Orientation and Ordering in Monomolecular Films of Sulfur-Modified

Sep 28, 2009 - characteristic features of the respective nucleobases and ssDNA backbone ..... characteristic regions of CdO, NH2, CdN, CsC, and phosph...
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J. Phys. Chem. C 2009, 113, 18312–18320

Orientation and Ordering in Monomolecular Films of Sulfur-Modified Homo-oligonucleotides on Gold Nirmalya Ballav,† Patrick Koelsch,‡ and Michael Zharnikov* Angewandte Physikalische Chemie, UniVersita¨t Heidelberg, Im Neuenheimer Feld 253, 69120 Heidelberg, Germany ReceiVed: July 21, 2009; ReVised Manuscript ReceiVed: September 9, 2009

The chemical integrity, packing density, orientation, and ordering in monomolecular films of sulfur-modified single-stranded DNA (ssDNA) on Au{111} were probed by a combination of X-ray photoelectron spectroscopy, angle-resolved near-edge absorption fine structure spectroscopy (at all relevant absorption edges), and infrared reflection absorption spectroscopy. As model systems, short chain (five nucleotides) thymine- and adeninebased homo-oligonucleotides were used. All ssDNA moieties were found to be bound to the substrate by the thiolate anchor, with no physisorbed ssDNA species but some contamination present. The density of the ssDNA moieties in the thymine- and adenine-based films was estimated at ∼4.8 × 1013 and ∼5.9 × 1013 mol/cm-2, respectively. At the same time, the former films exhibited a higher degree of the orientational order as compared to the latter ones. The lack of correlation between the packing density and orientational order is noteworthy and is assumed to be a specific property of sulfur-modified ssDNA films. Such behavior can be related to a special character of inter- and intramolecular interaction in these systems along with the influence of direct interaction between the nucleobases and substrate. 1. Introduction Immobilization of DNA strands on surfaces is an important issue for modern science and technology. The respective molecular assays can have controlled molecular recognition capabilities and serve as key components of biomedical nanoscale devices, such as, e.g., biochips and biosensors. In particular, films comprised of single-stranded DNA (ssDNA) can be used to monitor hybridization events with a target sequence of nucleobases, which is important for medicine, biology, and related fields. Different strategies for the attachment of ssDNA to the target surface can be pursued. In the most straightforward manner, ssDNA strands can be directly immobilized on a suitable surface (see e.g. refs 1-3). However, in this case, a strong interaction of ssDNA moiety with the surface can occur,2 with the basedependent binding strengths,1,4-6 making some parts of this moiety not available for hybridization with the target sequence.2 This problem can be partly avoided by using a segment of the ssDNA strand as a predetermined anchor;3 however, there are limitations regarding both the strength of such an anchor and the packing density of the molecules. An alternative strategy for the immobilization of ssDNA involves molecular selfassembly. On one hand, ssDNA strands can be chemically attached to specially designed self-assembled monolayers (SAM) prepared before on the target substrate and bearing specific docking moieties such as, e.g., amino or carboxylic acid groups.7,8 On the other hand, ssDNA strands can be modified by attachment of a functional group making strong bonding (chemisorption) to the target substrate. A suitable moiety in this * To whom correspondence should be addressed. E-mail: michael.zharnikov@ urz.uni-heidelberg.de. † Present address: Laboratory for Micro and Nanotechnology, Paul Scherrer Institut, 5232 Villigen, Switzerland. ‡ Present address: Institute of Toxicology and Genetics, Forschungszentrum Karlsruhe, Hermann-von-Helmholtz-Platz 1, 76344 EggensteinLeopoldshafen, Germany.

regard is thiol, which is probably the most popular headgroup in molecular self-assembly, making strong bonding to different metal and semiconductor surfaces.9-13 Consequently, different thiol-modified ssDNA and ssDNA-containing disulfides were synthesized and immobilized in a SAM-like fashion on suitable substrates (mostly on gold).2,3,5,14-17 In most studies, homooligonucleotides bearing just one selected nucleobase were used.2,3,15 The respective films are well suited as model systems for ssDNA layers, since they allow the effect of the individual nucleobases on the SAM structure to be untangled. In addition, they enable monitoring the ssDNA chain length effects, which were important for the orientation and ordering of the individual molecules within the films.2 A variety of different experimental techniques have been used to characterize films of thiol-modified ssDNA. Above all, X-ray photoelectron spectroscopy appeared to be quite useful to monitor the integrity and chemical identity of ssDNA films and to evaluate the packing density.2,3,7,14-17 However, with only a few exceptions,17 only the ssDNA matrix was monitored and no information on the SAM-substrate interface was provided. Further, infrared absorption spectroscopy in reflection mode (IRRAS) was applied and turned out to be quite useful for monitoring the presence of different functional groups in the films.2,3,7,14-16 In addition, near-edge X-ray absorption fine structure (NEXAFS) spectroscopy was utilized to monitor the orientation and ordering of the individual molecules within the films, with either the N K-edge2,18 or both C and N K-edges19 being addressed. Whereas the information provided by each of the above experimental techniques is important on its own, an extensive, many-side characterization of ssDNA SAMs requires a combination of these techniques, which are complementary to each other.2,14-16 Herein, we pursued further this approach and used a combination of XPS, IRRAS, and NEXAFS spectroscopy for the characterization of films comprised of two different sulfurmodified homo-oligonucleotides on Au{111}, viz. 5′-sulfur-

10.1021/jp906896w CCC: $40.75  2009 American Chemical Society Published on Web 09/28/2009

Monomolecular Films of S-Modified Homo-oligonucleotides

Figure 1. Chemical structure of the target molecules. The cleavage of the disulfide bond was expected upon adsorption onto the gold surface, resulting in the formation of mixed T5-S/MCH or A5-S/MCH film. The individual carbon, nitrogen, and oxygen atoms in the nucleobases are numbered (the numbering is performed in accordance with an assignment suggested in ref 20).

modified thymine and adenine homo-oligonucleotides with a short aliphatic linker between the homo-oligonucleotide moiety and sulfur atom (see Figure 1). We will abbreviate these systems as T5-S and A5-S below. Note that the films of different sulfurmodified thymine and adenine homo-oligonucleotides have been studied before,2,3,7,14-16 but no direct comparison between the layers formed from short-chain molecules bearing different nucleobases has been performed. In addition, we applied XPS to characterize not only the matrix of the target films but also the film-substrate interface. Further, the NEXAFS characterization was performed not only at the N K-edge as in most of the previous work,2,18 but at the relevant C and O K-edges as well. Moreover, the analysis of the NEXAFS data is additionally strengthened by direct comparison of the T5-S and A5-S spectra to the analogous curves for the respective nucleobases. In the following, we provide a brief description of the experimental procedure and setup. Thereafter, results are presented and discussed in Section 3, followed by a detailed discussion in Section 4. Finally, the results are summarized in Section 5. 2. Experimental Section The target compounds for the fabrication of ssDNA films were asymmetric disulfides shown in Figure 1; these HPLCpurified compounds were purchased from Operon Biotechnologies (Cologne, Germany) and VBC Genomics (Vienna, Austria). Such compounds are preferable as compared to thiols due to the higher stability of the disulfide moiety. The compounds were used as-received in view of previously published results,15 without further purification or preliminary removal of the -S(CH2)6OH (mercaptohexanol: MCH) stem (this moiety is sometimes described as a “protective group”).15 Other solvents and chemicals (see below), including dodecanethiol (DDT) and thymine and adenine in the powder form (stated purity >98%), were purchased from Sigma-Aldrich Chemie GmbH (Germany) and used as received. The gold substrates were prepared by thermal evaporation of 100 nm of gold (99.99% purity) onto polished single-crystal

J. Phys. Chem. C, Vol. 113, No. 42, 2009 18313 silicon (100) wafers (Silicon Sense) that had been precoated with a 5 nm titanium adhesion layer. Such evaporated films are polycrystalline in nature with a grain size of 20-50 nm as observed by atomic force microscopy. They are considered to be standard substrates for thiol- or disulfide-derived SAMs. The grains predominantly exhibit a (111) orientation, which is in particular supported by the observation of the corresponding forward scattering maxima in the angular distributions of the Au 4f photoelectrons and by the characteristic binding energy (BE) shift of the Au 4f surface component.21,22 The substrates were cleaned in warm piranha solution (70% H2SO4/30% H2O2) for 10 min and rinsed thoroughly with HPLC-grade water immediately prior to SAM preparation. The freshly cleaned gold wafers were placed in 2 mL of buffer solution (1 M KH2PO4/1 M K2HPO4/0.2 M NaCl) containing 5 µM concentration of the target molecules and incubated for 24 h at room temperature. After the incubation, all samples were rinsed with flowing HPLC-grade water to remove excess buffer salt and dried under flowing N2. The samples were either characterized immediately after the preparation or, in the case of synchrotron-based experiments, kept for several days in argon-filled containers until the characterization. DDT SAMs, used as a reference for the evaluation of the packing density, were formed by immersion of freshly prepared gold substrates into a 1 mmol solution of DDT in ethanol for 24 h at room temperature. After immersion, the samples were thoroughly rinsed with pure ethanol and blown dry with argon. The thymine and adenine powder films, used as references for the NEXAFS measurements, were pressed into a clean indium foil and thinned with a brush to suppress charging effects. This procedure has been optimized during our previous experiments on amino acids, peptides, and proteins; it results in homogeneous and contamination-free films with no traces of indium in the spectra.20,23-26 The fabricated ssDNA films were characterized by XPS, angle-resolved NEXAFS spectroscopy, and IRRAS. All experiments were performed at room temperature. The XPS and NEXAFS spectroscopy measurements were carried out under UHV conditions at a base pressure better than 1 × 10-9 mbar. The spectra acquisition time was selected in such a way that no noticeable damage by the primary X-rays occurred during the experiments.27-29 Most of the measurements were repeated several times on different samples, with good reproducibility of the results. The XPS measurements were performed in a custom-made UHV chamber, using a Mg KR X-ray source and a LHS 11 analyzer. The spectra acquisition was carried out in normal emission geometry with an energy resolution of ∼0.9 eV. The X-ray source was operated at a power of 260 W and positioned ∼1.5 cm away from the samples. The energy scale was referenced to the Au 4f7/2 peak of alkanethiolate-coated gold at a BE of 84.0 eV.30 For each sample, a wide scan spectrum and the Au 4f, P 2p, S 2p, C 1s, N 1s, and O 1s narrow scan spectra were measured. XPS spectra were fitted by symmetric Voigt functions, using a Shirley-type background. To fit the S 2p3/2,1/2 doublet we used two such peaks with the same full width at half-maximum (fwhm), a standard30 spin-orbit splitting of ∼1.18 eV (verified by fit), and a branching ratio of 2 (S 2p3/2/S 2p1/2). The fits were performed self-consistently; the same fit parameters were used for identical spectral regions. NEXAFS measurements were carried out at the bending magnet beamline HE-SGM of the synchrotron storage ring BESSY II in Berlin, Germany. The spectra acquisition was

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performed at the C, N, and O K-edges in the partial electron yield mode with a retarding voltage of -150, -300, and -350 V, respectively. Linear polarized synchrotron light with a polarization factor of ∼0.82 was used. The energy resolution of the whole setup was estimated to be on the order of 0.3 eV, slightly dependent on the photon energy. To monitor the orientational order in the films, the incidence angle of the light was varied from 90° (E-vector in the surface plane) to 20° (Evector near the surface normal) in steps of 10-20° (the angles are defined with respect to the surface plane).31 This approach is based on the dependence of the cross-section of the resonant photoexcitation process on the orientation of the electric field vector of the synchrotron light with respect to the molecular orbital of interest (so-called linear dichroism in X-ray absorption).31 This effect results in a characteristic dependence of an adsorption resonance intensity on the incidence angle of X-rays as far as there is an orientational order in the probed system. The photon energy (PE) scale was referenced to the pronounced π1* resonance of highly oriented pyrolytic graphite at 285.38 eV.32 The spectra of the T5-S and A5-S films were compared to the respective reference spectra of highly pure native polycrystalline powder films of thymine and adenine. These spectra were almost identical with those published by us previously.20 IRRAS measurements were performed with a dry-air-purged Vertex 70 FTIR spectrometer (Bruker Optics) equipped with a liquid-nitrogen-cooled MCT detector. All spectra were taken with use of p-polarized light incident at a fixed angle of 80° with respect to the surface normal. The spectra were measured at a resolution of 2 cm-1 and are reported in absorbance units A ) -log R/R0, where R is the reflectivity of the substrate with the monolayer and R0 is the reflectivity of the reference. Substrates covered with a perdeuterated hexadecanethiolate SAM were used as a reference. 3. Results The cleavage of the disulfide bond was expected upon the adsorption of the target molecules (see Figure 1), resulting in the formation of mixed T5-S/MCH or A5-S/MCH film. Because of the considerably larger molecular weight and characteristic volume of T5-S and A5-S as compared to MCH, the mixed character of such films is usually disregarded and they are described and considered as T5-S and A5-S layers.15 We will follow this approach below and mostly disregard the presence of MCH in the films. Note that according to the chemical structure of the target compounds, a 50-50% composition of the ssDNA and MCH constituents can be expected. However, this composition can be modified to some extent by the exchange reaction between the individual adsorbates and target molecules in solution, which frequently occurs in the case of asymmetric disulfides.33 Because of the latter process, the exact portion of the MCH moieties in the resulting films is elusive and the 50-50% composition can be considered as a coarse approximation only. Note also that the presence of the MCH moieties in the ssDNA films is believed to be not a negative but rather a positive factor for the film quality. In particular, backfilling ssDNA films with MCH species is frequently used to improve the orientational order and to control the packing density of the ssDNA moieties in the films.17 3.1. XPS. S 2p, N 1s, P 2p, C 1s, and O 1s XPS spectra of the T5-S and A5-S films are presented in Figure 2. The S 2p spectra of both films in Figure 2a exhibit a single doublet at a BE of 162.1-162.2 eV commonly assigned to the thiolates species bound to coinage metal surfaces.22,34,35 This suggests

Figure 2. S 2p (a), N 1s (b), P 2p (c), C 1s (d), and O 1s (e) XPS spectra of the T5-S and A5-S films. The experimental spectra (open circles) are decomposed into the individual contributions (solid lines) and fitted. See the text for details.

that these films have SAM-like character and all SAM constituents are bound to the substrate in the SAM-like fashion, with no physisorbed T5-S, A5-S, or MCH species available. The N 1s, P 2p, C 1s, and O 1s XPS spectra exhibit the characteristic features of the respective nucleobases and ssDNA backbone (sugar and phosphate groups).2,3,7,14-17,36 The N 1s, C 1s, and O 1s spectra are decomposed into the individual contributions based on the spectral shape and literature data for nucleobases36 and ssDNA films2,3,7,14-17 (see below for details). The N 1s XPS spectra are of particular importance since they are exclusively representative of the nucleobases, with the spectral shape characteristic of the unique nitrogen composition of these moieties. The N 1s spectra of the T5-S and A5-S films in Figure 2b correlate very well with the literature data for analogous systems.7 The N1s spectrum of the T5-S film exhibits an intense emission at ∼400.4 eV and a weak shoulder at ∼398.2 eV. The former emission can be assigned to NH groups of intact thymine bases (the nitrogen atoms N2 and N4 in Figure 1),2,3,7,16,36 which, due to the upright molecular orientation, are isolated from the substrate. A shoulder at ∼398.2 eV can be tentatively ascribed to the thymine bases distorted by the direct interaction with the substrate.2,37 The spectrum of the A5S film exhibits a characteristic asymmetric broad peak, which, in

Monomolecular Films of S-Modified Homo-oligonucleotides accordance with literature data,3,7,16 can be decomposed into two emissions at ∼399.0 and ∼400.8 eV, with a larger spectral weight for the former peak (similar spectral shape has been observed for analogous films).7 The assignment of both peaks is not straightforward, which may partly be related to tautomeric effects. Tentatively, the peak at 400.8 eV can be, analogous to the T5-S case, assigned to the nitrogen atoms N6 and N10 (Figure 1), whereas the peak at 399.0 eV can be assigned to the conjugated nitrogen (N2, N4, and N8 in Figure 1).7,16 The N1s spectral envelope for the A5-S film is presumably invariant with respect to adsorption conditions, i.e., unaffected by a possible direct interaction with the gold substrate.3 The P 2p XPS spectra are exclusively representative of the phosphate groups in the ssDNA backbone. The P 2p spectra of T5-S and A5-S films in Figure 2c exhibit a single peak at 133.6 and 133.8 eV, respectively, assigned to the phosphorus atom in the phosphate unity of the ssDNA backbone.14,15 This feature is common to all nucleotides. Due to a comparably small spin-orbit splitting (ca. 0.84 eV),30 only slight asymmetry of this peak is observed (similar to ref 16), and the individual P2p3/2 and P2p1/2 components of the P 2p doublet cannot be resolved. The C 1s and O 1s XPS spectra are representative of both nucleobases and ssDNA backbone, but can also be affected by the MCH species (see above) and contamination. The C 1s spectra of T5-S and A5-S films in Figure 1d exhibit broad features, which, in accordance with literature data,7,14-16,36 can be decomposed into several peaks related to the individual atoms in the nucleobases and ssDNA backbone.14-16,36 The exact assignments of these peaks are regretfully still elusive at the moment, in spite of some attempts of the classification.16 Highquality reference information for the thymine and cytosine nucleobases can be found in ref 36, in particular the C 1s spectrum of thymine could be decomposed into four components at 285.58, 286.73, 288.86, and 289.96 eV assigned to the carbon atoms C6, C5, C1, and C3, respectively (see Figure 1). A shift of the spectral weight from the low BE to the high BE side is observed on going from thymine to cytosine,7,36 following the introduction of the -NH2 group and the conjugated nitrogen atom. The latter moieties are also present in the case of adenine, which explains the observed difference between the C 1s spectra of T5-S and A5-S films. The O1s spectra of T5-S and A5-S films in Figure 1e exhibit similar spectral shapes and can be decomposed into two peaks at 531.1 and 532.8-532.9 eV (in accordance with previous work).14-16 Such a two-peak structure is common to all nucleotides and is not necessarily related to the presence of oxygen atoms in the nucleobases, but stems mostly from the ssDNA backbone.16 The peak at 531.1 eV can be assigned to the bridging oxygen atoms in phosphate groups, the oxygen atom in sugar, and the oxygen atoms in nucleobases.16,30 The peak at 532.8-532.9 eV can be assigned to the nonbridging oxygen atoms in phosphate groups.16,30 In addition, a contribution from residual oxygen-containing contamination at ca. 532.5 eV is possible. Such contamination is frequently removed during the formation of high-quality SAM (so-called self-cleaning effect), but persists to some extent if efficient molecular packing is not possible or limited. Apart from the spectral shape, the intensities of all the characteristic spectral features related to the nucleobases and ssDNA backbone are stronger for the A5-S film as compared to the T5-S one, whereas the intensity of the S 2p doublet exhibits the opposite behavior. This suggests a higher coverage in the case of the A5-S film than for the T5-S one. The lower intensity of the S 2p doublet in the former case is presumably

J. Phys. Chem. C, Vol. 113, No. 42, 2009 18315 related to the stronger attenuation of the respective signal by the denser (as compared to the T5-S film) ssDNA overlayer. Presumably, this effect overcompensates the higher density of the thiolate headgroups in the A5-S film as compared to the T5-S one. The above qualitative considerations were supplemented by quantitative analysis. In contrast to the previous work,14,15 we have exclusively relied on the intensity of the Au 4f signal from the substrate, taking a film of known molecular density as the direct reference; the respective sample was adjusted to the sample holder along with either T5-S or A5-S films and measured at the same conditions. As the reference film we used a DDT SAM on Au{111}, which has a molecular density of 4.63 × 1014 cm-2 (an area per molecule of 21.6 Å2)11 and an effective thickness of ∼15 Å. The effective thicknesses of the T5-S and A5-S films were estimated at 14.5 and 19.0 Å, based on the standard expression for the attenuation of the photoemission signal38 and the attenuation length reported for monomolecular films.39 The effective thickness values for DDT, T5-S, and A5-S films were related to the number of atoms (except for hydrogen) in the SAM constituents, which are 13, 120, and 125, respectively (we took into the account the MCH stem as well). The resulting molecular density in the T5-S and A5-S films was estimated at ∼4.8 and ∼5.9 × 1013 mol/cm-2, respectively. These values are close to analogous values of 4.4 × 1013 mol/cm-2 determined for pure thiol-ssDNA (long chains bearing different bases) adlayers on gold by radiometry40 and 3.7 × 1013 mol/cm-2 obtained for thymine-based long-chain thiol-ssDNA films on gold by the analysis of weighted XPS signal ratios.15 The higher density values in our case can, on one hand, be related to a smaller length of the ssDNA chains since, in contrast to standard SAMs, the molecular packing in the SAMs of thiolated ssDNA does not improve but rather worsen with increasing chain length.2 On the other hand, our analysis does not include any correction for possible contamination, which probably leads to a slight overestimate of the resulting values. In addition, there can be a difference in the exact values of the attenuation length for the reference system and ssDNA films. 3.2. NEXAFS Spectroscopy. C, N, and O K-edge NEXAFS spectra of the T5-S and A5-S films are given in Figures 3-5, respectively, along with the spectra of the respective nucleobases. In panels a, the spectra acquired at the so-called magic angle of X-ray incidence (55°) are presented; such spectra are not affected by any effects related to molecular orientation and give only information on the chemical identity of the investigated samples. In panels b, the difference between the spectra collected at X-ray incidence angles of 90° and 20° is shown; such difference curves are a convenient way to monitor the linear dichroism effects in the X-ray absorption experiments on the investigated films. The spectra in panels a are accompanied by the respective spectra of highly pure native polycrystalline powder films of thymine and adenine, which were used as direct references for the characteristic absorption structure; these spectra were acquired at the same conditions as the spectra of the ssDNA SAMs. They are almost identical with the spectra published by us earlier and analyzed in detail.20 The positions of the most prominent absorption resonances related to thymine and adenine nucleobases are compiled in Table 1. The C K-edge spectrum of the T5-S film in Figure 3a exhibits all four most prominent resonances of thymine nucleobase (see Table 1), labeled as a, b, c, and d. The intensity relation between the resonances a and b for the T5-S film is somewhat distorted in favor of a as compared to the thymine case. This is presumably related to the presence of residual contamination,

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Figure 3. C K-edge NEXAFS spectra of the T5-S and A5-S films acquired at an X-ray incidence angle of 55° (a), along with the difference between the spectra collected at X-ray incidence angles of 90° and 20° (b). The respective spectra of highly pure native polycrystalline powder films of thymine (T) and adenine (A) are shown in panel a as references. These spectra are scaled down to get comparable intensities of the characteristic absorption resonances. Only the PE range close to the absorption edge is shown. The most prominent resonances are marked (see the text and Table 1 for details). The dashed lines in panel b correspond to zero. The difference spectra achieve this line in the far postedge range.

Figure 4. N K-edge NEXAFS spectra of the T5-S and A5-S films acquired at an X-ray incidence angle of 55° (a), along with the difference between the spectra collected at X-ray incidence angles of 90° and 20° (b). The respective spectra of highly pure native polycrystalline powder films of thymine (T) and adenine (A) are shown in panel a as references. These spectra are scaled down to get comparable intensities of the characteristic absorption resonances. Only the PE range close to the absorption edge is shown. The most prominent resonances are marked (see the text and Table 1 for details). The dashed lines in panel b correspond to zero. The difference spectra achieve this line in the far postedge range.

which, among other effects, results in an appearance of a weak π* resonance at 285.0-285.1 eV associated with aromatic carbon and CdC species.34 This feature overlaps with the resonance a and results in a larger spectral weight at the respective PE position. A further difference between the spectra of the T5-S film and thymine is a substantial step-like intensity increase at ca. 287-288 eV in the former case, which is related to the presence of sugar and phosphate components in the ssDNA backbone.18 Finally, an additional resonance e at 288.5 eV is observed in the spectrum of the T5-S film. On one hand, this feature can be associated with the characteristic R* resonance of the alkyl linker connecting the thiol group and ssDNA backnone.31,34,41 On the other hand, it can be assigned to a σ* resonance of the carbon atoms attached to the oxygen in the sugar unit.18,31,42 A superposition of both contributions is also possible. The above discussion is fully applicable to the analysis of the C K-edge NEXAFS spectrum of the A5-S film. Similar to the T5-S case, this spectrum exhibits the most prominent absorption resonances of the respective nucleobase (b and c, see Table 1), the expected step-like intensity increase at ca. 287-288 eV, and an additional resonance e at 288.5 eV. The characteristic resonances of the nucleobase are even more pronounced than in the T5-S case. The weak resonance at 285.0 eV (d) is presumably related to contamination; no such

resonance was observed in the reference spectrum of adenine. Note, however, that this resonance has been observed for adenine by other authors,18 so that its presence or absence may, at least to some extent, be related to solid state effects or exact tautomeric state of adenine. The N K-edge spectra of the T5-S and A5-S films in Figure 4a exhibit the characteristic resonances of the respective nucleobases (b and c for T5-S and a and b for A5-S), which, similar to the C K-edge case, are more pronounced in the case of A5-S. In addition, the spectrum of the T5-S film reveals an additional weak feature at ∼399.0 eV (d), which was observed before for such films and attributed to the thymine bases which are in direct contact with the gold substrate.2 The O K-edge spectrum of the A5-S film in Figure 5 exhibits only the absorption structure (b and c) that is characteristic of the ssDNA backbone (phosphate and sugar) since adenine does not contain oxygen atoms. The spectral shape and the position of the dominant absorption maximum is similar to those of Na2HPO4,18 which assumes that this spectrum is dominated by the contributions from phosphate moieties. The O K-edge spectrum of the T5-S film represents a superposition of the spectrum of thymine, representative of the nucleobase, and the A5-S spectrum, representative of the ssDNA backbone. At the same time, the splitting of the resonance a in two peaks (a and a′) does not occur in the case of the T5-S film in contrast to the spectrum of thymine. A reason for this difference can be

Monomolecular Films of S-Modified Homo-oligonucleotides

J. Phys. Chem. C, Vol. 113, No. 42, 2009 18317 TABLE 1: Positions (eV) and Tentative Assignments of the Individual Peaks in the NEXAFS Spectra of the T5-S and A5-S Films Related to Thymine and Adenine Nucleotide Basesa positions assignments film

a

b

thymine 285.0 C6fLUMO adenine 286.1 C9fLUMO

c

d

285.8 287.8 289.4 C5fLUMO 286.6 287.3 C9fLUMO+1 C1,3,5,7fLUMO positions assignments

film

a

b

c 402.0 N2,4fLUMO+1

399.4 N2,4,6,8,10f LUMO

401.1 N2,4fLUMO 401.3 N2,4,6,8,10f LUMO+2

thymine adenine

positions assignments film

a

a′

thymine

531.2 (OH-C)fLUMO

532.8 (O)C)fLUMO

a See Figure 1 for the atom labels and Figures 3-5 for the peak labels. The assignments are performed in accordance with ref 20 (see this work for details).

Figure 5. O K-edge NEXAFS spectra of the T5-S and A5-S films acquired at an X-ray incidence angle of 55° (a), along with the difference between the spectra collected at X-ray incidence angles of 90° and 20° (b). The respective spectrum of highly pure native polycrystalline powder films of thymine (T) is shown in panel a as reference. This spectrum is scaled down to get comparable intensities of the characteristic absorption resonances. Only the PE range close to the absorption edge is shown. The most prominent resonances are marked (see the text and Table 1 for details). The dashed lines in panel b correspond to zero. The difference spectra achieve this line in the far postedge range.

a superposition of the resonances a and a′ of the nucleobase with the resonance b of the ssDNA backbone; the latter feature is located between the resonances a and a′. In addition, there are controversial literature data regarding the splitting of the a/a′ resonance: whereas it was observed in some cases for differently prepared samples,20,43,44 it was not observed in other cases.18 Probably, solid state effects, i.e., formation of a specific H-bonded configuration involving the oxygen atoms, play a role, depending on the identity of a particular sample containing thymine nucleobase.20 Along with the information on the chemical identity and integrity of the ssDNA films, NEXAFS data provide insight into molecular order and orientation in these systems by monitoring linear dichroism effects in X-ray absorption as mentioned above. The 90-20° difference spectra for the C, N, and O K edges are presented in Figures 3b, 4b, and 5b, respectively. These spectra exhibit negative peaks at the positions of the characteristic absorption resonances, which imply the presence of orientational order in T5-S and A5-S films. Considering that the respective molecular orbitals have π* character, i.e., are oriented perpendicular to the planes of the nucleobases, the negative sign of the difference peaks (i.e., higher absorption intensity at the grazing incidence) suggests that the nucleobases are preferably orientated parallel to the substrate surface. Apart from this qualitative conclusion, a quantitative evaluation of the NEXAFS data is possible,

following the general formalism of ref 31 in the specific form suggested in ref 2. For this purpose, in Figure 6, we have plotted the dependence of the normalized intensity of the most prominent π* resonances in the C and N K-edge NEXAFS spectra of the T5-S and A5-S films on X-ray incidence angle (the intensities were estimated by fitting the respective spectral regions). In the case of the C K-edge, these were a sum of the resonances a and b for T5-S and a sum of the resonances b and c for A5-S. In the case of the N K-edge, these were a sum of the resonances b and c for T5-S and a sum of the resonances a and b for A5-S. The experimental dependences are fitted by the theoretical curves for a vector-type orbital following the formalism of ref 31. The only fitting parameter was the average tilt angle (R) of the relevant molecular orbitals with respect to the surface normal. The resulting R values for the T5-S film are 26° and 22°, based on the C and N K-edge data, respectively. The analogous values for the A5-S film are 30° and 26°, based on the C and N K-edge spectra, respectively. For both T5-S and A5-S films, there is a good correlation between the results obtained on the basis of the C and N K-edge spectra, which underlines the reliability of the derived R values. Further, the resonance intensity dependence for the T5-S film in Figure 6b is very close to the analogous curve for the same film published earlier (only N K-edge has been measured).2 The respective intensity modulation amplitude (Aπ), i.e., the change of the normalized resonance intensity at going from grazing to normal incidence (see Figure 6), is 0.56 in our work (see Figure 6a) and 0.5 in ref 2. Taking into account the data at both absorption edges, we obtain Aπ values of 0.5 (exactly as in ref 2) and 0.35 for T5-S and A5-S films, respectively. The average R values for the T5-S and A5-S films are 24° and 28°. On one hand, one can consider these values as being representative of the orientation of the individual nucleobases in the film, assuming random azimuthal but ordered polar orientation (i.e., a definite value of their tilt with respect to the surface normal) of these moieties. A more sophisticated analysis,2 referring to the specific character of the ssDNA films,

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Figure 6. Dependence of the normalized intensity of the most pronounced absorption resonances at the N (a) and C (b) K-edge for the T5-S (filled circles) and A5-S (open circles) films on the X-ray incidence angle, along with the best theoretical fits (solid lines) according to the formalism of ref31 The plot follows the representation of ref2 to enable a direct comparison. In accordance with this representation, the incidence angles of X-rays are given with respect to the surface normal. Note also that we have measured only one branch of the presented curves (for the positive incidence angles) and symmetrized it around 0° (normal incidence) following the representation of ref 2.

Figure 7. IRRAS spectra of the T5-S and A5-S films in characteristic regions of CdO, NH2, CdN, CsC, and phosphate units (a) and methyl/ methylene (b) vibrational modes. The positions (in cm-1) of the most prominent absorption modes are marked. The dashed lines are guides for the eye.

suggests not only random azimuthal orientation of the individual nucleobases but also a definite distribution of their tilt angles. Approximating this distribution by the Gaussian formula and introducing so-called orientational disorder parameter δ, which is representative of the polar angle distribution width and is equal to zero for perfect ordering (see above), one can evaluate all possible combinations of R and δ and make some conclusions on the limitations of their range.2 We would rather abstain from such an analysis, but refer an attentive reader to ref 2, where the exact equations and respective theoretical curves can be found. Considering that adenine is oxygen-free, we limited our analysis to the C an N K-edge data only. We would like to note, nevertheless, that also the O K-edge spectra exhibit some linear dichroism both for the orbitals characteristic of the nucleobases (a for T5-S) and those representative of the ssDNA backbone (c). The latter difference peak is even more pronounced for the A5-S film as compared to the T5-S one, which can be associated with a better orientational order of the ssDNA backbones (in contrast to the nucleobases). 3.3. IRRAS. IRRAS spectra of the T5-S and A5-S films in characteristic regions of CdO, NH2, CdN, CsC, and phosphate (referred to below for brevity sake as CdO range) and methyl/ methylene vibrational modes are given in panels a and b of Figure 7, respectively; the positions of the most prominent features are marked. The spectra in Figure 7b exhibit C-H stretching modes of the methylene (2854 and 2929 cm-1 for T5-S) and methyl (2965 and 2894 cm-1 for T5-S) groups, characteristic of the aliphatic linker and the methyl moiety in the nucleobases (only for thymine), respectively, with, probably,

some contributions from C-H stretching mode of sugar.7 Alternatively, the low intense feature near 2894 cm-1 can be assigned to either a methine (subunit of the nucleobases) CH stretch or a CH2 Fermi resonance since its position is too high for CH3 (typically ∼2880-2885 cm-1). The presence of the C-H stretching modes of methyl in the spectrum of the A5-S film can be tentatively related to contamination. Accordingly, both absolute and especially relative intensity of these modes is noticeably lower in the case of A5-S as compared to T5-S. The positions of the asymmetric and symmetric methylene stretching bands for both T5-S and A5-S films differ significantly from the typical values for all-trans conformation of the alkyl chain (2920 and 2852 cm-1, respectively)45,46 but are quite close to the values characteristic of a liquid alkane (2928 and 2856 cm-1, respectively).45 This suggests that the aliphatic foots of the T5-S and A5-S chains in the respective films are disordered, so that the order is only present in the ssDNA part of the film (see Section 3.2). The CdO region of the IRRAS spectra is characteristic of the ssDNA part of the T5-S and A5-S moieties. This region has been frequently used for the characterization of substituted and nonsubstituted ssDNA films by IRRAS1-3,7,14 and highresolution energy loss spectroscopy.16 The prominent bands between 1020 and 1160 cm-1 and between 1180 and 1290 cm-1 originate mostly from the stretching vibration modes of phosphate units (PdO, PsOH, and PsOsC stretches).7,14,16 These bands are more intense in the spectra of the A5-S SAM than that of T5-S, which, in agreement with the XPS data, suggests a higher packing density of the ssDNA chains in the former

Monomolecular Films of S-Modified Homo-oligonucleotides films. The absorption band centered at ca. 1459 cm-1 is presumably related to base deformations coupled to vibrations of the sugar-phosphate backbone;7 this band is much stronger for A5-S. The most prominent band at 1704 cm-1 in the spectrum of the T5-S film in Figure 7a is assigned to the CdO stretching vibration of the unperturbed thymine nucleobase.2,3 An additional band between 1550 and 1620 cm-1, appearing as a shoulder of the 1704 cm-1 feature, is frequently assigned to thymine nucleobases perturbed by direct interaction with the substrate.2,3 Note that the presence of such species in the T5-S film was also implied by the XPS and NEXAFS data (see Sections 3.1 and 3.2, respectively). At the same time, an analogous shoulder was observed in the films of thymine-based ss-DNA oligomers covalently attached to aminosilane SAM on Al2O3, and assigned to the ring deformation (C-C) stretch.7 This suggests that the shoulder might contain some contributions from the nonperturbed thymine moieties as well. The IRRAS spectrum of the A5-S film in Figure 7a exhibits a broad band between 1550 and 1770 cm-1 with most prominent peaks at ∼1601, ∼1648, and ∼1695 cm-1. All these three features are characteristic of adenine, even though the latter peak is usually not that pronounced and has a shoulder-like shape.1,3,7 Two former peaks can be assigned to a combination of NH2 bending and CdN stretching modes and to NH2 bending modes, respectively.7 The assignment of the ∼1695 cm-1 peak is not clear at the moment. Interestingly, the relative intensity distribution between the absorption peaks within the 1550-770 cm-1 band seems to depend on the bonding situation. In the case of unperturbed (i.e., upright-oriented) adenine-based ssDNA oligomers, a higher absorbance is observed at higher wavenumbers (∼1648 cm-1),7 whereas for somewhat perturbed (by the interaction with the substrate) adenine-based ssDNA oligomers a higher absorbance is exhibited at lower wavenumbers (∼1601 cm-1).1,3 The 1550-1770 cm-1 band for the A5-S film of this study in Figure 7a seems to represent a superposition of these two cases, which suggests, similar to the T5-S case, that some adenine nucleobases in the A5-S film are perturbed by the direct interaction with the gold substrate. 4. Discussion The available XPS, NEXAFS, and IRRAS data give complementary information on the chemical identity, integrity, structure, and molecular orientations in the T5-S and A5-S films. According to these data, the target asymmetric disulfides comprised of the MCH and either T5-S or A5-S stems build SAM-like mixed T5-S/MCH or A5-S/MCH films on the Au{111} substrate. All the molecules in these films are bonded to the substrate by the thiolate-type anchor. The films do not contain any physisorbed MCH, T5-S, or A5-S species but are somewhat contaminated, which is understandable in view of the fact that self-cleaning of the substrate, occurring upon molecular assembly in densely packed and well-ordered SAMs, does not work to the full extent because of the bulky character of the ssDNA moieties and specific character of self-assembly in the ssDNA films (see below). The density of the T5-S and A5-S moieties in the respective films was estimated at ∼4.8 × 1013 and ∼5.9 × 1013 mol/cm-2, which is close but slightly higher than the analogous values for ssDNA films reported in the literature, viz. 2-3 × 1013 mol/cm-2,7 3.7 × 1013 mol/ cm-2,15 and 4.4 × 1013 mol/cm-2,40 with the latter value being probably most accurate. Possible reasons for the slightly higher values in our case are discussed in Section 3.1. Apart from possible differences in the film quality, our values contain a

J. Phys. Chem. C, Vol. 113, No. 42, 2009 18319 contribution from contamination and should be lowered to some extent after the respective correction. The higher packing density of the T5-S film as compared to the A5-S one is an interesting finding. Apart from the respective numerical values, which are course estimates only, the difference in the packing density follows directly from the comparison of the respective N 1s and P 2p XPS spectra (Figure 2), characteristic exclusively of the nucleobases and ssDNA backbone, respectively, and IRRAS spectra in the region of CdO vibrational modes (Figure 7a). The latter spectra exhibit pronounced bands related to phosphate units, which are more intense in the spectrum of the A5-S film as compared to the T5-S case. The packing densities of the T5-S and A5-S films do not correlate with the orientational order in these systems. According to the C and N K-edge NEXAFS data for these films (see Section 3.2) and in agreement with previous results for the T5-S system,2 there is a preferable alignment of the individual nucleobases in the parallel-to-the-substrate-surface fashion. This alignment is, however, not perfect but accompanied by a definite extent of disordering, which is noticeably higher in the case of the A5-S films as compared to the T5-S ones. The difference is directly seen from the modulation amplitude of the characteristic NEXAFS resonances (Figure 6) and tentatively given by the respective average tilt angles of the nucleobase planes, which were estimated at 24° and 28° (with respect to the substrate surface) for the T5-S and A5-S films, respectively (see the discussion on Section 3.2 for details). The lack of the correlation between the packing density and molecular orientation is unusual for SAM-like systems, in which higher packing density is, as a rule, accompanied by a smaller molecular inclination. There can be several reasons for this behavior in ssDNA films in general and in the films of this study in particular. First of all, whereas self-assembly in standard SAM systems is driven by attractive interaction (van der Waals or electrostatic) between the individual molecular chains,13 immobilization of ssDNA is subject to strong electrostatic repulsion,15 which changes completely the balance of structurebuilding interactions. The major organization factor is then nucleobase stacking,15 which, at the given length of the ssDNA strand, can be different for different nucleobases, and thymine and adenine in particular. Second, ssDNA strands are usually longer and more flexible than standard rod-like molecules, capable of building SAMs, which hamper achieving high orientational order in these systems. Third, whereas standard SAM-constituents either interact weakly with the substrate (aliphatic chain) or are hindered in this regard due to the molecular rigidity (e.g., in the case of oligophenyl chain), flexible ssDNA strands can contact the substrate surface and interact directly with it even in densely packed films. The evidence of such direct interaction was observed both for the T5-S and A5-S films, in the N K-edge NEXAFS spectrum (Figure 4) and CdO range IRRAS spectrum (Figure 7a), respectively. The direct interaction between the nucleobases and metal substrate is relatively strong5 and results in the change of their electronic structure47 as, e.g., evidenced by the N K-edge NEXAFS spectra of the single-stranded DNA on gold.2 The strength of the nucleobase-gold interaction depends on the identity of the nucleobase and is, according to literature, noticeably stronger for adenine than for thymine.1,4,6,47 This difference can influence the immobilization process in the T5-S and the A5-S films of this study5 and, along with the repulsive interaction between the ssDNA strands and nucleobase stacking,

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be responsible for the observed differences in the packing density and orientational order in these films. Note that, due to the bulky character of the ssDNA strands, the aliphatic linkers of the T5-S and A5-S moieties in the respective films are separated from each other far beyond the characteristic, “equilibrium” spacing occurring in SAMs of nonsubstituted alkanethiolates on Au(111).11,13 Therefore, the interaction between these linkers is presumably very weak and does not contribute noticeably to the molecular ordering and to the ordering of the aliphatic linkers themselves in particular. This is presumably the reason for the large percentage of gauche defects in these moieties, as suggested by the IRRAS data. 5. Conclusions Using asymmetric disulfides comprised of either T5-S and MCH or A5-S and MCH stems as target materials we have prepared SAM-like mixed T5-S/MCH and A5-S/MCH films on Au{111} substrate. The chemical integrity, packing density, orientation, and ordering in these films were probed by a combination of XPS, angle-resolved NEXAFS spectroscopy at all relevant absorption edges, and IRRAS in characteristic regions of CdO, NH2, CdN, CsC, phosphate units, and methyl/ methylene vibrational modes. According to the experimental data, all ssDNA moieties in the resulting films are bound to the substrate over the thiolate anchor, with no physisorbed MCH, T5-S, or A5-S species but some contamination available. The density of the ssDNA moieties in the T5-S and A5-S films was estimated at ∼4.8 × 1013 and ∼5.9 × 1013 mol/cm-2, respectively. At the same time, T5-S films exhibited a higher degree of orientational order as compared to the A5-S films, with an average tilt angle of the nucleobase planes with respect to the substrate surface of 24° and 28°, respectively (these values can be considered as representative parameters only). The lack of correlation between the packing density and orientational order can be a specific property of thiolated ssDNA films and be related to a special character of inter- and intramolecular interaction in these systems along with the influence of direct interaction between the nucleobases and substrate. Acknowledgment. We thank M. Grunze for support of this work, R. Schmidt for help in the sample preparation, A. Nefedov and Ch. Wo¨ll (Ruhr-Universita¨t Bochum) for technical cooperation at BESSY II, and BESSY II staff for technical support. This work was supported by the DFG (ZH 63/9-3 and KO 3618/ 1-1). References and Notes (1) Kimura-Suda, H.; Petrovykh, D. Y.; Tarlov, M. J.; Whitman, L. J. J. Am. Chem. Soc. 2003, 125, 9014–9015. (2) Petrovykh, D. Y.; Perez-Dieste, V.; Opdahl, A.; Kimura-Suda, H.; Sullivan, J. M.; Tarlov, M. J.; Himpsel, F. J.; Whitman, L. J. J. Am. Chem. Soc. 2006, 128, 2–3. (3) Opdahl, A.; Petrovykh, D. Y.; Kimura-Suda, H.; Tarlov, M. J.; Whitman, L. J. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 9–14. ¨ stblom, M.; Zhang, H.; Jang, N.-H.; Liedberg, (4) Demers, L. M.; O B.; Mirkin, C. A. J. Am. Chem. Soc. 2002, 124, 11248–11249. (5) Wolf, L. K.; Gao, Y.; Georgiadis, R. M. Langmuir 2004, 20, 3357– 3361. ¨ stblom, M.; Liedberg, B.; Demers, L. M.; Mirkin, C. A. J. Phys. (6) O Chem. B 2005, 109, 15150–15160. (7) Saprigin, A.; Thomas, C.; Dulcey, C.; Patterson, C.; Spector, M. Surf. Interface Anal. 2004, 36, 24–32. (8) Wu, Yi-Te.; Liao, J.-D.; Lin, Je.-I.; Lu, C.-C. Biocojugate Chem. 2007, 18, 1897–1904. (9) Ulman, A. Chem. ReV. 1996, 96, 1533–1554.

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