XPS and AFM Characterization of Oligonucleotides Immobilized on

J. Redondo-Marugan , M.D. Petit-Dominguez , E. Casero , L. Vázquez , T. García , A.M. Parra-Alfambra , E. Lorenzo. Sensors and Actuators B: Chemical 2...
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XPS and AFM Characterization of Oligonucleotides Immobilized on Gold Substrates E. Casero,† M. Darder,† D. J. Dı´az,‡ F. Pariente,† J. A. Martı´n-Gago,§ H. Abrun˜a,‡ and E. Lorenzo*,† Departamento de Quı´mica Analı´tica y Ana´ lisis Instrumental, Facultad de Ciencias, Universidad Auto´ noma de Madrid, Canto Blanco, 28049 Madrid, Spain, Department of Chemistry and Chemical Biology, Baker Laboratory, Cornell University, Ithaca, New York 14853-1301, and Instituto de Ciencia de Materiales de MadridsCSIC, Cantoblanco, 28049 Madrid, Spain Received February 21, 2003. In Final Form: April 25, 2003 The adsorption processes of oligonucleotides onto gold substrates have been investigated in aqueous phosphate buffer solutions using X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM) in conjunction with the quartz crystal microbalance. The hybridization of a thiol-modified, singlestranded oligonucleotide (HS-polyA), anchored to a gold surface via the thiol group, with its complementary sequence (polyT) has been observed and characterized via XPS and AFM techniques, which provide semiquantitative information about the amount of the material deposited and its surface morphology, making possible the monitoring of the hybridization process. The use of these techniques represents a complementary approach to those generally employed, such as spectrophotometry and surface plasmon resonance.

Introduction 1,2

The study of DNA on surfaces is driven both by the inherent interest in understanding different aspects of this molecule and by its importance for medical diagnostics using DNA biosensor or biochip arrays. Compared to amino acids and protein adsorption, much less has been done regarding basic adsorption studies of nucleic acids and DNA, except as related to DNA biochips. The direct adsorption of DNA on surfaces has not been as intensely studied as the adsorption on preadsorbed spacer layers or premodified DNA. This is based, at least in part, on the fact that direct adsorption can give rise to interactions that are too strong for the DNA to retain its full function. For example, it is possible, if not likely, that nonhybridized DNA segments will bind to the surface and thus be unable to bind to the complementary matching segments. The retention of the full recognition function of single-stranded DNA segments is crucial for diagnostic applications such as biosensors or biochips. Thus, there is a great deal of interest in the understanding of the basic aspects involved in the immobilization of single-stranded DNA on solid supports and its subsequent hybridization for many biotechnological applications, particularly for the development of DNA biosensors/biochips with potential use in medical diagnostics and genome sequencing.3 The common principle of most biosensor/biochips is that “detector molecules” are attached/immobilized on a solid surface in such a way that a specific signal is obtained from the sensor when such detector molecules selectively react with the biomolecules they are designed to detect. * To whom correspondence should be addressed.E-mail: [email protected]. † Universidad Auto ´ noma de Madrid. ‡ Cornell University. § Instituto de Ciencia de Materiales de Madrid-CSIC. (1) Fink, H. W.; Scho¨nenberger, C. Nature 1999, 398, 407. (2) Braun, E.; Eichen, Y.; Sivan, U.; Ben-Yoseph, G. Nature 1998, 391, 775. (3) Zhou, X. C.; Huang, L. Q.; Li, S. F. Biosens. Bioelectron. 2001, 16, 85.

That is, the detection relies on the biorecognition between predesigned native or synthetic reagent molecules and unknown sample molecules. In the case of DNA biosensors, a single-stranded DNA segment (i.e. nucleic acid chain, without cross-linking) is attached to the surface so that all groups of the nonhybridized, single strand are exposed and available for binding to the complementary DNA strand. When the sensor is exposed to a DNA sample to be analyzed, the intent is to obtain a specific response when there is perfect matching between the detector molecule and the complementary matching sequence with no response, or a highly attenuated response, being obtained in the case of one or more mismatches. The desire to design increasingly selective and sensitive DNA-based biosensors has stimulated a great deal of interest in the understanding of its basic physicochemical behavior. As a result, particular interest has been paid to the study of the immobilization, characterization, and hybridization of DNA on surfaces. Surface science has played a key role in furthering these studies. For example, X-ray photoelectron spectroscopy (XPS),4 surface plasmon resonance (SPR),5 scanning tunneling microscopy (STM),6 and atomic force microscopy (AFM)7 have been extensively employed for characterizing these processes. However, although these are very powerful techniques, they generally encounter difficulties with monitoring adsorption processes in real time, an aspect which is very important for the immobilization of DNA on surfaces. The quartz crystal microbalance (QCM) technique has been employed as a highly sensitive detector for measuring in-situ changes in mass with nanogram resolution.8 It is an effective technique for monitoring the adsorption process and, in fact, has been employed to study the adsorption kinetics (4) Herne, T. M.; Tarlov, M. J. J. Am. Chem. Soc. 1997, 119, 8916. (5) Yang, M. S.; Yau, H. C. M.; Chan, H. L. Langmuir 1998, 14, 6121. (6) Zhang, Z.; Pang, D.; Zhang R. Bioconjugate Chem. 2002, 13, 104. (7) Casero, E.; Va´zquez, L.; Martı´n-Benito, J.; Morcillo, M. A.; Lorenzo, E.; Pariente, F. Langmuir 2002, 18, 5909. (8) Ward, M. D.; Buttry, D. A. Science 1990, 249, 1000.

10.1021/la034308q CCC: $25.00 © 2003 American Chemical Society Published on Web 07/01/2003

Oligonucleotides Immobilized on Gold Substrates

of thiols and thiol-substituted viologens, dendrimers, and electropolymerization reactions in real time.9a-d As mentioned above, the immobilization of DNA on a transducer is the first step in biosensor design. It can be achieved using different strategies including entrapment, adsorption, and chemical binding. Despite the large body of work devoted to this topic in recent years, there is still a great deal of interest in the development of methods of immobilization that will not alter the nucleic acid structure. Once a single-stranded DNA segment probe is immobilized on a surface, there are important parameters that need to be determined, such as surface coverage and orientation. In particular, imaging of immobilized DNA can provide valuable information and insight about the structure of the molecules. In this sense, AFM has emerged as an especially suitable and attractive technique for the visualization of DNA immobilized on surfaces since it is able to provide morphological information of biological material at the molecular level without damaging the sample surface.10,11 This makes AFM (as well as related techniques) a powerful technique in the study of biological samples. On the other hand, XPS is capable of providing qualitative and quantitative information about the presence of different elements on the surface, and it has been previously used to characterize DNA probes immobilized onto gold substrates.4 In the present paper, we have employed XPS as an effective method for following the hybridization process based on the analysis of XPS spectra from the surface where the DNA was immobilized and hybridized. Since the development of DNA-based biosensors requires efficient immobilization of single-stranded DNA on the sensor surface with optimum coverage and orientation, the main objective of this work was the study of the immobilization and hybridization of a thiol-modified, single-stranded oligonucleotide (HS-polyA) with its complementary sequence (polyT) by means of surface characterization techniques including XPS and AFM. These techniques can provide semiquantitative information about the amount and morphology (structure) of the material deposited on a surface, making possible the monitoring of the hybridization process. This can result in an alternative method to those generally used, such as spectrophotometry12a,b and surface plasmon resonance.13 Moreover, to assess the extent of nonspecific adsorption, we have characterized the immobilization/adsorption process of a single-stranded oligonucleotide (polyA) on gold surfaces using QCM and XPS techniques. Whereas, in the majority of work on immobilized DNA, short (typically less than 50 bases), synthetic oligonucleotides are generally used, we have employed a somewhat longer (relative to the above) 200-base polyA strand. The choice of a longer chain was not arbitrary but rather driven by our interest and desire in using redox-active materials that are sensitive to hybridization events (typically via intercalation) in amperometric DNA biosensors. Such an approach is based on the electrochemical transduction of (9) (a) De Long, H. C.; Buttry, D. A. Langmuir 1992, 8, 2491. (b) De Long, H. C.; Buttry, D. A. Langmuir 1990, 6, 1319. (c) Karpovich, D. S.; Blanchard, G. J. Langmuir 1994, 10, 3315. (d) Frobo¨se, C.; Doblhofer, K. J. Chem. Soc., Faraday Trans. 1995, 13, 1949. (10) Thomson, N. H.; Smith, B. L.; Almqvist, N.; Schmitt, L.; Kashlev, M.; Kool, E. T.; Hansma, P. K. Biophys. J. 1999, 76, 1024. (11) Forbes, J. G.; Jin, A. J.; Wang, K. Langmuir 2001, 17, 3067. (12) (a) Fang, X.; Li, J. J.; Perlette, J.; Tan, W.; Wang, K. Anal. Chem. 2000, 72, 747A. (b) Squirrel, D. J. Measurement of Nucleic Acid Hybridation by Total Internal Reflection Fluorescence. PCT Int. Pat. W09306241, 1993. (13) Peterson, A. W.; Heaton, R. J.; Georgiadis, R. M. Nucleic Acids Res. 2001, 29, 163.

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the hybridization event and combines the high specificity of DNA hybridization reactions with the excellent sensitivity and simplicity of electrochemical transducers. The hybridization is usually detected by the increase in the electrochemical response, associated with the redox active material, arising from its enhanced affinity for the formed surface duplex relative to a single DNA strand. This enhancement is dependent on the length of the DNA employed and generally increases with chain length. For this purpose, a 200 base pair chain was chosen as representing a compromise between enhanced affinity on one hand and kinetic limitations on the other. Experimental Section Materials. Sodium phosphate (Sigma Chemical Co., Saint Louis, MO) was employed for the preparation of buffer solutions. Four different DNA chains were used in this study. PolyA (adenine) was purchased from Sigma (P-9403). Thiolated singlestranded DNA [HS-polyA], purchased from Isogen Bioscience (Maarssen, The Netherlands), was a 200-adenine oligonucleotide with a HS-(CH2)6 group attached at the 5′ end. The complementary and noncomplementary single-stranded DNA [polyT] and [polyC] had 200-thymine and 200-cytosine oligonucleotides, respectively, and were obtained from Isogen. All oligonucleotides were dissolved in 10 mM, pH 7 phosphate buffer. Water was purified with a Millipore Milli-Q-System. All solutions were prepared just prior to use. Experimental Techniques. QCM Measurements. AT-cut quartz crystals (5.0 MHz) of 25 mm diameter with Au electrodes deposited over a Ti adhesion layer (Maxtek Inc., Santa Fe Springs, CA) were used for QCM measurements. An asymmetric keyhole electrode arrangement was used, in which the circular electrode geometrical areas were 1.370 (front side) and 0.317 cm2 (backside). The electrode surfaces were overtone polished. Prior to use, the quartz crystals were cleaned by immersion in piranha solution, H2SO4/H2O2 (3:1 (v/v)). Caution: Piranha solution is extremely reactive! They were subsequently rinsed with water and acetone and dried in air. The quartz crystal resonator was set in a probe (TPS-550, Maxtek) made of Teflon in which the oscillator circuit was included, and the quartz crystal was held vertically. The flow system included a flow cell (FC-550, Maxtek) made from Kynar, which has two stainless steel inlet and outlet tubes with a 0.047 in. i.d. and 0.062 in. o.d. A Viton O-ring provided a seal between the cell and the face of the sensor crystal. The cell was used in place of the crystal retainer ring in the Teflon probe (TPS-550, Maxtek). Once installed in the probe, the cell created a flow chamber of approximately 100 µL, where only one side of the quartz crystal was in contact with the flow solution. The assembly, as well as the buffer (10 mM sodium phosphate + 0.4 M NaCl) and the sample reservoirs were immersed in a waterjacketed beaker thermostated at the assay temperature with a Haake F6 digital temperature controller. The buffer or the sample stream were pumped through 0.8 mm i.d. Tygon tubes at a flow rate of 80 µL min-1 with a Gilson Minipuls 3 peristaltic pump. The frequency of the quartz crystal oscillator was monitored with a plating monitor (PM-740, Maxtek) and simultaneously recorded by a personal computer. XPS Measurements. The XPS spectra were recorded using a double pass cylindrical mirror electron energy analyzer and a twin-cathode X-ray source set to Mg KR excitation (1253 eV). The base pressure in the ultrahigh vacuum chamber during measurements was lower than 10-9 mbar. The gold surfaces used for DNA immobilization consisted of glass substrates (11 × 11 mm) covered with a chromium layer (1-4 nm thick) onto which a gold layer (200-300 nm thick) was deposited (Metallhandel Schro¨er GmbH, Lienen, Germany). The spectra, presented as raw data, were corrected for emission from satellite radiation. AFM Measurements. Contact-mode atomic force microscopy images were obtained using a Digital Instruments nanoscope E controller with a Molecular Imaging 8 µm scanner and inside a Molecular Imaging isolation chamber. A Au(111) disk electrode (grown from the melt, cut, and polished at the Materials Preparation Facility of the Cornell Center for Materials Research, CCMR) was mounted on the cell using a custom (homemade)

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Teflon mount. All images shown are unfiltered, and no off-line zoom was used. Values for the detector set points used were kept low (in the range of 0.0-1.0 V) to avoid excessive pressure by the tip against the film. Scan rates between 1 and 5 Hz at 512samples resolution were used without further modification. Procedures. QCM Experiments. The immobilization of polyA by direct adsorption onto the QCM electrodes was carried out by immersing a freshly cleaned QCM-Au resonator in a thermostated bath. Prior to adsorption, the thermostated buffer solution (10 mM phosphate buffer + NaCl 0.4 M at different pH values) was pumped through the system and the frequency was monitored as a function of time. When the frequency had reached a constant value, a 100 µg/mL polyA solution was pumped through the flow cell in place of the buffer solution. The immobilization process was followed by the decrease in frequency until equilibrium was reached. XPS Experiments. For XPS measurements the adsorption was carried out by immersing freshly cleaned gold substrates into a solution of HS-polyA (5.4 µg/mL in 0.1 M phosphate buffer at pH 7.0). Prior to analysis or hybridization, the samples were rinsed thoroughly with deionized water to remove any loosely bound material. The samples corresponding to the hybridization experiments were prepared by immersing the HS-polyA/Au substrates in a solution of polyT (5.8 µg/mL in 0.1 M phosphate buffer at pH 7.0) or polyC (10 µg/mL in 0.1 M phosphate buffer at pH 7.0), respectively. Hybridization was performed for 4 h at 39 °C. AFM Experiments. Prior to modification, the Au(111) disk electrode was flame annealed for 5 min. and quenched in MilliPore water (which had been previously degassed for 15 min. with prepurified nitrogen). The freshly annealed electrode was immersed in a HS-polyA solution (5.4 µg/mL in 0.1 M phosphate buffer at pH 7.0) for 4 h, rinsed with buffer solution, and mounted on the microscope cell for analysis. The sample corresponding to the hybridization experiment was prepared by immersing the HS-polyA/Au substrate in a solution of polyT (5.8 µg/mL in 0.1 M phosphate buffer at pH 7.0). Hybridization was performed for 4 h at 39 °C.

Results and Discussion Immobilization of HS-PolyA on Gold Surfaces. Even though the direct adsorption of polyA on gold surfaces is a very simple method of immobilization which gives rise to a strongly adsorbed layer,4 it is quite likely that some of the immobilized nucleotides will be unavailable for hybridization.14 In an attempt to avoid this problem, we have used a different immobilization methodology based on chemisorption, which involves the modification of the polyA chain with a HS-(CH2)6 group attached at the 5′ end. In principle, two general mechanisms can contribute to the immobilization of HS-polyA on gold surfaces: a chemisorption process involving the thiolate group and nonspecific adsorption through the nitrogencontaining nucleotide side chains. Since nonspecific adsorption is generally a pH-dependent process, we investigated the influence of pH on the adsorption of polyA in order to establish the conditions under which the nonspecific contributions were minimized. Four different pH values (1.9, 3.5, 7.0, and 8.5) were employed in order to encompass a wide pH range and specifically to straddle the pKa of adenoside, which has been reported to be 3.52.15 At pH values above (below) the pKa, the poyA will be negatively charged (neutral). These studies were carried out using the QCM technique, which allows the measurement of mass changes at surfaces through changes in the resonant frequency of the quartz crystal resonator. These measurements allow (14) Henke, L.; Piunno, P. A. E.; MCClure, A. C.; Krull U. J. Anal. Chim. Acta 1997, 344, 201. (15) Fasman, G. D., Ed. Handbook of Biochemistry and Molecular Biology, Vol. 2, Nucleic Acids; Chemical Rubber Co.: Cleveland, OH, 1976; p 76 206.

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Figure 1. Time dependence of the frequency changes of a quartz crystal resonator associated with the immobilization process of polyA in a 0.1 M/0.4 M phosphate buffer/NaCl at four different pH values. The solid lines represent data fitted to a first-order kinetic equation. Table 1. Surface Coverage (Γ) and Frequency Changes (∆F) for a Quartz Crystal Resonator in Contact with a Solution Containing 100 µg/mL of PolyA in 0.1 M/0.4 M Phosphate Buffer/NaCl at Different pH Values pH ∆Fmax (Hz) Γ (mol cm-2) 1.9 3.5

-2 -54.4

2.8 × 10-12

pH ∆Fmax (Hz) Γ (mol cm-2) 7.0 8.5

-3.2 -3.1

1.5 × 10-13 1.4 × 10-13

one to obtain kinetic information about the adsorption process as well as the surface coverage. Figure 1 shows the resulting frequency changes associated with the immobilization process of polyA on the gold surface in a phosphate buffer solution at the four different pH values (pH ) 1.9, 3.5, 7.0, and 8.5) selected. For all pH values studied, upon the addition of polyA, the frequency decreased gradually during the first 10-20 min and then a steady state was reached. Assuming that the decrease in frequency was due only to changes in mass arising from the adsorption of DNA, one can calculate the amount of material adsorbed through the Sauerbrey equation (eq 1), where ∆m is the mass change (ng cm-2) and Cf (17.7 ng Hz-1 cm-2) is a proportionality constant for the 5.0 MHz crystals used in this study.

∆m ) -Cf∆F

(1)

The frequency decreases (∆F) and the surface coverage (Γ) values obtained for the immobilization of polyA at different pH values are summarized in Table 1. Γ values were calculated assuming a molecular mass of 380 000 Da. From the data presented in Table 1, one can conclude that the amount of polyA immobilized on a gold surface is strongly pH-dependent with the highest coverage being obtained at values around pH 3.5. However, at values above and below pH 3.5, the amount of immobilized DNA was greatly reduced. It is evident that polyA is adsorbed through the nitrogen-containing nucleotide side chains. Therefore, the relative charges of adenine and gold play an important role in the adsorption process; both being strongly dependent on pH. As mentioned earlier, adenosine has been reported to have pKa 3.52 15 and the ζ-potential of the gold surface at this pH value is negative.16 Therefore, at pH 3.5 the polyA chain will be partially protonated, whereas the gold surface will be negatively (16) Weiser, H. B.; Merrifeld, P. J. Phys. Chem. 1950, 54, 990.

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Figure 2. XPS spectra of the N(1s) peak for a gold surface after being immersed in a 0.1 M phosphate buffer solution (pH ) 7.0) (A) without oligonucleotide, (B) containing 5.4 µg/mL HS-polyA, and (C) after hybridization of B with polyT (5.8 µg/ mL). The intensities have been normalized to the corresponding Au(4f) emission.

Figure 3. XPS spectra of the N(1s) peak for (A) a gold surface after being immersed in a 0.1 M phosphate buffer solution (pH ) 7.0) containing 5.4 µg/mL of HS-polyA and (B) A after being exposed to a noncomplementary sequence (10 µg/mL polyC). The intensities have been normalized to the corresponding Au(4f) emission.

charged, thus enhancing electrostatic attraction and consequently adsorption.17 On the other hand, at pH 7.0 or above, most of the adenine bases will be deprotonated so that direct adsorption is not favored. Similarly, at low pH values (1.9) the gold surface will be positively charged, again giving rise to electrostatic repulsion and thus low coverages. As mentioned above, the shape of the frequency-time profile can be employed to study the kinetics of adsorption. The adsorption process can be controlled by either transport (diffusion-controlled) or kinetics (activationcontrolled), which predict time dependencies of t1/2 and exp(t), respectively. Assuming that the immobilization process is kinetically controlled, we fit the data (Figure 1, solid line) to a first-order kinetics equation: ∆F ) ∆Fmax(1 - e-kt), where ∆F is the frequency change (Hz), ∆Fmax is the frequency change between the initial and the final steady-state frequencies, and k is the first-order rate constant (min-1). The k values obtained from the fit of experimental data at values of pH 3.5 7.0, and 8.5 were found to be 0.13, 0.68, and 0.27 min-1, respectively. These values seem to indicate that whereas the process at high pH gives rise to a lower surface coverage, it is more rapid than the process at low pH. Although largely speculative, it is possible that, at the different pH values studied, the polyA chains have different configurations with chains more or less exposed which will alter the kinetics of adsorption. On the basis of the above-mentioned results, we selected conditions intended to minimize the nonspecific adsorption of polyA in order to optimize surface binding through the thiol groups in the thiolated derivatives. Thus, pH 7.0 was chosen since it represents a good compromise in terms of minimizing nonspecific adsorption and having the added benefit of being physiologically relevant. The immobilization of HS-polyA onto gold surfaces was characterized with XPS by monitoring the nitrogen 1s peak intensities. Figure 2 shows the N(1s) core level photoemission peak from different samples. To make meaningful comparisons, the integrated intensities (area) of the N(1s) peaks were normalized relative to the corresponding Au(4f) peak. Thus, the relative amount of DNA adsorbed on the surface can be determined by comparison of their respective areas. To ensure that the N(1s) signal originated from immobilized DNA and not from other sources, a gold substrate was

subjected to the entire immobilization procedure but in the absence of DNA. As is evident in Figure 2A. there was no peak associated with a N(1s) signal, indicating that the presence of such a peak would have to necessarily arise from adsorbed DNA. The N(1s) core level peak recorded from HS-polyA immobilized on a gold surface is shown in Figure 2B. The presence of this peak indicates that the procedure for immobilization of the HS-polyA is effective. We have also carried out the same procedure using non-thiol-derivatized polyA (not shown) and have found that the normalized N(1s) peak area is approximately 50% of that measured for HS-polyA, in good agreement with the intensities reported in ref 4. Hybridization of HS-PolyA with PolyT. The hybridization of surface-immobilized HS-polyA with polyT was studied by XPS and AFM. Figure 2C shows the N(1s) peak obtained from the sample corresponding to the hybridization of the immobilized HS-polyA with its complementary sequence polyT. The N(1s) peak area obtained for the immobilized HS-polyA was approximately 60% of that obtained after hybridization. The relative number of nitrogen atoms contained in adenine and thymine bases are 5 and 2, respectively. Therefore, the expected ratio of N atoms in polyA with respect to polyA hybridized with polyT should be 70%. The difference between these values (10% is within experimental error. Furthermore, a small amount of polyT could be nonspecifically absorbed through the nucleotide bases to the surface, reducing the experimentally found percentage. To confirm that the increase in the N(1s) peak area is mainly due to the hybridization process and not to nonspecific adsorption of polyT, a HS-polyA/Au substrate was exposed to a solution containing a noncomplementary sequence (polyC). In this case, the N(1s) peak area from the polyA immobilized on the surface (Figure 3A) remained essentially unaltered after exposure to poly C (Figure 3B), indicating the absence of nonspecific adsorption. The results found by XPS were complemented by AFM studies. Figure 4 shows a 2.25 µm × 2.25µm AFM image of the bare Au(111) single-crystal surface following the flame annealing and quenching procedures described in the Experimental Section. As can be ascertained, the surface appears smooth with large-area terraces separated by well-defined steps. Figure 5 shows a 400 nm × 400 nm image of the Au(111) surface following adsorption of the thiol-terminated polyA. Contrary to the bare Au(111) surface described above, the image in this case exhibits a textured

(17) Ito K.; Hashimoto, K.; Ishimori, Y. Anal. Chim. Acta 1996, 327, 29.

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Figure 4. 2.25 µm × 2.25 µm contact mode AFM image of a bare Au(111) single-crystal surface in contact with a 0.1 M phosphate buffer solution (pH ) 7.0).

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Figure 6. 750 nm × 750 nm contact mode AFM image of a Au(111) surface in contact with a 0.1 M phosphate buffer solution (pH ) 7.0) after modification with an adsorbed layer of a HS-polyA and subsequently hybridized with polyT (5.8 µg/mL). Inset: 1.25 µm × 1.25 µm image under otherwise identical imaging conditions.

Figure 7. Typical linear scans along the scan direction obtained from images of Figures 5 (lower curve) and 6 (upper curve).

Figure 5. 400 nm × 400 nm contact mode AFM image of a Au(111) surface in contact with a 0.1 M phosphate buffer solution (pH ) 7.0) after modification with an adsorbed layer of a HS-polyA.

morphology with features that we ascribe to the presence of polyA ostensibly adsorbed through the thiol end onto the gold surface. The average size of these features is estimated to be about 20 nm in diameter. It should be mentioned that even after extensive rinsing, the features remained over the gold surface. Figure 6 presents an image of the Au(111) surface after modification with polyA (as above) and after allowing for the hybridization reaction to take place with polyT for 4 h at 39 °C, as described in the Experimental Section. As in Figure 5 the image also shows a textured morphology, but it is decidedly much more globular with an average size of about 50 nm. This is especially evident in the inset, which provides an image over a wider field (1.25µm × 1.25µm) that stress the differences with the gold surface modified only with polyA. Moreover, the image (in the

inset) exhibits features reminiscent of the steps observed on gold surfaces (Figure 4), indicating the absence of an adsorbed thick layer of oligonucleotides. Figure 7 represents typical linear scans taken from images of Figures 5 and 6. These curves were extracted along the fast-scan direction. In this figure the increase in the average feature size after hybridization is evident. The average apparent size of the HS-polyA inmobilized is around 20 nm which suggests that the HS-polyA loses its linear configuration, folding into small globular aggregates that do not overlap with each other. However, after hybridization with polyT, the average size increases significantly, to about 50 nm. The enhancement of the lateral dimension of the average feature size is consistent with hybridization while still maintaining a globular texture. It should also be mentioned that the appearance of features with an apparent globular morphology is a known artifact in AFM that could be induced by several factors [see ref 18 for a detailed description], including the convolution of the tip shape over the surface. The apparent height of both structures (Figure 7) was found to be around 0.5 nm instead of the expected 2.0 nm that (18) Erts, D.; Polyakov, B.; Olin, H.; Tuite, E. J. Phys. Chem. B 2003, 107, 3591.

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corresponds to the height of a DNA duplex. However, given the tight packing of the immobilized material on the surface, the tip is unable to penetrate sufficiently so as to reflect the true height of the features and hence the values appear to be underestimated. Other factors, such as adhesion of the oligonucleotides to the tip apex and/or the flexibility of the chains, could also influence the apparent height and diameter of the surface features. Since the immobilized oligonucleotides are homogeneously distributed over the surface and they have a regular size, we conclude (somewhat speculatively) that the HS-polyA immobilized layer self-organizes onto the gold surface. Furthermore, and in good agreement with the XPS and QCM results presented above, this also shows the absence of nonspecific adsorption, which would lead to a heterogeneous aggregate size distribution. Conclusions The chemisorption of a thiol-modified, single-stranded, 200-base oligonucleotide (HS-polyA) onto a gold electrode surface has been investigated via the QCM, XPS, and AFM techniques. We have also characterized the immobilization process of a single-stranded, 200-base oligonucleotide (polyA) on gold substrates in order to assess the extent of the nonspecific adsorption as well as optimize

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the adsorption conditions. The pH dependence of the adsorption was studied via the QCM technique, and it was found that the maximum was reached around pH 3.5, which represented a compromise between the charge on the oligonucleotide (pKa ) 3.52) and the surface charge on the gold surface. A value of pH 7.0 was found to be optimal for minimizing nonspecific adsorption. The hybridization of HS-polyA adsorbed onto a gold surface, by both complementary (polyT) and noncomplementary (polyC) oligonucleotides, was followed by XPS and AFM and was found to be highly specific. Moreover, from AFM images obtained before and after the hybridization process, we have been able to determine that oligonucleotides immobilized on the surface fold into globular structures of around 10 nm in diameter, which enlarged to about 50 nm upon hybridization. The procedure employed could represent an alternative method to those generally employed such as spectrophotometry and surface plasmon resonance. Acknowledgment. This work has been partially supported by MCYT (Spain) Grant Nos. BQU2002-02406, PB98-0082, and MAT2002-395 and by NSF Grant No. INT-0122759. LA034308Q