Determination of the Optimized Conditions for Coupling

Bioconjugate Chem. 2007, 18, 1897–1904

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Determination of the Optimized Conditions for Coupling Oligonucleotides with 16-Mercaptohexadecanoic Acid Chemically Adsorbed upon Au Yi-Te Wu,† Jiunn-Der Liao,*,†,‡ Je-Inn Lin,† and Cheng-Chan Lu§ Department of Materials Science and Engineering, Center for Micro/Nano Science and Technology, and Department of Pathology, National Cheng Kung University, No. 1, University Road, Tainan 70101, Taiwan. Received June 18, 2007; Revised Manuscript Received September 17, 2007

A specific 5′-modified amino group oligonucleotide (Primer 1), 15-mers in length, is selectively coupled with the carboxyl terminated 16-mercaptohexadecanoic acid (MHDA) chemically adsorbed on Au and subsequently hybridized with Antisense Primer. The amide-coupling process is of significance to create an intermediate structure for the purpose of adding Primer 1, while the hybridization reaction is relevant to various diagnostic purposes to determine the presence in nucleic acids for a target sequence. In this work, the coupling setting was particularly emphasized by varying commonly used temperatures and pH values with a definite concentration of coupling agents (i.e., 10 mM). The recombination with analogous hybridization treatment was investigated using high resolution X-ray photoelectron spectroscopy and a 75° grazing angle Fourier transform infrared spectrometer. On the basis of the spectroscopic studies, the optimized conditions for the coupling process that is also correlated with the molecular density of subsequent hybridization process on MHDA/Au have been proposed at 37 °C and a pH value of 4.5. Therefore, it is pertinent to intensify the joining of short-chain DNA strands by complementary base pairing in diagnostic applications such as the identification of single nucleotide polymorphisms.

1. INTRODUCTION Single nucleotide polymorphisms (SNPs) are common DNA sequence variations that cause phenotypic differences among individuals. Most SNPs do not lead to physical changes in humans; however, a small part of them may predispose individuals to disease and even influence their complex response to medical treatments or drug regimens (1–3). A considerable effort to decode the human genome or to further the understanding of human genetics has been promoted to identify SNPs, for example, SNPs related to drug-metabolizing enzymes (4), secretion disorder (5), allele-specific extensions (6). In particular, only a small percentage of a person’s DNA sequence code is necessary for the production of proteins or those involved in transcription regulation (7). SNPs found within a coding sequence are therefore of special interest when they are highly associated with the structural and functional properties of proteins. Recent advances in biotechnology have significantly enhanced the process of disease or protein detection (4–10) and the practice of preventative and curative medicine. In particular, association study (11, 12) can detect and indicate which pattern is most likely associated with the disease-causing genes or complex human disorders. In a recent verification process (13), SNP finding is automatically achieved using a combined approach between sequence-specific pattern matching of flanking sequence and a quality assessment of inconsistencies on the average once every kilo-base pair in the human genome. The use of specific oligonucleotides as therapeutic agents or biomarkers (14), for example, lies upon their ability to interfere with the molecular machinery of protein synthesis either by binding to the mRNAs transcribed from a gene or by binding directly to a target gene. In recent years, oligonucleotide * Corresponding author. Phone: (886) 6 2757575ext. 62971. Fax: (886) 6 2346290. E-mail: [email protected]. † Department of Materials Science and Engineering. ‡ Center for Micro/Nano Science and Technology. § Department of Pathology.

hybridization has become relevant to various diagnostic purposes to determine the presence in nucleic acids for a target sequence that is complementary to the oligonucleotide probe (15–17). Hybridization on a solid support involves the immobilization of one of the interacting nucleic acids on the surface, while the other is free in solution. For the former, high-density DNA microarrays with the immobilization of aptamers (short, known single-stranded DNA or RNA sequences) are a well-established method capable of measuring gene expression levels (8, 16). The chip-based optical detection of the molecular interactions occurring on the immobilized and labeled DNA sequence or functional protein as a probe has been promptly developed (18–20). This process combined with a recent technique on microelectronics and rich SNP profiles meets the demands for rapid screening with reduced sample amounts. For the latter, the use of colloidal Au as a color label as bridged between the attached DNA and the oligonucleotides with sequences complementary to either ends of the target DNA is successfully delivered for the detection of DNA hybridization in solution (21). However, specific DNA probes attached on magnetic particles are also used for the detection of viable bacteria (22). A perceptive detection of specific molecular binding that is based on optical visualization of the labeled molecules on a microstructured chip surface is still improving. One of the major problems relies on the stability of molecular interactions in varying conditions such as the variation of ionic strengths (23, 24), pH values (23), and temperatures (25). In this study, selfassembled monolayers (SAMs) chemically adsorbed on Au substrates have been introduced for the construction of molecular additions. SAMs are well-ordered and densely packed twodimensional ensembles of long-chain molecules, which have attracted tremendous attention because of the potential applications in various fields, such as DNA chips, microelectronics, sensors, and nanopatterned SAMs (26–28). The SAMs treatment is also one of the proposed methods for extending lithography down to the nanometer scale by applying electron-beam patterning for a new kind of lithography resist (29, 30). Much

10.1021/bc700217n CCC: $37.00  2007 American Chemical Society Published on Web 10/31/2007

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Figure 1. For a practical SAM application, a tailored surface containing -CH3 and carboxyl tail groups can be patterned for the subsequent addition of short-chained oligonucleotides.

attention is devoted to aliphatic SAMs and, above all, to films of n-alkanethiol on noble metal substrates. In this study, alkanethiolate monolyers with the carboxyl tail group are chemically adsorbed on Au. A synthesized oligonucleotide with 5′-modified -NH2 or 3′-modified -OH ends on the respective sequences, in association with an allelic polymorphism within the human tumor necrosis factor alpha (TNF-R) promoter region (31–34), is subsequently immobilized on SAMs/Au. Optimized conditions for DNA hybridization based on the combination of temperatures, pH values, and the concentration of coupling agents are particularly investigated. As compared with previous measurements using 2D electrospray ionization tandem mass spectroscopy (35), this study utilizes surfacesensitive measurements for the identification of chemical bindings in the respective reactions and possibly for subsequent SNPs diagnostic applications.

2. EXPERIMENTAL PROCEDURES 2.1. Preparation of SAMs/Au with Carboxyl Tail Groups. The substrates for thiol-derived SAM fabrication were prepared by thermal evaporation of ≈50 nm of Au onto the polished single crystal silicon (100) wafers (Silicon Sense) primed with a ≈5 nm titanium adhesion layer. The octadecanethiol (ODT, HS-(CH2)17-CH3) SAMs were formed by standard immersion procedure (36), resulting in the homogeneous ODT SAMs on Au (Figure 1a). For the practical applications, a particular part of the ODT SAMs can be easily removed by low-energy electron beam irradiation (Figure 1b), followed by immersion into an ethanolic 1 mM solution of 16-mercaptohexadecanoic acid (MHDA, HS-(CH2)15-COOH, Aldrich) for 1–2 h. A tailored surface with mixed -CH3 and carboxyl tail groups is produced for subsequent treatments (Figure 1c). To investigate the succeeding reactions on the surfaces, a uniform MHDA/Au representing the MHDAmodified part was employed, followed by coupling and hybridizing with the respective oligonucleotides. 2.2. Preparation of the Oligonucleotides. Four types of the oligonucleotides, 15-mers in length, were employed for the experiments. A digested probe, 5′-GGGCATGGGGACGGG (MDBio Inc., type-1) as an aptamer, with a concentration of 5 µM was synthesized by means of polymerase-mediated single-base primer extension. It was first verified by Dot Hybridization method using Digoxigenin (DIG)-labeled Sequence Specific Oligonucleotide Probe (SSOP, 5′-CCCGTCCCCATGCCC, type-2) with a concentration of 100 µM, followed by dilutions to different concentrations. The modified amino group at the 5′-end, H2N-(CH2)6-5′GGGCATGGGGACGGG-3′ (MDBio Inc., type-3) as Primer

Wu et al.

Figure 2. (a) Oligonucleotides were verified by 100 µM SSOP diluted to five different concentrations (i.e., 100, 10, 1.0, 0.1, 0.01, and 0.001 µM, for marks 1–6). (b) Oligonucleotides were assessed in different pH values and reaction times. The pH values of marks 1–4 were 4.5, 5.5, 6.5, and 7.4, respectively (buffered for 6 h). Those of marks 5–8 were analogous in pH values but buffered for 4 h. Those of marks 9–12 were also analogous in pH values but buffered for 2 h. Mark 13 was a control for reference.

1, was expected to be amide-bonded on MHDA/Au by adding the coupling agents, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC, Sigma) and N-hydroxysuccinimide (NHS, Sigma), in PBS or MES buffer solution with various concentrations (1, 10, or 100 mM) and various pH values (4.5, 5.5, 6.5, or 7.4). The Primer 1/MHDA/Au was annealed at 42 °C and thereafter hybridized with the 5′-CCCGTCCCCATGCCC-3′ (MDBio Inc., type-4) as Antisense Probe. The concentration of Primer 1 or Antisense Probe was ≈5 µM, which was first dissolved in deionized water and preserved at -20 °C. 2.3. Surface Characterization. Water contact angles were measured immediately after preparing MHDA/Au substrates. The measurements were performed under an argon atmosphere at 22 ( 1 °C. The sessile drop method was used, and a JVCTK1200 microscope with processing software took the droplet image. A droplet of ≈5 µL was used. For each sample, 10 measurements with a standard deviation below 1° were carried out, and an average value was calculated. Water contact angles of the pristine Au and a uniform MHDA/Au were ≈83.4° and ≈23.2°, respectively. The decrease of contact angle is expected in the presence of the hydrophilic group. Fourier-transform infrared (FTIR, Bomem DA8.3) with 75° grazing incident angle reflectance (Harrick) was utilized for identifying chemical species in the specific range of 1000–2000 cm-1 with 0.5 cm-1 resolution and precision of a wavenumber no less than 0.01 cm-1. In this range, positions of the band frequencies were assigned to major IR-active groups in the asprepared oligonucleotides (37). Synchrotron-based high-resolution X-ray photoelectron spectroscopy (HRXPS) was utilized to characterize the chemical structures of MHDA on Au, amide bonds with Primer 1, and subsequent hybridization with Antisense Probe. HRXPS measurements were carried out at the U5 Undulatory Beam Line of the National Synchrotron Radiation Research Center in Hsinchu, Taiwan. The time for the acquisition of the entire set of the HRXPS spectra for an individual sample was selected as a compromise between spectral quality and the damage induced by X-rays. Details about HRXPS measurements can be found elsewhere (36, 38). To determine the elemental compositions at the respective surface, the C 1s, S 2p, O 1s, N 1s, and P 2p core level spectra were measured and calculated from HRXPS peak area with correction algorithms and atomic sensitivity factors. The spectra were fitted using Voigt peak profiles and a Shirley background (38). The stability of Au–S bonds subsequent to the treatments

Oligonucleotides with 16-Mercaptohexadecanoic Acid

Bioconjugate Chem., Vol. 18, No. 6, 2007 1899

Figure 3. IR absorption spectra of Primer 1/MHDA/Au prepared in different concentrations of EDC and NHS at a pH value of 4.5 at 37 °C. The as-measured surfaces were (a) MHDA/Au and (a) with the concentrations of EDC and NHS at (b) 1 mM, (c) 10 mM, and (d) 100 mM.




was correlated with the S 2p doublet with a binding energy (BE) of ≈162.0 eV. The decrease of the intermediate amide bonds due to the addition of the oligonucleotides was calculated by comparing the intensity of the amide group with that of -N*) from the oligonucleotides (39).

3. RESULTS AND DISCUSSION 3.1. Preliminary Tests on the Oligonucleotides Using SSOP Method. The oligonucleotides, Primer 1 and Antisense Probe, with a concentration of 5 µM were first verified by 100 µM SSOP diluted to five different concentrations (Figure 2a). The indications revealed a good relationship with the diluted concentrations. The SSOP diluted to 0.1 µM was still distinguishable (mark 4 in Figure 2a). In Figure 2b, the quality of the oligonucleotides was assessed in different buffer solutions, that is, with MES at pH 4.5, 5.5, and 6.5 or PBS at pH 7.4, with a reaction time of 2, 4, or 6 h. The variations of the buffer solution and the reaction time did not cause structural damage or hydrolysis in the oligonucleotides.

3.2. Chemical Structures Examined by FTIR with 75° Grazing Incident Angle Reflectance. An analogous FTIR curve of the pristine surface was taken as the reference and is shown in the bottom curves of Figures 3–6. The characteristic IR peaks for MHDA/Au at 1714.4 cm-1 (i.e., C)O in the carboxyl acid) and 1468.5 cm-1 (i.e., CH2 in aliphatic chains) were identified. The carboxyl tail group of MHDA was anticipated to create an amide bond with the NH2-modified Primer 1 using EDC and NHS as the coupling agents (35, 40). In the case of the acidic environment at pH 4.5 and 37 °C (Figure 3), the addition of 1 mM EDC and NHS buffered in MES solution (Figure 3b) mainly created ester compounds (1265.5 cm-1 and 1108.4 cm-1). The NHS-ester (1744.3 cm-1) was formed transitionally, whereas the coupling of the NH2-modified Primer 1 was not found. As the buffered concentration of EDC and NHS was increased to 10 mM (Figure 3c), IR absorptions of C-O in deoxyribose of nucleoside (1068.8 cm-1), phosphodiester bond (1117.5 and 1213.9 cm-1), C)O in NHS-ester (1749.1 cm-1), and C)O in Guanine of Purine (1696.1 cm-1), in association with Primer 1, were detected (40). Nevertheless, IR absorptions in the range of 1500–1650 cm-1, correlating with the presence


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Figure 7. HRXPS spectra of Primer 1/MHDA/Au prepared in 10 mM EDC and NHS at 37 °C. The as-measured surfaces were (a) MHDA/Au, (b) Primer 1 coupled with MHDA/Au at pH 4.5, (c) Primer 1 from (b)/MHDA/Au hybridized with Antisense Probe, (d) Primer 1 coupled with MHDA/ Au at pH 7.4, and (e) Primer 1 (from d)/ MHDA/Au hybridized with Antisense Probe. The changes of the intensities of the elements are illustrated in Figure 10.

Figure 8. HRXPS spectra of Primer 1/MHDA/Au prepared in 10 mM EDC and NHS at 4 °C. The as-measured surfaces were (a) MHDA/Au, (b) Primer 1 coupled with MHDA/Au at pH 4.5, (c) Primer 1 from (b)/MHDA/Au hybridized with Antisense Probe, (d) Primer 1 coupled with MHDA/ Au at pH 7.4, and (e) Primer 1 (from d)/MHDA/Au hybridized with Antisense Probe. The changes of the intensities of the elements are illustrated in Figure 11.

of amide bonds, were considerably weak. It is likely that the sensitivity of the measurement is insufficient to interpret a minor quantity of the intermediate amide bonds. Furthermore, with the addition of 100 mM EDC and NHS in the buffered solution (Figure 3d), the characteristic amide group at 1648.7 (amide I) and 1537.9 cm-1 (amide II) and most of the analogous species associated with Primer 1 were simultaneously found. Analogous results were obtained in the case of the acidic environment at pH 4.5 and 4 °C (Figure 4) and the alkaline environment at 37 °C (Figure 5) and 4 °C (Figure 6). By comparison, the NH2-modified Primer 1 was well coupled with MHDA/Au in the case of 100 mM EDC and NHS buffered in MES solution at pH 4.5 and 37 °C, while IR intensity of the characteristic amide group and most of the analogous species associated with Primer 1 were clearly found. From the above measurements, as the coupling agents were buffered to a concentration of 10 mM, the covalently bonded Primer 1 could be identified, whereas the IR intensity correlated

with the amide group, the intermediate structure, was relatively insignificant. 3.3. Chemical Bonds Examined by HRXPS. For the pristine MHDA/Au, the characteristic structures of MHDA were determined by HRXPS and presented in the bottom curves of Figures 7 and 8. After carrying out the decompositions of the C 1s, O 1s, and N 1s spectra illustrated in Figure 9, the C 1s spectrum exhibited a major emission peak at a BE of ≈284.8 eV (i.e., C*-C or C*-H) with a fwhms of ≈0.9 eV and chemical shifts at BEs of 285.9 eV (C*-O) and ≈289.2 eV (i.e., O)C*OH). The S 2p spectrum exhibited a single S 2p doublet with a BE of ≈162.0 eV for the S 2p3/2 component. This doublet is commonly related to the thiolate species bonded to Au surface (36, 38, 41, 42). The O 1s spectrum exhibited the emission peaks at BEs of 532.1 eV (C-O) and 533.4 eV (C)O), resulting from the presence of MHDA. No traces of nitrogen or phosphorus were found.



Figure 9. Curve-fitted HRXPS spectra for Primer 1/MHDA/Au prepared in 10 mM of EDC and NHS at 37 °C. The as-measured surfaces were (a) MHDA/Au, (b) Primer 1 coupled with MHDA/Au at pH 4.5, and (c) Primer 1 from (b)/MHDA/Au hybridized with Antisense Probe at 42 °C. The decomposition of the respective C 1s, O 1s, and N 1s spectra is described in section 3.4.

Interfacing of surface characterization with analytical data is not well-developed. Particularly, chemical species of covalent attachment presenting in the multiple linkages are difficult to be defined by configurations. In this section, the respective samples with the coupling agents of 10 mM are purposely studied using HRXPS to gain information of the aptamers upon MHDA/Au under different pH values and coupling or annealing temperatures. The HRXPS spectra for Primer 1/MHDA/Au, prepared at different pH values or temperatures, are shown in Figures 7b and d and 8b and d, which exhibit analogous BEs. Followed by hybridization with Antisense Probe, their C and O intensities and BEs exhibited variable data (shown in Figures 7c and 7e and 8c and e). Among them, the HRXPS spectra for Primer

1/MHDA/Au prepared in 10 mM EDC and NHS at 37 °C were curve-fitted and studied. In Figure 9, the decompositions of the C 1s spectra were assigned: (1) 284.8 eV, C*-C, (2) 285.9 eV, C*-O, (3) 286.7 eV, amide group or NHS-ester, (4) 287.9 eV, from the oligonucleotide G or C, and (5) 289.1 eV, O)C*-OH or from the oligonucleotide C. The decompositions of the O 1s spectra were assigned: (1) 532.1 eV, C-O*-C from the oligonucleotide G or C, (2) 532.8 eV, amide group or (N)2C)O* from the oligonucleotide G, (3) 533.4 eV, C)O* from the oligonucleotide, (4) 534.4 eV, O*)C-OH or C-O-P from the oligonucleotide and (5) 535.6 eV, NHS-ester. The decompositions of the N 1s spectra were assigned: (1) 399.0 eV, -N*) from the oligonucleotide G or C, (2) 400.0 eV, O)C-N*-H from the amide group, (3) 400.5 eV, -N*-, -N*H-, -N*H2 from the


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Figure 10. Variations of the content of the elements on Primer 1/MHDA/Au prepared in 10 mM EDC and NHS at 37 °C. The as-measured surfaces were (a) MHDA/Au, (b) Primer 1 coupled with MHDA/Au at pH 4.5, (c) Primer 1 from (b)/MHDA/Au hybridized with Antisense Probe, (d) Primer 1 coupled with MHDA/Au at pH 7.4, and (e) Primer 1 (from d)/ MHDA/Au hybridized with Antisense Probe.

Figure 11. Variations of the content of the elements on Primer 1/MHDA/Au prepared in 10 mM of EDC and NHS at 4 °C. The as-measured surfaces were (a) MHDA/Au, (b) Primer 1 coupled with MHDA/Au at pH 4.5, (c) Primer 1 from (b)/MHDA/Au hybridized with Antisense Probe, (d) Primer 1 coupled with MHDA/Au at pH 7.4, and (e) Primer 1 (from d)/ MHDA/Au hybridized with Antisense Probe.

oligonucleotide G or C, and (4) 402.5 eV, -N*-O from NHSester. For the P 2p spectra shown in Figures 7 and 8, the emission peak at a BE of 133.2 eV was associated with the presence of oligonucleotides. In Figure 9, the ratio of peak 1 over peak 3 at b of N1s spectra was close to 2/3, which is identical to the presence of Primer 1 (i.e., mainly from oligonucleotide G), while that at c of N 1s spectra was close to 3/5 (39), which is approximately the product of Primer 1 in hybridization with Antisense Probe. On the basis of these qualitative measurements, the hybridization between Primer 1 and Antisense Probe noticeably occurred as a result of the particular treatments. 3.4. Semiquantitative Measurements for S–Au and Amide-Coupled Bonds. The addition of Antisense Probe in hybridization with Primer 1 also resulted in decreasing the HRXPS intensity of the amide group, which was associated with the assignments of peak 3 of C 1s, peak 2 of O 1s, and peak 2 of N 1s (Figure 9). In the case of b of Primer 1 coupled on MHDA/Au at pH 4.5 and c of surface b in hybridization with Antisense Probe, the relative intensities of peak 2 over peak 1 in b and in c were calculated as ≈0.57 for b and ≈0.22 for c. The spectroscopic result estimated that the intermediate amide

bonds significantly decreased from an intensity ratio of ≈0.57 to that of ≈0.22 (or -61.4%) due to the hybridization with Antisense Probe that increased the packing density of the oligonucleotide molecules on Primer 1/ MHDA/Au. The average thickness of MHDA uultrathin film on Au is ≈19.4 Å, in comparison with ≈18.9 Å for the CH3 terminated thiols with analogous chain length on Au, whereas a relatively disordered carboxyl terminated SAM on Au is anticipated (43). In our previous calculations, the intermolecular spacing of the headgroup S–S for MHDA/Au is presumably larger than that for CH3 terminated thiols on Au (≈4.97 Å) (44) due to the functions and/or the interactions of hydrogen bonds between the tail groups (43). As a result, the headgroup (i.e., S adsorbed on Au) of MHDA/Au is not as closely packed as that of CH3terminated thiols on Au. As illustrated in Figures 7 and 8, the S–Au bonds kept nearly intact during the coupling and subsequent hybridization processes. It implied that the MHDA uultrathin film on Au was suitable as a molecular support. For a stable coupling process, the bond angle for O)C*-O (≈120°) and that for O)C*-N (≈109°) is changed, which will alter the molecular arrangement as well as the packing density of the oligonucleotide on MHDA/Au. With these calculations, the


photoelectron intensity of the intermediate amide bonds continuously decreases because of the completion of hybridization naturally increases the packing density of the oligonucleotides on MHDA/Au. The element’s ratios calculated from Figures 7 and 8 with respect to the pristine MHDA/Au surface are shown in Figures 10 and 11, respectively. Note that an equal concentration (i.e., 10 mM) of the coupling agent was used. The intensity ratios of the C 1s, O 1s, N 1s, and P 2p spectra increased with the addition of Primer 1 and subsequent to the hybridization of Antisense Probe. Those of the S 2p spectra decreased with the addition of the oligonucleotides because of the increase of molecular density on MHDA/Au. No significant traces of other sulfur-derived species (45, 46) were found during the processes. The coupling process in the acid environment (i.e., pH 4.5) either at 37 or 4 °C steadily decreased the S 2p intensities, that is, c or e with respect to b or d of S 2p spectra in Figures 10 and 11. In combination with the calculation of molecular density on MHDA/Au, an optimized coupling condition was first suggested at pH 7.4 and 37 °C. However, the increased packing density of the oligonucleotides on MHDA/Au resulted in increasing the elements’ ratios of c or e with respect to b or d in C 1s, O 1s, N 1s, and P 2p spectra. In combination with the elements’ ratio of the oligonucleotide G, C, A, or T, an optimized coupling condition was suggested at pH 4.5 and 37 °C. Comparing the case of the coupling temperature at 37 °C with that at 4 °C, the former exhibited proficiency in increasing the quantity of the oligonucleotides on MHDA/Au, particularly on the basis of the increased intensity ratios of N 1s and P 2p spectra and the decreased intensity ratios of S 2p spectra. However, comparing the pH value for amide coupling at 4.5 with that at 7.4, the former exhibited values in correlation with the elements’ ratio of the oligonucleotide. From these measurements, the optimized coupling conditions in this study were presumably placed at pH 4.5 and 37 °C.

CONCLUSIONS The use of tailored SAMs chemically adsorbed upon Au is increasingly important for recent techniques in microelectronics. In combination with specific short-chain oligonucleotides along with rich SNP profiles on the microelectronic device, the invention meets the demands for rapid screening with reduced sample amounts. The coupling of the carboxyl tailed MHDA (≈2 nm) with a 5′-modified amino group oligonucleotide 15mers in length forms an ultra-thin layer. Such a coupled oligonucleotide is relevant to determine the presence of nucleic acids and to act as a diagnostic probe for a target sequence. A perceptive detection for the specific molecular binding on a microstructured chip surface has been highly improved. Therefore, this study particularly emphasizes the stability of molecular interactions under treatment conditions such as pH values and temperatures for the coupling or annealing process. On the basis of the high-resolution spectroscopic studies and the definite concentration of coupling agents (i.e., 10 mM), the optimized conditions for the coupling process have been proposed at pH 4.5 and 37 °C. At the same time, the S–Au bonds and the intermediate amide-coupled structure are considerably resistant to subsequent treatments. Thus, this work provides a method to intensify the joining of short-chain DNA strands with the tailored SAMs/Au prior to completing a hybridization process. It is also promising in creating nanopatterned SAMs/Au for advanced DNA chips, in particular for the detection of SNPs.

ACKNOWLEDGMENT This work has been supported by the National Science Council of R.O.C under grant No. 95–2621-Z-006–002. The authors would like to thank the Center for Micro/Nano Science


and Technology and the Sustainable Environment Research Center, National Cheng Kung University, and National Synchrotron Radiation Research Center, Hsinchu, Taiwan for access to equipment, technical support, and partial financial support.

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