Stress and DNA Assembly Differences on Cantilevers Gold Coated by

Aug 20, 2009 - Instituto de Microelectr´onica de Madrid, IMM-CNM-CSIC, Isaac Newton 8, ... Changes in the sign of differential surface stress of gold...
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Stress and DNA Assembly Differences on Cantilevers Gold Coated by Resistive and E-Beam Evaporation Techniques M. Arroyo-Hernandez,* J. Tamayo, and J. L. Costa-Kr€amer Instituto de Microelectr onica de Madrid, IMM-CNM-CSIC, Isaac Newton 8, PTM, 28760 Tres Cantos, Madrid, Spain Received February 26, 2009. Revised Manuscript Received July 10, 2009 Changes in the sign of differential surface stress of gold-coated cantilevers produced by thiol-derivatized singlestranded DNA immobilization are observed, depending on the method used to deposit the gold. While the DNA immobilization on e-beam gold-coated cantilevers produces a compressive differential surface stress in the metallic layer, the opposite is observed for resistively coated cantilevers under the same immobilization conditions. The gold films exhibit quite a similar morphology, and the immobilization differences seem to be related to the charge state of the metallic layer surface. This in turn produces a different distribution of the orientation of the DNA strands on the gold layer. A tentative explanation for the observed effect is proposed.

Introduction Genomics and DNA sequencing are emerging fields that require the detection of specific oligonucleotide sequences. The development of DNA arrays and biosensors are based in the detection of the DNA hybridization using different transduction mechanisms. For that purpose, a single-stranded DNA (ss-DNA) that is complementary to the sequence to be detected (target) has to be immobilized on a surface. Most of the immobilization techniques are based on the silanization and the formation of self-assembled monolayers of thiolated molecules on gold. A family of biosensors that is reaching great relevance for DNA biosensing is nanomechanical, as it can detect DNA sequences and single base mismatches with high sensitivity and specificity.1-3 In addition, they have the advantage of allowing labelfree detection, which is time consuming and can modify the original molecular recognition signal. The transduction mechanism is the change in the cantilever resonance frequency (dynamic mode) and/or the vertical deflection (static mode). In this scheme, the cantilevers used as nanomechanical biosensors are similar to those used in atomic force microscopy (AFM), i.e., fabricated with silicon, silicon nitride, or polymers.4,5 For biosensing applications, the cantilever surface requires a prior functionalization, usually made with a gold thin-film coating for selective immobilization of thiol modified DNA. This metallic layer is usually deposited by physical vapor deposition techniques. The most common techniques are two types of thermal evaporation in which the gold is placed in a crucible and heated by either a resistive heating (from now on referred as “resistive evaporation”) or an electron beam (hereby termed “e-beam evaporation”). In both techniques, the deposition rate is controlled by the ingot temperature, and the energy and the deposition rates of the *Corresponding author. E-mail: [email protected].

(1) Fritz, J. Analyst 2008, 133(855), 863. (2) McKendry, R.; Zhang, J.; Arntz, Y.; Strunz, T; Hegner, M; Lang, H. P.; Baller, M. K.; Certa, U; Meyer, E; Guntherodt, H. J.; Gerber, C. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 9783–9788. (3) Goeders, K. M.; Colton, J. S.; Bottomley, L. A. Chem. Rev. 2008, 108, 522– 542. (4) McFarland, A. W.; Poggi, M. A.; Bottomley, L. A.; Colton, J. S. Rev. Sci. Instrum. 2004, 75, 2756–2758. (5) Nordstr€om, M.; Keller, S.; Lillemose, M.; Johansson, A; Dohn, S; Haefliger, D; Blagoi, G; Havsteen-Jakobsen, M; Boisen, A. Sensors 2008, 8, 1595–1612.

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atomic species arriving to the surface corresponds to the thermal energies, and therefore, cannot be adjusted separately. When cantilevers are used as sensors in the static mode, the changes in the deflection are related to the variations of the surface stress. These changes in the surface stress are produced by an asymmetrical molecule adsorption and recognition, happening only on the coated side of the cantilever. Thus, the origin of the surface stress must be understood and correlated to the molecular process taking place. First it has to be considered that a surface is a system where the charge symmetry is broken. The surface stress is understood in terms of charge redistribution in the vicinity of the surface due to the absence of atoms above it, i.e., a different surface atomic coordination. This charge redistribution is also influenced by other factors, such as the surface reconstruction, epitaxial growth, or bulk relaxation of the gold thin-film coating.6 The adsorption of atoms or molecules re-establishes the bonds between the surface atoms and the atomic entities above them and produces a new charge redistribution that modifies the surface stress accordingly.7 Besides the metal charge redistribution, the entities at the surface can interact among themselves and also contribute to the surface stress. As such, the thiolated-DNA immobilization on gold-coated cantilevers and the DNA hybridization produce a differential surface stress. As previously mentioned, the understanding of the origin of the measured stress is necessary to correctly translate it into molecular recognition information. The origin and sign of the surface stress produced by the DNA hybridization is controversial, while the stress produced by DNA immobilization is understood and has always been reported as compressive. To explain the former, different proposals, based on the interactions between the DNA molecules, have been made: (i) electrostatic interaction and entropy configuration changes,8 (ii) steric hindrance interactions,2 (iii) hydration forces,9 and (iv) flexoelectric effects.10 (6) Koch, R. J. Phys.: Condens. Matter 1994, 6, 9519–9550. (7) Haiss, W. Rep. Prog. Phys. 2001, 64, 591–648. (8) Wu, G.; Ji, H.; Hansen, K.; Thundat, T; Datar, R; Cote, R; Hagan, M. F.; Chakraborty, A. K.; Majumdar, A. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 1560– 1564. (9) Hagan, M. F.; Majumdar, A; Chakraborty, A. K. J. Phys.Chem. B 2000, 106, 10163–10173. (10) Liu, F.; Zhang, Y.; Ou-Yang, Z. Biosens. Bioelectron. 2003, 18(655), 660.

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The latter has been explained in terms of the charge transfer from the surface when the Au-S-DNA bond forms11 and the additional repulsion between the DNA molecules.12-14 Nevertheless, a deviation of this standard behavior is presented and investigated in this work. Our experiments show that the gold evaporation technique influences the differential surface stress measured after the DNA immobilization process. In particular, a tensile surface stress has been obtained for cantilevers coated by resistive evaporation. In order to identify the origin of this tensile component of the stress, different gold properties and their influence in the DNA immobilization have been investigated. Our results include a statistical study that suggests that the final stress of the cantilever depends on a delicate balance between different contributions and the balance depends on the coverage and the DNA molecule orientation and distribution.

Experimental Section Arrays of five silicon cantilevers were purchase from Micromasch. The cantilevers are 400 μm in length, 100 μm in width, and 1 μm thick. The top side of the cantilevers was coated using thermal and e-beam evaporators. In both cases, a 2 nm chromium layer was deposited before a 20 nm gold layer to enhance the metal adherence. The cantilevers were mounted on a steel holder and held using tungsten clips. The cantilever and holder were in contact with an electrically grounded bar transfer through the whole deposition process. The evaporation rate was 0.2 A˚/s for both Cr and Au. The base pressure was in the 10-8 mTorr range, and the pressure during deposition was in the 10-7 mTorr range. The target to substrate distance was around 40 cm for both of the evaporation chambers. The cantilever profiles were measured immediately after the gold-coating process. The immobilization procedure was also performed immediately after the profile measurements. The DNA immobilization was performed by the cantilever immersion in 200 μL of a 1 μM thiolated ssDNA (50 -HSCTACCTTTTTTTTCTG-30 ) solution for 24 h at room temperature. Subsequently, they were cleaned by soaking in three subsequent baths: a PBS buffer solution (137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, 2 mM KH2PO4; pH = 7.5) and twice in deionized water. The cantilevers were dried under a nitrogen flux. To study the cantilevers response to the DNA hybridization, the gold-coated cantilevers were sequentially immersed in 200 μL of a 1 μM noncomplementary DNA sequence (50 -AGCTTCCGTACTCGAT-30 ) solution and 200 μL of a 1 μM complementary-DNA (50 -CAGAAAAAAAAGGTAG-30 ) solution for 1 h at room temperature. The cleaning procedure was the same as in the immobilization step. The noncomplementary DNA was used to block the surface against further unspecific interaction of the complementary DNA. Thiolated ssDNA (SH-ssDNA), noncomplementary DNA, and complementary-DNA were purchased from Microsynth after a HPLC purification and a subsequent desiccation. For the solution preparation, DNA samples were diluted in a PBS buffer. The cantilever profiles were measured using a read out technique that combines optical beam deflection with the scanning of a 3 mW laser that is mounted on two perpendicular voice-coil actuators.15 (11) Ibach, H. Surf. Sci. Rep. 1997, 29, 193–263. (12) Godin, M.; Williams, P.; Laroche, O.; Tabard-Cossa, V.; Beaulieu, L.; Lennox, R. B.; Grutter, P. Langmuir 2004, 20, 7090–7096. (13) Tabard-Cossa, V.; Godin, M.; Burgess, I.; Monga, T.; Lennox, R. B.; Grutter, P. Anal. Chem. 2007, 79, 8136–8143. (14) Watari, M; Galbraith, J; Lang, H. P.; Sousa, M; Hegner, M; Gerber, C; Horton, M. A.; McKendry, R. A. J. Am. Chem. Soc. 2007, 129, 601–609.  (15) Mertens, J; Alvarez, M; Tamayo, J. App. Phys. Lett. 2005, 87, 234102.

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The longitudinal and the transversal axes of the cantilevers were scanned allowing the simultaneous measurement of the displacement along the cantilever (profile) and the arrays of cantilevers. The profile was obtained from the displacement of the reflected laser on the photodetector. The stress was calculated by the fitting of the cantilever profile in the position 0-200 nm, using Stoney0 s equation: 1 1 -ν ¼6 ðΔσ t -Δσ b Þ ð1Þ R ET 2 Where R is the curvature radius of the cantilever, (Δσt - Δσb) is the differential surface stress between the opposite cantilever sides (top and bottom), T is the thickness, and E and ν are the Young’s modulus and the Poisson ratio, respectively. All the measurements shown in this work were made under 0% controlled relative humidity (RH). No variation in the sign of the stress was observed depending on the RH, although small variations of its magnitude were found. The relative displacements used to calculate the differential surface stress corresponding to each recognition essay have been calculated by the subtraction of the profile after and before each step. This way, any contribution to the stress different from the DNA immobilization and hybridization is removed. The DNA layer thickness was measured using both the white light reflectance spectroscopy (WLRS) and the atomic force microscopy (AFM) methods. The WLRS methodology resembles swept wavelength interferometry (SWI) but involves, instead of a laser (single wavelength), a VIS-NIR light source and a PC-driven VIS-NIR miniaturized spectrometer instead of a photodetector. The beam from the light source interacts with the sample and produces a reflectance signal in the VIS-NIR spectrum, which is continuously recorded from the spectrometer. Depending on the number of films over the substrate, a number of internal reflections are taking place providing the final signal at each wavelength of the incident light. First, the optical thicknesses of the Cr/Au layers deposited on the Si were measured. The interference spectra were recorded and fitted in the 500-700 nm range. The values obtained using standard refraction indexes of the deposited metallic layers did not separate significantly from the nominally deposited, i.e., 2 nm Cr and 20 nm Au. After the DNA immobilization and assuming optical properties of the DNA close to the PMMA standard lithographic resin (its refractive index is equal to the crystallized DNA one), the optical thickness of the DNA layer was obtained by fitting the recorded spectrum using the Au and Cr thickness values measured before the immobilization. An independent thickness measurement was performed using AFM. The AFM images were recorded in air in both the tapping and the contact modes using a Veeco and a Nanotec microscope. The cantilevers used were purchased from Olympus. The tapping mode measurements were made using Si tips that were aluminum coated with a nominal radius of 7 nm, a spring constant of 1.8 N/m, and a resonance frequency of 70 kHz. The contact mode measurements were made using a gold-coated Si nitride tip with a nominal radius of 15 nm, a spring contact of 0.1 N/m, and a resonance frequency of 21 kHz.

Results and Discussion Figure 1 shows the bending produced by the thiolated ssDNA (SH-ssDNA) immobilization for the two arrays of cantilevers that have been gold coated by an e-beam (solid line) and a resistive evaporation (dotted line), respectively. The cantilevers were immersed in a 1 μM SH-ssDNA solution for 24 h at room temperature. As mentioned above, the data show always the differential profile, i.e., the difference obtained by the subtraction of the profile after and before each step. It can be observed that Langmuir 2009, 25(18), 10633–10638

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Figure 1. Cantilever differential profile due to SH-ssDNA immobilization for the two arrays of cantilevers that have been gold coated on one side by an e-beam (solid line) and a resistive evaporation (dotted line). The inset depicts a scheme of the experiment.

the DNA immobilization on the e-beam gold-coated cantilevers produces a negative differential surface stress that results in a cantilever bending against the gold side. The stress sign could be explained in terms of two different compressive contributions. One is the charge transfer from the surface atoms during the Au-S-DNA bond formation due to the larger S electronegativity. This is what is expected from depleting the negative charge from a metallic layer.7 The other negative contribution is the repulsion between the DNA molecules. This contribution arises from the steric hindrance and the electrostatic interaction and has been reported extensively in the literature.12-14 Somehow surprisingly, the resistively coated cantilevers bend toward the gold side after the DNA immobilization. This behavior points to a tensile stress contribution produced after the DNA immobilization, and that is influenced by some change in the gold thin film related to the deposition technique. The surface stress produced by the cantilever gold coating on one side was compared depending on the evaporation technique. Both of the coatings, the resistive and the e-beam deposited gold films produce cantilevers that bend toward the gold side. Nevertheless, the magnitude is slightly different: (0.3 ( 0.8) and (0.7 ( 0.1) N/m for both the resistive and the e-beam, respectively. Based on previous works,16,17 the first difference to be considered is the morphology of the gold coating. AFM measurements show that the grain size and rms roughness are 38.6 and 1.03 nm, respectively, for the e-beam evaporation and 32.2 and 0.94 nm, respectively, for the resistive evaporation. The insignificant differences observed in the gold coating morphology are not large enough to explain an opposite differential stress due to the immobilization process. However, as the surface stress is related to a charge redistribution, a plausible explanation to be considered is a different surface charge state. Notice that the chemisoption process itself is an example of a charge redistribution that has, as a consequence, a variation of the surface stress.6,7 To test our hypothesis of an influence of the surface charge distribution in the DNA immobilization, two arrays of cantilevers were coated simultaneously by each deposition technique. The cantilevers were electrically grounded during the (16) Weissm€uller, J; Duan, H. Phys. Rev. Lett. 2008, 101, 146103. (17) Mertens, J; Calleja, M; Ramos, D; Taryn, A; Tamayo, J. J. Appl. Phys. 2007, 101, 034904.

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Figure 2. Cantilever differential profile due to SH-ssDNA immobilization for the charged (solid line) and the discharged (dotted line) cantilevers gold coated by (a) e-beam and (b) resistive evaporation. The inset depicts a scheme of the experiment. Charged/ discharged refers to the electrically ungrounded/grounded gold state during the immobilization.

deposition. During the subsequent DNA immobilization process, one of them was electrically grounded (termed “discharged” from now on), while the other was ungrounded (termed “charged” from now on). The results of the experiments are summarized in Figure 2. The e-beam coated cantilevers bend against the gold side independently of the “charge” state, although the stress magnitude decreased somehow after discharging. The resistively coated cantilevers, on the other hand, show an opposite stress sign depending on the charge: a compressive stress if they were connected to the ground during the immobilization process and a tensile stress if they were not. The accuracy and trustworthiness of these (somehow surprising) results are shown in the statistical study, corresponding to a set of 47 charged (Figure 3a) and 19 discharged (Figure 3b) cantilevers resistively gold coated. The average differential surface stress value confirms an opposite sign after the discharge process. In particular, the charged cantilevers bend toward the gold side (average surface stress = 0.3 ( 0.2 N/m; tensile stress), while the discharged ones bend against it (average surface stress = -0.032 ( 0.03 N/m; compressive stress). Note also that the measurement value dispersion is smaller in the discharged gold resistively coated cantilevers. This is consistent with the fact that the charged state is, in principle, not as well-defined as the discharged state. DOI: 10.1021/la900696f

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Figure 4. Evolution of the differential surface stress with the SH-ssDNA coverage for the gold-coated resistively evaporated cantilevers that have been SH-ssDNA immobilized under ungrounded conditions. The surface coverage has been varied by changing the following immobilization conditions: the immobilization time and the SH-ssDNA solution concentration.

Figure 3. Statistics on the differential surface stress due to SH-ssDNA immobilization on the (a) charged and (b) discharged resistively gold coated cantilevers. The Gaussian shape illustrates the average and the dispersion values. Charged/discharged refers to the electrically ungrounded/grounded gold state during the immobilization.

As explained above, the mechanisms involved in the SHssDNA immobilization through the formation of the AuS-ssDNA bonds are known to produce a compressive surface stress. This contribution, together with the repulsion between the DNA molecules, explains in principle the downward bending of the cantilevers. Nevertheless, the tensile stress produced on the charged gold surfaces should have a different origin than the formation of the Au-S-ssDNA bond and the repulsion between the DNA molecules. To find a tentative explanation of the origin of the tensile component, the influence of the charge state in the DNA immobilization process should be considered. The different charge state of the surface influences the electrical double layer in which the electrical potential drops near the gold surface surrounded by the saline buffer solution containing the DNA molecules. This in turn influences both the kinetics of the molecule immobilization, since the DNA molecules are affected by a different interface potential, and the DNA molecule orientation toward the surface, since the backbone DNA is negatively charged. Both effects are intimately related since in the initial stages of the immobilization, when the coverage is low, the DNA molecules are essentially laying down on the surface, while for the (18) Petrovykh, D. Y.; Kimura-Suda, H; Whitman, J; Tarlov, M. J. J. Am. Chem. Soc. 2003, 125, 5219–5226.

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high coverage, the molecules tend to stand up perpendicular to the surface.18 The influence of the molecule coverage and orientation in the tensile component of the surface stress was further studied experimentally for the resistively evaporated cantilevers. First of all, the surface stress was studied when the surface coverage was modified by varying the immobilization conditions. In particular, two approaches were considered: (i) the reduction of the immobilization time (3 h instead of 24 h), which in turn reduces the surface density, and (ii) the increase of the DNA concentration (4 μM SH-ssDNA and 10 μM SH-ssDNA instead of 1 μM SHssDNA for a 24 h immobilization time), which increases the surface density. The results are shown in Figure 4. As expected, for the lower surface concentration (experiment i), the differential surface stress is tensile (1.6 ( 0.2 N/m) with the higher magnitude than that of the longer immobilization time and, thus, the higher surface density (0.3 ( 0.2 N/m). On the other hand, as the surface density increases (experiment ii), the compressive component of the stress becomes larger and, thus, the differential surface stress turns to compressive. In particular, the values obtained are the following: (-0.09 ( 0.05) and (-0.27 ( 0.08) N/m for 4 and 10 μM SHssDNA, respectively. This clearly shows that the higher the surface concentration, the higher the compressive component in the resulting differential surface stress. This last behavior can be explained as the large DNA coverage increases the compressive component due to the larger number of formed Au-S-ssDNA bonds, and the repulsive interactions between the DNA molecules. The fact that the molecules are standing up implies that the Au-DNA chain interaction is significantly reduced, and hence, the possible tensile stress induced by this kind of interaction also decreases. In order to check the proposed relation between the stress sign and the orientation of the DNA molecules as a function of the surface charge, the thickness of the DNA layers formed on both the charged and the discharged gold surfaces coated by resistive evaporation was studied by WLRS and AFM. Figure 5a presents the results corresponding to the DNA layer apparent thickness measured by WLRS. The data show that the DNA layer immobilized on the discharged gold surface is about 2.32 times thicker than that of the layer formed in the charged gold surfaces. This ratio has been obtained by assuming a refractive index Langmuir 2009, 25(18), 10633–10638

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Figure 5. Apparent DNA layer thickness measured by a) WLRS and b) AFM for the SH-ssDNA layers formed on both the charged and the discharged resistively gold-coated cantilevers.

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Figure 6. Statistics on the differential surface stress due to the DNA hybridization on (a) charged and (b) discharged gold-coated cantilevers by thermal evaporation. The Gaussian shape illustrates the average and the dispersion values. Charged/discharged refers to the electrically ungrounded/grounded gold state during the immobilization and the hybridization.

n = 1.46 for λ = 633 nm19 for the DNA immobilized on both the charged and the discharged gold surfaces. This value is the theoretical index for a homogeneous crystallized DNA layer. Nevertheless, a different molecule orientation and coverage would result in the formation of the DNA layers with different optical properties, so the obtained values must be taken as indicative that a qualitative thickness difference does exist. To obtain a quantitative value of the DNA height, noncontact AFM images were also obtained. For that purpose, the DNA was immobilized on a pattern of gold stripes deposited on Si. The step height before and after the DNA immobilization was measured and subtracted to obtain the DNA layer thickness. The results are shown in Figure 5b. The thickness measured for the DNA layers immobilized on the charged gold surfaces is basically zero, so it can be considered as a lying down orientation of the DNA molecules. The height of DNA layers formed on the discharged gold surfaces is 4.2 nm. As the theoretical length of the SHssDNA is 6.7 nm, this would imply a molecule orientation of 39° with respect to the surface. This orientation is consistent with previous works.20 Our thickness measurements then confirm the hypothesis of a relation between the surface charge state and the molecule orientation distribution. A candidate for the mechanism by which the charge state of the resistively evaporated gold coating induces a horizontal

orientation of the DNA molecules is presented in what follows. Once the SH-ssDNA has been immobilized, a further Au-DNA interaction can proceed through the phosphate backbone and/or the bases. In principle, the interaction through the bases could be discarded based on the low adsorption affinity of T bases on gold.21 Remember that the experiments were performed using the DNA sequence: 50 -HS-CTACCTTTTTTTTCTG-30 , mainly formed by T bases. Regarding the interaction through the negatively charged backbone, previous works22 have reported the formation of positively charged clusters during the resistive gold evaporation. This together with the heating mechanism of the e-beam evaporator (electrons accelerated toward the source) suggests that the surface charge of the resistively evaporated gold is more positive than the surface of the e-beam evaporated gold. Moreover, preliminary experiments have been made using a PNA (peptide nucleic acid), which is a nonchiral and an uncharged DNA mimic, with a backbone composed of repeating N-(2aminoethyl)-glycine units linked by peptide bonds, instead of the negatively charged DNA backbone. These results do not show a tensile differential surface stress when immobilized on the charged resistively evaporated gold. For these experiments,

(19) Moiseev, L; Unlu, M. S.; Swan, A. K.; Goldberg, B. B.; Cantor, C. R. Proc. Natl. Acad. Sci. U.S.A. 2006, 108, 2623–2628. (20) Legay, G; Markey, L; Meunier-Prest, R; Finot, E. Ultramicroscopy 2007, 107, 1111–1117.

(21) Kimura-Suda, H; Petrovykh, D. Y.; Tarlov, M. J.; Whitman, L. J. J. Am. Chem. Soc. 2003, 125, 9014–9014. (22) Barnes, M. C.; Jeon, I. D.; Kim, D. Y.; Hwang, N. M. J. Cryst. Growth 2002, 242, 455–462.

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the PNA sequence used was Cys-O-O-ATCCCGCAT.23 It is interesting to note that, apart from the absence of the phosphate backbone, the A, C, and G bases have a larger adsorption affinity on Au than that of T. This result also points to the interaction through the phosphate backbone as a possible explanation of the DNA molecules tendency to orientate horizontally. The results presented confirm that the charge state of the Au surface affects the subsequent immobilization process, making possible the different orientational distributions of the SHssDNA bonded molecules that produce a net tensile stress. This net tensile stress could be only explained by a tensile component that overcomes the well-known compressive ones, i.e., the charge transfer from the surface atoms and the repulsion between the DNA molecules. The tensile contribution to the stress could arise from the interactions between the different kinds of DNA orientations among themselves and the interaction of the phosphate DNA backbone with the metal layer. Anyway, it is important to keep in mind that the stress is related to a different pattern of charge redistribution, not only the net charge transfer. Recent experiments point out along this line: cantilevers respond to the collective in-plane molecular interactions rather than to the individual events.24 Finally, the cantilever response to the DNA hybridization was studied. Figure 6 shows the surface stress associated to the DNA hybridization essay, measured after blocking the surfaces with a noncomplementary DNA sequence. Thus, the differential surface stress has been calculated from the subtraction between the profiles obtained for the complementary and the noncomplementary DNA immersed cantilevers. The data in Figure 6 show that the surface stress due to the hybridization essay is larger for the discharged gold surfaces. This is in agreement with the fact that (23) Briones, C; Mateo-Marti, E; Gomez-Navarro, C; Parro, V; Roman, E; Martı´ n-Gago, J. A. Phys. Rev. Lett. 2004, 93, 208103. (24) Norman, L. L.; Badia, A. J. Am. Chem. Soc. 2009, 131, 2328-2337.

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when the DNA molecules are standing up both the surface coverage reached and the hybridization efficiency are increased. It is interesting also to note that the statistical dispersion is lower for the discharged gold surfaces. This is what was previously observed in the immobilization results, and what could be anticipated, since a discharged surface state is better defined than a charged (floated) one. In conclusion, an opposite sign of the differential surface stress of gold-coated cantilevers after DNA immobilization is reported as a function of the deposition technique. The effect is most probably related to the surface charge state of the deposited gold. The compressive stress (the cantilever bending against the goldcoated side) can be explained in terms of the Au-S-ssDNA bond formation and the repulsion between the DNA molecules. It is proposed that the tensile stress (the cantilever bending toward the gold-coated side) is obtained when the DNA molecules are lying down on the surface. The mechanism proposed to explain this tensile stress is based on the interactions Au-DNA and lying down DNA-standing up DNA. The tensile component becomes dominant when the surface charge state favors a preferential lying down orientation of the DNA molecules. It is interesting to note that, although both evaporation techniques are thermal and, in principle, equivalent from the point of view of the energy of the arriving species, the present results demonstrate that the DNA immobilization and the hybridization efficiency are influenced by the metal deposition technique. Acknowledgment. Financial support from the Spanish Ministry project TEC2006-10316 is gratefully acknowledged. M.A.H. acknowledges a postdoctoral fellowship from the Spanish Juan de la Cierva program. J.L.C.-K. acknowledges the University of Minnesota and the MRSEC program for the use of nanofabrication facilities. The authors acknowledge C.Briones for providing the PNA molecules and J. Mertens, M. Martı´ n-Gonzalez and M. Calleja for their technical support.

Langmuir 2009, 25(18), 10633–10638