of dye-labeled nucleic acids from the surface results in extremely intense, yet transient fluorescence, which is emitted from DNA molecules floating in solution before they diffuse out of the detection volume (the dye luminescence can increase by more than 2 orders of magnitude upon desorption).11 No photodegradation of the Cy3 dye was observed during the measurements.12
Results and Discussion Figure 1 depicts the fluorescence intensity together with the electrode potential, which is left floating (open circuit potential) during the adsorption of MCH. Before injecting MCH (1 mM in buffer solution containing [Tris] ) 10 mM and [NaCl] ) 50 mM, pH ) 7.3) into the solution covering the immobilized DNA layer (region A), the surface potential is comparably positive, while the extremely low fluorescence signifies that the oligonucleotides are lying on the substrate. The adsorption of MCH causes a drop in the surface potential,13 which is characteristic for an oxidative adsorption process, that is, an injection of electrons into the substrate upon the formation of the S-Au bond.14 In the initial stage of the MCH adsorption, the potential drop is accompanied by a peak in the fluorescence intensity, which originates from the desorption of a fraction of the immobilized nucleic acids from the surface (B). After a few hundred seconds, the fluorescence signal from released oligonucleotides has vanished (due to out-diffusion), yet significantly increased (10) Rant, U.; Arinaga, K.; Fujita, S.; Yokoyama, N.; Abstreiter, G.; Tornow, M. Langmuir 2004, 20, 10086-10092. (11) Rant, U.; Arinaga, K.; Fujiwara, T.; Fujita, S.; Tornow, M.; Yokoyama, N.; Abstreiter, G. Biophys. J. 2003, 85, 3858-3864. (12) Owing to the presence of the metal surface (proximity quenching effect), photobleaching reactions are substantially slowed and not observed for the relatively short time during which the Cy3 dyes are exposed to laser illumination (∼10 min; see also ref 2). As long as samples are kept in the dark, Cy3 can be stored for a few days at room temperature without showing significant signs of degradation. (13) The initial, steplike decrease is caused by electrostatic discharge effects when immersing the pipet into solution. (14) (a) Ulman, A. Chem. ReV. 1996, 96, 1533-1554. (b) Hatchett, D. W.; Stevenson, K. J.; Lacy, W. B.; Harris, J. M.; White, H. S. J. Am. Chem. Soc. 1997, 119, 6596-6606.
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emission compared to state A can be observed from the remaining DNA layer (C). This elevated fluorescence represents a “standing” DNA layer, for which the marker-metal distance is maximal, and thus the nonradiative energy transfer (quenching) is weak. Subsequently, the solution was exchanged for fresh buffer (without MCH) and the sample was stored overnight. During that time, the sample was kept in the dark and installed in the optical setup to avoid possible photobleaching and ensure quantitative comparability of the fluorescence measurements, respectively. When resuming the measurement after 30 h, the negative surface charge induced by the MCH adsorption had equilibrated, and the surface potential had leveled out to its initial value (D). At the same time, the fluorescence emission decreased, indicating a less upright orientation of the strands. Nevertheless, the still pronounced fluorescence emission confirms the passivation effect of the MCH sublayer: after an MCH treatment, direct contact and hence unspecific interactions between immobilized nucleic acids and the gold surface are largely suppressed, owing to the function of MCH as a spacer layer. A second MCH adsorption step yet again induces negative surface charge and hence results in the reorientation of the DNA layer (E). Remarkably, desorption is just observed for the first, but not for subsequent MCH adsorption steps, suggesting that most of the weakly adsorbed nucleic acids are removed during the first step. As evident from changes in the potential as well as the fluorescence, adsorption takes place all over again when exposing the surface to MCH for the second time; thus, vacancies must have developed in the DNA/MCH layer during long storage (cf. schematic D in Figure 1). In addition, we find a larger interfacial capacitance, which indicates holes in the molecular layer (data not shown). Besides degradation of the MCH layer, desorption of DNA during long storage might contribute to the formation of holes: DNA surface coverage measurements (according to the method of Steel et al.15) point to desorption of a small fraction (roughly 10%) of the layer overnight. The initial DNA coverage plays a crucial role: while the coadsorption of MCH to densely packed layers results in the desorption of nucleic acids, in the case of low-density layers (
