Structural Properties of Oligonucleotide Monolayers on Gold Surfaces

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Structural Properties of Oligonucleotide Monolayers on Gold Surfaces Probed by Fluorescence Investigations Ulrich Rant,*,† Kenji Arinaga,†,‡ Shozo Fujita,‡ Naoki Yokoyama,‡ Gerhard Abstreiter,† and Marc Tornow*,† Walter Schottky Institut, Technische Universitaet Muenchen, Am Coulombwall 3, 85748 Garching, Germany, and Fujitsu Laboratories Limited, 10-1 Morinosato-Wakamiya, Atsugi 243-0197, Japan Received March 17, 2004. In Final Form: July 29, 2004 We present optical investigations on the conformation of oligonucleotide layers on Au surfaces. Our studies concentrate on the effect of varying surface coverage densities on the structural properties of layers of 12- and 24mer single-stranded DNA, tethered to the Au surface at one end while being labeled with a fluorescent marker at the opposing end. The distance-dependent energy transfer from the marker dye to the metal surface, which causes quenching of the observed fluorescence, is used to provide information on the orientation of the DNA strands relative to the surface. Variations in the oligonucleotide coverage density, as determined from electrochemical quantification, over 2 orders of magnitude are achieved by employing different preparation conditions. The observed enhancement in fluorescence intensity with increasing DNA coverage can be related to a model involving mutual steric interactions of oligonucleotides on the surface, as well as fluorescence quenching theory. Finally, the applicability of the presented concepts for investigations of heterogeneous monolayers is demonstrated by means of studying the coadsorption of mercaptohexanol onto DNA-modified Au surfaces.

Introduction Self-assembled monolayers of oligonucleotides tethered to gold surfaces have attracted considerable attention in recent years due to their use in various fields of DNArelated research. Fundamental investigations have been devoted to the complex behavior of DNA adsorption on metal surfaces and the conformational structure of the assembled layers.1,2 In contrast to electrically insulating substrates such as glass, the use of a metal as the supporting surface for the immobilized DNA layer opens a variety of possibilities for techniques employing control over the electrode potential in order to, for instance, conduct electrochemical measurements probing charge transport across DNA,3 induce conformational changes in the layer structure,4 influence the adsorption of DNA during the assembly process, or trigger the release of DNA into solution by electrical pulses.5 In the context of experiments involving oligonucleotides which have been labeled with a marker dye to enable fluorescence detection, a metallic substrate imposes a noteworthy aspect to the optical properties of dyes within its close proximity. Energy transfer of the excited dye molecule to the metal facilitates nonradiative transitions * To whom correspondence should be addressed. Ulrich Rant: phone, +49 89 28912776; e-mail, [email protected]. Marc Tornow: phone, +49 89 28912772; fax, +49 89 3206620; e-mail, tornow@ wsi.tum.de. † Technische Universitaet Muenchen. ‡ Fujitsu Laboratories Ltd. (1) Levicky, R.; Herne, T. M.; Tarlov, M. J.; Satija, S. K. J. Am. Chem. Soc. 1998, 120, 9787. (2) Wackerbarth, H.; Grubb, M.; Zhang, J.; Hansen, A. G.; Ulstrup, J. Langmuir 2004, 20, 1647. (3) Treadway, C. R.; Hill, M. G.; Barton, J. K. Chem. Phys. 2002, 281, 409. (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 (24), 6781. Zhang, Z.-L.; Pang, D.-W.; Zhang, R.-Y. Bioconjugate Chem. 2002, 13, 104. (5) Rant, U.; Arinaga, K.; Fujiwara, T.; Fujita, S.; Tornow, M.; Yokoyama, N.; Abstreiter, G. Biophys. J. 2003, 85, 3858.

for relaxation to the dye’s electronic ground state, causing a “quenching” of the observed fluorescence;6,7 this process shows a pronounced dependence on the distance of the dye to the surface within typically a few tens of nanometers, a length scale characteristic of the size of many biomolecules. In our studies, we are taking advantage of this effect as the fluorescence intensity emitted from dyes attached to the top end of tethered oligonucleotides changes as the DNA strands take different orientations on the Au surface. Therewith, we are able to investigate the influence of the coverage density of the adsorbed oligonucleotide layer on its conformational properties; furthermore we analyze the obtained data within a simple steric model treating the immobilized oligonucleotides as gyrating rigid rods, tethered to the surface at one end. The presented results and their agreement with the steric model support the notion that the dynamic behavior of the tethered DNA strands on the microscopic scale determines coalesced layer properties. We point out that a profound understanding and control of the properties of DNA layers on solid substrates are very important with respect to applications, as for instance the hybridization efficiency of immobilized single-stranded probe oligonucleotides to target oligonucleotides in solution depends strongly on the accessibility and hence the structural conditions of the immobilized strands. As our studies are based on conventional methods of fluorescence detection, we expect an effortless transferability of the presented results due to the widespread use of optical techniques in DNA-related research and applications in particular. Materials and Methods Materials. 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 12- and 24mer single-stranded (ss) oligonucleotides were 5′ (6) Chance, R. R.; Prock, A.; Silbey, R. Adv. Chem. Phys. 1978, 37, 1. (7) Persson, B. N. J. J. Phys. C 1978, 11, 4251.

10.1021/la0492963 CCC: $27.50 © 2004 American Chemical Society Published on Web 10/13/2004

Oligonucleotide Monolayers on Gold HS-(CH2)6-TAG TCG GAA GCA-Cy3 3′ and 5′ HS-(CH2)6TAG 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. single crystalline sapphire wafers, by subsequently depositing Ti(10 nm)/Pt(40)/ Au(200) using standard optical lithography and metallization techniques. The substrates were cleaned in boiling 2 M KOH solution and prior to DNA adsorption exposed to HNO3 (60%) for 1 h, 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). To obtain a wide range of DNA surface coverage densities, many different samples were prepared, varying the following parameters: time of exposure to the solution containing the DNA (5 s-2 h); DNA concentration (0.05-10 µM); concentration of added salt (monovalent salt for low to medium DNA coverage ([NaCl] ) 3 mM-1 M); alternatively divalent salt for highest DNA coverage ([MgCl2] ) 0.4 and 1 M for 12- and 24mer DNA, respectively)). Generally, the coverage density of the self-assembled DNA layers on Au increases with increasing assembly time, higher DNA concentrations, and increasing concentration as well as valence of the added salt.8,9 After the adsorption process, the electrodes were thoroughly rinsed with buffer solution ([Tris] ) 10 mM, pH ) 7.3, [NaCl] ) 50 mM]). Following DNA adsorption, the modified Au surfaces were subjected to a second adsorption step by exposing them to mercaptohexanol (MCH, [MCH] ) 1 mM, exposure time ∼ 2 h), which leads to the formation of a mixed ssDNA/MCH monolayer. Here, MCH is used as a spacer molecule which specifically binds to Au by its thiol group. As has been reported in the literature8 and is also evident from the results of this work, the adsorption of MCH removes loosely bound DNA from the surface and fills the space between the remaining ssDNA strands which are bound to the surface by their thiol linker. In that way, it prevents unspecific DNA-Au interactions. DNA coverage was quantified using electrochemical methods as introduced by Steel et al.10 Briefly, the DNA layer is exposed to a low ionic strength electrolyte solution ([Tris] ) 10 mM) containing a multivalent redox cation, hexaammineruthenium(III) chloride (RuHex, [RuHex] ) 0.1 mM), obtained from Sigma-Aldrich. Under these conditions, the negative charge of the DNA’s anionic phosphate groups is compensated by electrostatically trapping RuHex to its backbone, thereby confining to the Au surface a number of redox markers that is proportional to the number of DNA molecules in the immobilized monolayer. Upon application of a potential step from +0.1 to -0.4 V (vs Ag/AgCl reference) at the Au electrode, three contributions add up to the accumulated charge in a chronocoulometric measurement: (i) electrical double layer charge, (ii) charge from reduction of RuHex molecules diffusing to the surface from bulk solution, and (iii) surface excess charge from reduction of surface confined, DNA-bound RuHex. The surface excess charge, which is proportional to the number of DNA molecules at the surface, can be extracted from the accumulated charge when accounting for contributions i and ii, as described by Steel et al. A prerequisite of the described measurement is that no redox marker adsorption occurs at the surface between DNA strands, since it would contribute parasitically to the determined surface excess charge. Therefore the use of MCH as a passivating layer becomes obligatory and for that reason also MCH adsorption was performed routinely prior to electrochemical measurements.11 Apparatus. After preparation, the samples were installed in an uncapped cell filled with buffer solution ([Tris] ) 10 mM, pH ) 7.3, [NaCl] ) 50 mM, continuously purged with argon gas) (8) Herne, T. M.; Tarlov, M. J. J. Am. Chem. Soc. 1997, 119, 8916. (9) Petrovykh, D. Y.; Kimura-Suda, H.; Whitman, L. J.; Tarlov, M. J. J. Am. Chem. Soc. 2003, 125, 5219. (10) Steel, A. B.; Herne, T. M.; Tarlov, M. J. Anal. Chem. 1998, 70, 4670. (11) A second or third MCH adsorption step did not result in any further release of DNA from the surface (as confirmed by optical measurements) but improved the insulating electrical properties of the ssDNA/MCH layer.

Langmuir, Vol. 20, No. 23, 2004 10087 which allowed for optical as well as electrochemical measurements. For electrochemical experiments, a potentiostat (Autolab PGSTAT30, Eco Chemie, The Netherlands) was utilized to control the potential of the Au working electrodes with respect to a Ag/ AgCl reference electrode, using a Pt-wire counter electrode. Fluorescence measurements of the immobilized Cy3-labeled ssDNA were conducted by positioning a special optical fiber mount over the electrode. Here, green light from an Ar+ laser (λ ) 514 nm) is guided onto the electrode surface at an angle of ∼45°, whereas fluorescence from 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 equipped with a cooled photomultiplier operating in single-photon-counting mode. Reference measurements of unmodified Au surfaces were used for background correction.

Experiments and Discussion The discussion of the presented experiments and results will be structured as follows: In the first and main part, we will elucidate the effect of varying surface densities on the structural properties of an immobilized DNA layer, as probed by fluorescence methods. Investigations are conducted with singlestranded oligonucleotides of different lengths, 12- and 24mer, to compare the experimental results to a simple steric model treating the DNA strands as rigid rods featuring only one significant free parameter: the rod length. In the second part, the introduced concepts will be applied to study a heterogeneous monolayer system comprising ssDNA and MCH. We are able to directly confirm desorption and structural reorientation of ssDNA strands, induced by coadsorption of MCH at the DNAmodified Au surface. To discuss the influence of varying surface coverage on the structural properties of DNA layers, we start by focusing on the experimental observations for singlestranded 12mer DNA, depicted in Figure 1 (solid circles). Three regimes can be identified when plotting the fluorescence intensity (F) versus the surface coverage (σ) in a double logarithmic plot. Two linear regions at low and high coverage values, respectively, are separated by a distinct transition region below approximately σ ≈ 1016 m-2. The additive offset in the logarithmic diagram below and above the transition region resembles a substantially increased slope for F in the regime of high σ in a linear plot. Before elucidating the presented experiments in detail, let us first qualitatively depict how the observed fluorescence is expected to change upon increasing the number density of molecules on the surface. In that context, it is necessary to consider the consequences of nonradiative energy transfer from the excited dye molecule to the metal surface, since this process brings about a dependence of the dye’s fluorescence quantum yield on its distance to the Au surface. As a result, the fluorescence emitted from the dye attached to the top end of a DNA strand, tethered to the metal surface at its opposing end, will increase as its distance to the metal increases. Provided that the DNA strands are homogeneously distributed on the surface,12 with their “sticky” thiol ends tethered to Au, we anticipate three distinct regimes in the behavior of the observed (12) In electrolyte solution containing monovalent salt, the DNA’s intrinsic negative charge is not fully compensated by surrounding ions. Therefore electrostatic repulsion between adjacent strands on the surface is expected to prevent cluster formation.

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Figure 1. Fluorescence intensity of Cy3-labeled singlestranded oligonucleotides tethered to a Au surface vs the number density of surface coverage (σ). Solid circles are 12mer and open squares 24mer DNA. Solid lines are calculated according to the model presented in the text. The inset illustrates three different regimes of surface coverage as discussed in the text: before the onset of steric interactions (region I), at the onset of steric interactions (region II), and at high packing densities at which movement of the strands is considerably restricted (region III). Error bars are evaluated from measuring σ on four equally prepared surfaces.

fluorescence intensity as the DNA number density on the surface varies (cf. inset of Figure 1): (I) If the average spacing between two neighboring DNA strands (2r) is larger than 2 times the DNA length (2l), that is, at very low σ, no steric interactions occur between strands on the surface. The average distance dye-metal (zavg) and hence the dye’s quantum yield are minimal. Increasing the number of dye-labeled molecules on the surface within this regime results in a linear increase of the observed fluorescence intensity, since zavg and hence the quantum yield remain constant. (II) For higher molecule densities on the surface, at which the DNA-DNA spacing becomes smaller than 2l, collisions will arise between the ends of neighboring oligonucleotides. Considering the rigidity of short oligonucleotides, these steric interactions induce a restriction to the possible strand orientations on the surface, defining a minimal accessible angle of the DNA’s top end to the surface (θmin), cf. Figure 2. Figuratively speaking, the DNA strands are forced to “stand up” on the surface, thereby increasing zavg (a mechanism also known from the physics of polymer brushes13). As a consequence, an enhancement in the fluorescent dye’s quantum yield superimposes on the linear dependence of the observed fluorescence with σ. The quantum yield shows a pronounced dependence on the coverage density in that regime, since zavg is sensitive to increasing DNA coverage. (III) As σ becomes larger and approaches its theoretical limit of a densely packed monolayer of “standing” DNA strands, the considerations of region II remain in principle valid; however, we may adapt a simplified view: if the orientation of the oligonucleotides on the surface is relatively upright already, further growth of σ will not result in any substantial increase in zavg, so the dependence of the observed fluorescence on the DNA coverage becomes proportional to σ again. In contrast to region I, though, the (approximately constant) fluorescence quantum yield (13) Netz, R. R.; Andelman, D. Phys. Rep. 2003, 380, 1.

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Figure 2. Schematic of DNA strands (represented as rigid rods of length l) randomly gyrating around their hexagonally packed anchoring points on a Au surface covered by MCH. Steric interactions among neighboring strands impose restrictions to the range of accessible angles between the strands and the surface. Averaged over time, the distance from the top strand end to the surface is zavg. For clarity, only three strands are depicted.

of the dye will be enhanced, since zavg is larger and corresponds roughly to the DNA length l. Provided with these qualitative concepts, we return to the discussion of Figure 1, still focusing on the experimental data obtained for 12mer ssDNA. In accordance with the discussion of the previous paragraphs, we observe a distinct increase of the fluorescence intensity at coverage values of ca. 1016 molecules/m2, which we attribute to the onset of steric interactions. Generally, the trends below and above the transition region are found to be linear to a good approximation. Note that the different slopes (appearing as a distinct offset in the logarithmic plot) for regions below and above the onset of steric interactions (corresponding to region II) can also be explained by the arguments of cases I and III; moreover, this behavior denotes the different structural properties of the layer at low and high DNA coverage. A comparison of the 24mer to the 12mer oligonucleotides for σ > 1016/m2 shows enhanced fluorescence emitted from dyes attached to the longer 24mer oligonucleotides, which is in agreement with the arguments of region III in the last paragraph, that is, an increased quantum yield for dyes at larger distances from the metal surface. In addition, the parallel trends of the 12- and 24mer oligonucleotides in region III denote the same functional dependence of the emitted fluorescence on the surface coverage in that regime. Furthermore, it is instructive to relate the onset of steric interactions (osi) of neighboring DNA molecules on the surface to their length: assuming that the DNA strands form a 2D hexagonally packed monolayer while the individual strands gyrate freely around their tethering points, one can easily calculate that the observed onset for 12mer DNA, σosi,12 ∼ 8.7 × 1015/m2, corresponds to a DNA length of 58 Å, that is, an internucleotide spacing of 4.8 Å. This value interpolates to the internucleotide spacings reported for stacked (poly(dA), 3.2 Å)14 and unstacked (poly(dT), 5.2 Å)15 polynucleotides, preferably tending to the latter. Recall that the used DNA comprises diverse nucleotides for which an unstacked conformation would be expected. Due to the increased length of 24mer ssDNA, the presented data in Figure 1 do not cover the range of surface densities below the onset of steric interactions (region I), even if an internucleotide spacing as short as the value of double-stranded (ds) DNA (3.4 Å) is considered. (14) Stannard, B. S.; Felsenfeld, G. Biopolymers 1975, 14, 299. (15) Mills, J. B.; Vacano, E.; Hagerman, P. J. J. Mol. Biol. 1999, 285, 245.

Oligonucleotide Monolayers on Gold

We note that for both 12mer and 24mer DNA layers the fluorescence intensities of the samples featuring the highest surface coverage turn out to be smaller than expected. However, these samples were prepared differently from all the others as MgCl2 was added to the buffer solution in order to achieve the assembly of densely packed molecule layers. Divalent ions (Mg2+) can mediate attractive interactions between DNA strands which facilitate dense packing of the DNA strands and at the same time might lead to the formation of clusters on the surface. In that case, the assumption of homogeneously distributed adsorption sites made in the introduction of the model is not valid. Additionally, quenching effects of the dye label can come into play; although the fluorescence of the used Cy3 dye is not quenched by nucleotide bases,16 it is known to exhibit self-quenching by formation of nonfluorescent aggregates at high degrees of labeling,17 which could apply to monolayers of high packing densities and, particularly, to patches of clustered molecules. Following the preceding qualitative discussion, a simple model will be developed in the next sections involving energy transfer theory in conjunction with considerations regarding the conformation of the DNA layer. It is intended to represent a most straightforward approach, to be compared deliberately with experimental data. However, despite its simplicity and few adjustable parameters it provides a remarkably quantitative description of the effects observed for molecule surface densities above the onset of steric interactions. Tarlov and co-workers1,8 showed that the coadsorption of MCH onto Au surfaces featuring immobilized ssDNAs has a profound effect on the conformation of the DNA layer: by forming a dense sublayer, MCH causes the remaining oligonucleotides to be bound to the surface solely at their sticky thiol end and prevents further contact of the strands (and hence unspecific binding) to the Au surface. According to this, and by considering the mechanical rigidity of short oligonucleotides, we treat the DNA strands as rigid rods, tethered to the surface at one end while the opposing end is extending into solution. In addition, we assume that the strands are gyrating freely around their tethering points;18 moreover, we expect this rotational movement to be highly dynamic, driven by Brownian motion (a crude estimation neglecting friction effects illustrates the fast dynamics of this rotation: equating the thermal energy at room temperature with the rotational energy of a rod of the mass and length of a 12mer single strand yields less than a nanosecond for the time period of revolution). Averaging over time, a particular strand thus spatially requires the volume of a cone with a spherical cap, as depicted in Figure 2. The cone boundaries, that is, the lateral radius of free gyration r, are defined by collisions between the top ends of neighboring rods, which impose steric interactions on the layer conformation. For simplicity, we assume a hexagonal close packing of the tethering points on the surface and hence find a relation between r and the absolute number density of molecules on the surface, σ: r ) (2x3σ)-1/2. Furthermore, the minimal accessible angle between the DNA rods and the surface, θmin, adjusts according to r, as given by cos θmin ) r/l (l being the rod length). Resuming the argument of thermal agitation, one can conclude that (16) Torimura, M.; Kurata, S.; Yamada, K.; Yokomaku, T.; Kamagata, Y.; Kanagawa, T.; Kurane, R. Anal. Sci. 2001, 17, 155. (17) Berlier, J. E.; Rothe, A.; Buller, G.; Bradford, J.; Gray, D. R.; Filanoski, B. J.; Telford, W. G.; Yue, S.; Liu, J.; Cheung, C.-Y.; Chang, W.; Hirsch, J. D.; Beechem, J. M.; Haugland, R. P.; Haugland, R. P. J. Histochem. Cytochem. 2003, 51 (12), 1699. (18) Note that the length of the thiol linker that tethers the DNA to the Au surface features the same length as MCH.

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the time-averaged angle θ between the strands and the surface will be the mean value of θmin and θmax ) 90°. Therefore, θ can range from 45° (isolated strands) to 90° (close packing), as the strands are forced to stand up with increasing surface coverage. Let us now quantitatively address the dependence of the observable fluorescence on the surface coverage and the orientation of the strands on the surface. The measurable fluorescence intensity, F, is emitted by dyes attached to the top DNA end and can be written as

F ) cσ QY

(1)

where QY is the dye’s fluorescence quantum yield (depending on the distance of the dye to the Au surface) and c is a constant comprising measurement parameters such as the detection area, factors related to the optical geometry, and instrument sensitivity. The fluorescence quantum yield19 is defined by the fraction of fluorophores that, upon excitation by absorption of incident photons, relax to the ground state through emission of photons. It adjusts according to the competing processes of radiative and nonradiative transitions, characterized by their respective lifetimes, τr and τnr. While for isolated molecules the lifetimes are determined by intrinsic molecular properties, energy transfer from the excited fluorophore to acceptors in its vicinity can open up a highly efficient channel for nonradiative relaxation, marked by a considerably diminished τnr. In the case of τnr , τr, the quantum yield is primarily governed by the nonradiative lifetime and can be approximated as QY(τnr) ≈ τnr/τr. To calculate τnr for a fluorophore in proximity to a metal surface (kz , 1, k is the magnitude of the wave vector at the emitted frequency ω), we employ an expression20 derived by Persson and co-workers,7,21 which relates τnr to the fluorescence lifetime at infinite distance from the metal, τ∞, and the fluorophore’s distance to the metal surface, z:

(

τnr-1(z) ≈ τ∞-1 1 + β)

(

)

m -  1 η 2 Im 3 m + 1 8k

γ)

)

β γ + z3 z4

(

(2)

)

ωF η 1 ω 6ξ + 18 3 k ω ωp 8k F p (3)

where η is an orientational parameter (η ) 3/4 for parallel dipole orientation with respect to the surface plane), and 1 and m are the (complex) dielectric constants of the medium in which the dipole is embedded and the metal, respectively. The remaining constants are electron gas parameters of the metal; kF is the Fermi wave vector, ωF is the Fermi frequency, ωp is the plasma frequency, and ξ ≈ 1 and depends on the electron gas density.7 Numerical values used in the calculations are kF ) 1.21 × 1010 m-1, ωF ) 8.40 × 1015 rad/s,22 ωp ) 1.36 × 1016 rad/s,23 1 ) 1.15, and m ) -5.3 + 1.54i.24 k ) 1.112 × 107 m-1 and ω ) 3.334 × 1015 rad/s were calculated from the peak emission wavelength of Cy3 (565 nm). This yields β ) 2.465 × 10-23 m3 and γ ) 2.365 × 10-32 m4 for the coefficients of the (19) Lackowicz, J. R. Principles of Fluorescence Spectroscopy, 2nd ed.; Kluwer Academic/Plenum: New York, 1999. (20) Cnossen, G.; Drabe, K. E.; Wiersma, D. A. J. Chem. Phys. 1992, 98 (7), 5276. (21) Avouris, P.; Persson, B. N. J. J. Phys. Chem. 1984, 88, 837. (22) Ashcroft, N. W.; Mermin, N. D. Solid State Physics; Brooks/ Cole: Monterey, CA, 1976. (23) Innes, R. A.; Sambles, J. R. J. Phys. F 1987, 17, 277. (24) American Institute of Physics Handbook, 3rd ed.; McGraw-Hill: New York, 1972.

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distance-dependent terms in eq 2. Knowledge of absolute values for the time constants τ∞ and τr is not compulsory, since eq 1 can be rewritten as eq 1′: F ) c′σ QY′, in which the time constants are extracted from the term denoting the quantum yield in eq 1 and are merged with c to give QY′ and c′, respectively. Therewith, they contribute to the fitting parameter c′, without giving rise to additional parameters. Finally, we combine the proposed monolayer model with the theory of fluorescence quenching as follows: the experimentally determined number density of molecules σ is used to sequentially calculate the free radius of gyration, the minimum angle of strands to the surface, the resulting time-averaged angle, and ultimately the average distance of the dye at the DNA top end to the surface, zavg, which includes a constant offset due to the “spacer” MCH layer (assumed to have a thickness of 1 nm). Note that zavg is a function of σ containing only a single parameter, that is, the rod (DNA) length l. Subsequently, zavg is inserted into eq 2 which accounts for fluorescence quenching by energy transfer, to eventually calculate the observable fluorescence intensity F using eq 1′. We point out that the expression derived for F(σ) contains merely two free parameters, with the factor c′ remaining constant for given measurement conditions. Consequently, monolayer properties are described by solely one adjustable parameter, namely, the rod length, which simplifies the discussion of experimental observations within the model and evinces clearly when more complex effects begin to dominate the layer properties. Solid lines in Figure 1 were generated by fitting the described model to the observed fluorescence of 12mer and 24mer single-stranded oligonucleotide monolayers, respectively. Numerical evaluation was conducted simultaneously for both data sets, optimizing the parameters on the condition of finding a common c′ (according to the arguments of above) and individual rod lengths for the different DNA samples.25 The obtained rod lengths are l12 ) 58 Å and l24 ) 78 Å for 12- and 24mer ssDNA, respectively. We find fairly good agreement between theory and experiment, especially in the range of surface coverage values between 1016 and 6 × 1016 m-2. The approximately linear increase at high σ for both oligonucleotide monolayers features a larger slope for the 24mer DNA (marked by a positive offset in the logarithmic plot). In the case of 12mer ssDNA, the rod length obtained from analyzing the data using the described model matches well the value of the DNA length estimated from the steep rise in fluorescence at σosi,12 ∼ 8.7 × 1015 m-2 (interpreted as the onset of steric interactions). According to the expectation, the fit line foretells a bend in this region; however, the calculation underestimates the steepness of the rise. It is important to bear in mind that the assumption of a 2D hexagonal packing of the DNA tethering points, as stated in the mathematical formulation above, may be plausible for moderate to high packing densities, but it remains unclear if the comparably weak interactions at the onset of steric collisions (region II) can impose close-packing conditions to the monolayer already or if the increase of σ in this region is simultaneously accompanied by a reordering of the layer. Given a certain number density of molecules on the surface (σ ∼ σosi,12), a departure from a regular 2D hexagonal packing would result in a decreased average distance between neighboring strands,

more effective steric interactions, and hence an earlier and steeper onset in the observed fluorescence. Evaluation of F using the equations described above can only be performed for values of σ above the onset of steric interactions, that is, regions II and III. No strand to strand interaction takes place at lower surface coverage (region I); hence F should be linearly dependent on σ, featuring a diminished slope compared to F(σ > σosi,12). This anticipated trend is depicted in Figure 1 by the dotted extrapolation of the solid fit line calculated for ss12mer DNA. In contrast to their shorter counterparts, the 24mer oligonucleotides exhibit an apparent length (78 Å) which is considerably smaller than their expected molecular length (∼115 Å). We attribute this to a bending of the flexible strands (the persistence length of ssDNA is reported to be in the range of approximately 1-4 nm),26 which reduces the effective end to end distance. Obviously, the rigid-rod concept starts to forfeit its justification for single strands of 24 or more nucleotides. An oversimplified explanation of the undersized apparent length by a reduced internucleotide spacing (equivalent to 3.3 Å) can be excluded, since the discrepancy of the fit line with respect to the data at low σ points to a more complex steric behavior of longer single strands, possibly due to an effect of surface coverage and mutual strand interaction on the bending properties of the individual strand. In the discussion so far, the diameter of the considered rods has been neglected implicitly, that is, the rods were assumed to be infinitely thin. However, we also derived expressions for steric interactions of rods featuring a finite thickness, not only to account for the diametral dimensions of the oligonucleotides but also in view of possible implications of the counterion clouds which are associated with the charged DNA backbones. Concerning the quality of the obtained fits, for diameters up to ∼10 Å (corresponding to the molecular diameter) the refined model is equivalent to the basic model, whereas for choices of larger diameters the agreement to experimental data worsens. Since the employment of the more refined treatment does not lead to any improvement when describing experimental results, we refrain from elucidating it in detail here; yet it is worth mentioning that its shortcoming for large “effective” diameters indicates that long-range interactions normal to the molecular axis, which could for instance be mediated by counterion cloud correlations, do not seem to have a dominant influence on the observed effects. In the following section, we demonstrate the applicability of the described concepts to the study of mechanisms involved in the assembly of heterogeneous monolayers by means of fluorescence investigations. Remarkably, the employed technique allows monitoring of processes at the surface in situ and in real time. As described in the materials section, the coadsorption of MCH onto DNAmodified Au surfaces has been utilized regularly during the standard sample preparation. For that reason, we elucidate this process in further detail in view of gaining complementary information on the layer properties and underlining the usefulness of this procedure. Figure 3 depicts the course of fluorescence intensity versus time upon injection of MCH (t ) 0) into the buffer solution covering a DNA layer (ss12mer) on Au. We observe a rapid increase in fluorescence, followed by a decrease and subsequent relaxation to a stable level. The steadystate fluorescence intensities observed before and after

(25) Data points with surface coverage of >1017 m-2 were omitted in the fits for reasons of possible CY3 self-quenching.

(26) Kuznetsov, S. V.; Shen, Y.; Benight, A. S.; Ansari, A. Biophys. J. 2001, 81, 2864.

Oligonucleotide Monolayers on Gold

Figure 3. Observed fluorescence intensity of Cy3-ssDNA (12mer) vs time upon initiating a MCH adsorption at t ) 0 (resulting DNA surface coverage, ∼1016 molecules/m2). The prominent peak immediately after admixing MCH indicates desorption of DNA from the Au surface (see the text). Inset: Ratio of the fluorescence measured after and before MCH adsorption vs the number density of ssDNA on the Au surface (σ) determined after MCH adsorption. The solid line is a guide to the eye.

MCH adsorption differ considerably. In accordance with the previous sections, we attribute this fluorescence offset to changes in the conformation of the DNA layer on the Au surface. The peak in fluorescence intensity which is triggered by the MCH injection can be assigned to the release of labeled oligonucleotides from the surface into solution. Elsewhere, we published details about the employed technique to sensitively detect the desorption of labeled oligonucleotides from Au surfaces;5 briefly, upon release from the surface the DNA floats within the volume of optical detection above the surface. Due to the increased distance to the metal, fluorescence quenching is no longer effective and bright fluorescence is emitted from dyes attached to oligonucleotides in that state (e.g., the fluorescence quantum yield increases by more than 2 orders of magnitude when increasing the distance of the fluorophore to the metal from 5 to 100 nm). Diffusion of the released oligonucleotides out of the volume of optical detection causes the observed intensity to decrease again until finally only the fraction of DNAs remaining on the surface are still detected. Given that, it is evident that the MCH treatment initiates two phenomena: desorption of oligonucleotides from the surface and conformational changes in the DNA layer remaining on the surface. By noting that DNA exhibits a complex adsorption behavior on gold surfaces, characterized by a variety of possible interaction mechanisms and binding sites (e.g., binding through bases, electrostatic “image charge” attraction, etc.), we are able to give the following explanation of the effect of coadsorbing MCH, largely in agreement with the work of other groups: 1,27 While MCH assembles to a chemically bound monolayer on Au, linked by its thiol end, it displaces weakly bound DNA strands from the surface and forms a dense sublayer. Thereby, unspecifically adsorbed oligonucleotides are removed from the surface, while strands that were chemically attached to the Au surface by their thiol linker beforehand remain tethered to the Au surface.28 Fur(27) Georgiadis, R.; Peterlinz, K. P.; Peterson, A. W. J. Am. Chem. Soc. 2000, 122, 3166. (28) Repeated exposures to MCH did not result in further desorption of DNA from the surface.

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thermore, the MCH sublayer detaches the backbones of the linked DNA strands from the surface and prevents unspecific interactions to the Au. After that, these DNA strands are able to perform free gyrations around their tethered end and zavg adjusts according to the arguments discussed in the first part of this work. The effect of coadsorbing MCH onto Au surfaces featuring varying DNA surface coverage can now be discussed within the framework of the preceding paragraphs: the inset of Figure 3 depicts the ratio of the steadystate fluorescence intensities (for ss12mer DNA) measured after and before the MCH treatment versus the number density of molecules on the surface (measured after the MCH adsorption). At low values of surface coverage, the data suggest constant values for FDNA-MCH/FDNA whereas the fluorescence ratio rises quickly commencing at 6 × 1015 m-2. After reaching its peak value at ∼1016 m-2, the ratio decreases constantly until it falls even below 1 at ∼1017 m-2, which signifies an effective decrease of fluorescence caused by the adsorption of MCH. This behavior can be understood in terms of the interplay of the two effects mentioned above: desorption of DNA from the surface and changes in the structural conformation of the layer tethered to the surface. At low σ, that is, in the absence of steric interactions, FDNA-MCH/FDNA is expected to be constant as the relative fraction of specifically versus nonspecifically bound oligos is σ-independent in this regime. Then, the constant fraction of released molecules (negative contribution to the observed fluorescence) and the constant fraction of detached but still tethered molecules (positive contribution) lead to a σ-independent FDNA-MCH/FDNA. With the onset of steric interactions, the average orientation of the tethered oligonucleotides after MCH treatment becomes more upright, accompanied by a pronounced increase in fluorescence intensity which dominates over the decrease caused by the desorption of molecules. As the number density of molecules on the surface continues to grow, the contribution from the structural reorientation of the oligonucleotides becomes less important, since surface coverage values significantly higher than 1016 m-2 require a considerable fraction of oligonucleotides to be relatively upright even before MCH adsorption. Hence with increasing surface coverage the desorption of molecules from the surface becomes the prevailing factor in the relative change in fluorescence which results in a decrease of FDNA-MCH/FDNA to values even smaller than unity. Conclusion In summary, we have investigated structural properties of oligonucleotide monolayers tethered to Au surfaces by means of fluorescence methods. We elucidated how energy transfer theory can be utilized to access information about the orientation of the labeled DNA strands relative to the surface. In combination with a basic geometric model, treating the DNA strands as rigid rods gyrating around their tethering point, we presented a quantitative description that relates the fluorescence intensity observed from the oligonucleotide monolayer to the number density of molecules on the surface. In that context, it is essential to consider that thermal agitation brings about a highly dynamical behavior of the individual strands which influences the space that is occupied by a single strand and in turn determines the experimentally accessible timeaveraged angle of the strands relative to the surface. The remarkable agreement of the model which features only a single relevant parameter, namely, the DNA length, to

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the experimental observations emphasizes that (i) the orientation of strands within the monolayer is primarily dominated by mutual steric interactions (collisions) between neighboring strands and (ii) the treatment of single-stranded DNA within the rigid rod approximation is limited to short oligonucleotides, as our results designate a rigid character for the 12mer but yet a significant flexibility for the longer 24mer ssDNA, indicated by a short effective length. However, we point out that due to the greater persistence length of double-stranded DNA, our considerations are expected to provide a sound description of the structural properties and their influence

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on fluorescence measurements for layers comprising double-stranded polynucleotides up to substantial lengths. Acknowledgment. We gratefully acknowledge Mr. Yoshitaka Yamaguchi for preparation of the wafer substrates and Dr. Tsuyoshi Fujihara for fruitful discussions. We also acknowledge financial support by Fujitsu Laboratories Ltd. and by the Deutsche Forschungsgemeinschaft (SFB 563). LA0492963