Determination of the Optimized Conditions for Coupling Oligonucleotideswith 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 ofPathology, 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 thecarboxyl terminated 16-mercaptohexadecanoic acid (MHDA) chemically adsorbed on Au and subsequentlyhybridized with Antisense Primer. The amide-coupling process is of significance to create an intermediate structurefor the purpose of adding Primer 1, while the hybridization reaction is relevant to various diagnostic purposes todetermine the presence in nucleic acids for a target sequence. In this work, the coupling setting was particularlyemphasized by varying commonly used temperatures and pH values with a definite concentration of couplingagents (i.e., 10 mM). The recombination with analogous hybridization treatment was investigated using highresolution X-ray photoelectron spectroscopy and a 75° grazing angle Fourier transform infrared spectrometer. Onthe basis of the spectroscopic studies, the optimized conditions for the coupling process that is also correlatedwith the molecular density of subsequent hybridization process on MHDA/Au have been proposed at 37 °C anda pH value of 4.5. Therefore, it is pertinent to intensify the joining of short-chain DNA strands by complementarybase pairing in diagnostic applications such as the identification of single nucleotide polymorphisms.

1. INTRODUCTION

Single nucleotide polymorphisms (SNPs) are common DNAsequence variations that cause phenotypic differences amongindividuals. Most SNPs do not lead to physical changes inhumans; however, a small part of them may predisposeindividuals to disease and even influence their complex responseto medical treatments or drug regimens (1–3). A considerableeffort to decode the human genome or to further the understand-ing of human genetics has been promoted to identify SNPs, forexample, 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 isnecessary for the production of proteins or those involved intranscription regulation (7). SNPs found within a codingsequence are therefore of special interest when they are highlyassociated with the structural and functional properties ofproteins. Recent advances in biotechnology have significantlyenhanced the process of disease or protein detection (4–10) andthe practice of preventative and curative medicine. In particular,association study (11, 12) can detect and indicate which patternis most likely associated with the disease-causing genes orcomplex human disorders. In a recent verification process (13),SNP finding is automatically achieved using a combinedapproach between sequence-specific pattern matching of flankingsequence and a quality assessment of inconsistencies on theaverage once every kilo-base pair in the human genome.

The use of specific oligonucleotides as therapeutic agents orbiomarkers (14), for example, lies upon their ability to interferewith the molecular machinery of protein synthesis either bybinding to the mRNAs transcribed from a gene or by bindingdirectly to a target gene. In recent years, oligonucleotide

hybridization has become relevant to various diagnostic purposesto determine the presence in nucleic acids for a target sequencethat is complementary to the oligonucleotide probe (15–17).Hybridization on a solid support involves the immobilizationof one of the interacting nucleic acids on the surface, while theother is free in solution. For the former, high-density DNAmicroarrays with the immobilization of aptamers (short, knownsingle-stranded DNA or RNA sequences) are a well-establishedmethod capable of measuring gene expression levels (8, 16).The chip-based optical detection of the molecular interactionsoccurring on the immobilized and labeled DNA sequence orfunctionalproteinasaprobehasbeenpromptlydeveloped(18–20).This process combined with a recent technique on microelec-tronics and rich SNP profiles meets the demands for rapidscreening with reduced sample amounts. For the latter, the useof colloidal Au as a color label as bridged between the attachedDNA and the oligonucleotides with sequences complementaryto either ends of the target DNA is successfully delivered forthe detection of DNA hybridization in solution (21). However,specific DNA probes attached on magnetic particles are alsoused for the detection of viable bacteria (22).

A perceptive detection of specific molecular binding that isbased on optical visualization of the labeled molecules on amicrostructured chip surface is still improving. One of the majorproblems relies on the stability of molecular interactions invarying conditions such as the variation of ionic strengths (23, 24),pH values (23), and temperatures (25). In this study, self-assembled monolayers (SAMs) chemically adsorbed on Ausubstrates have been introduced for the construction of molecularadditions. SAMs are well-ordered and densely packed two-dimensional ensembles of long-chain molecules, which haveattracted tremendous attention because of the potential applica-tions in various fields, such as DNA chips, microelectronics,sensors, and nanopatterned SAMs (26–28). The SAMs treatmentis also one of the proposed methods for extending lithographydown to the nanometer scale by applying electron-beampatterning for a new kind of lithography resist (29, 30). Much

* Corresponding author. Phone: (886) 6 2757575ext. 62971. Fax:(886) 6 2346290. E-mail: jdliao@mail.ncku.edu.tw.

† Department of Materials Science and Engineering.‡ Center for Micro/Nano Science and Technology.§ Department of Pathology.

Bioconjugate Chem. 2007, 18, 1897–1904 1897

10.1021/bc700217n CCC: $37.00 2007 American Chemical SocietyPublished on Web 10/31/2007

attention is devoted to aliphatic SAMs and, above all, to filmsof n-alkanethiol on noble metal substrates. In this study,alkanethiolate monolyers with the carboxyl tail group arechemically adsorbed on Au. A synthesized oligonucleotide with5′-modified -NH2 or 3′-modified -OH ends on the respectivesequences, in association with an allelic polymorphism withinthe human tumor necrosis factor alpha (TNF-R) promoterregion (31–34), is subsequently immobilized on SAMs/Au.Optimized conditions for DNA hybridization based on thecombination of temperatures, pH values, and the concentrationof coupling agents are particularly investigated. As comparedwith previous measurements using 2D electrospray ionizationtandem mass spectroscopy (35), this study utilizes surface-sensitive measurements for the identification of chemicalbindings in the respective reactions and possibly for subsequentSNPs diagnostic applications.

2. EXPERIMENTAL PROCEDURES

2.1. Preparation of SAMs/Au with Carboxyl TailGroups. The substrates for thiol-derived SAM fabrication wereprepared by thermal evaporation of ≈50 nm of Au onto thepolished single crystal silicon (100) wafers (Silicon Sense)primed with a ≈5 nm titanium adhesion layer. The octade-canethiol (ODT, HS-(CH2)17-CH3) SAMs were formed bystandard immersion procedure (36), resulting in the homoge-neous ODT SAMs on Au (Figure 1a). For the practicalapplications, a particular part of the ODT SAMs can be easilyremoved by low-energy electron beam irradiation (Figure 1b),followed by immersion into an ethanolic 1 mM solution of16-mercaptohexadecanoic acid (MHDA, HS-(CH2)15-COOH,Aldrich) for 1–2 h. A tailored surface with mixed -CH3 andcarboxyl tail groups is produced for subsequent treatments(Figure 1c). To investigate the succeeding reactions on thesurfaces, a uniform MHDA/Au representing the MHDA-modified part was employed, followed by coupling and hybrid-izing with the respective oligonucleotides.

2.2. Preparation of the Oligonucleotides. Four types of theoligonucleotides, 15-mers in length, were employed for theexperiments.

A digested probe, 5′-GGGCATGGGGACGGG (MDBio Inc.,type-1) as an aptamer, with a concentration of 5 µM wassynthesized by means of polymerase-mediated single-baseprimer extension. It was first verified by Dot Hybridizationmethod using Digoxigenin (DIG)-labeled Sequence SpecificOligonucleotide Probe (SSOP, 5′-CCCGTCCCCATGCCC,type-2) with a concentration of 100 µM, followed by dilutionsto different concentrations.

The modified amino group at the 5′-end, H2N-(CH2)6-5′-GGGCATGGGGACGGG-3′ (MDBio Inc., type-3) as Primer

1, was expected to be amide-bonded on MHDA/Au by addingthe coupling agents, 1-ethyl-3-(3-dimethylaminopropyl) carbo-diimide (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 thereafterhybridized with the 5′-CCCGTCCCCATGCCC-3′ (MDBio Inc.,type-4) as Antisense Probe. The concentration of Primer 1 orAntisense Probe was ≈5 µM, which was first dissolved indeionized water and preserved at -20 °C.

2.3. Surface Characterization. Water contact angles weremeasured immediately after preparing MHDA/Au substrates.The measurements were performed under an argon atmosphereat 22 ( 1 °C. The sessile drop method was used, and a JVC-TK1200 microscope with processing software took the dropletimage. A droplet of ≈5 µL was used. For each sample, 10measurements with a standard deviation below 1° were carriedout, and an average value was calculated. Water contact anglesof the pristine Au and a uniform MHDA/Au were ≈83.4° and≈23.2°, respectively. The decrease of contact angle is expectedin the presence of the hydrophilic group.

Fourier-transform infrared (FTIR, Bomem DA8.3) with 75°grazing incident angle reflectance (Harrick) was utilized foridentifying chemical species in the specific range of 1000–2000cm-1 with 0.5 cm-1 resolution and precision of a wavenumberno less than 0.01 cm-1. In this range, positions of the bandfrequencies were assigned to major IR-active groups in the as-prepared oligonucleotides (37).

Synchrotron-based high-resolution X-ray photoelectron spec-troscopy (HRXPS) was utilized to characterize the chemicalstructures of MHDA on Au, amide bonds with Primer 1, andsubsequent hybridization with Antisense Probe. HRXPS mea-surements were carried out at the U5 Undulatory Beam Line ofthe National Synchrotron Radiation Research Center in Hsinchu,Taiwan. The time for the acquisition of the entire set of theHRXPS spectra for an individual sample was selected as acompromise between spectral quality and the damage inducedby X-rays. Details about HRXPS measurements can be foundelsewhere (36, 38).

To determine the elemental compositions at the respectivesurface, the C 1s, S 2p, O 1s, N 1s, and P 2p core level spectrawere measured and calculated from HRXPS peak area withcorrection algorithms and atomic sensitivity factors. The spectrawere fitted using Voigt peak profiles and a Shirley background(38). The stability of Au–S bonds subsequent to the treatments

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

Figure 2. (a) Oligonucleotides were verified by 100 µM SSOP dilutedto five different concentrations (i.e., 100, 10, 1.0, 0.1, 0.01, and 0.001µM, for marks 1–6). (b) Oligonucleotides were assessed in differentpH 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 5–8were analogous in pH values but buffered for 4 h. Those of marks9–12 were also analogous in pH values but buffered for 2 h. Mark 13was a control for reference.

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was correlated with the S 2p doublet with a binding energy(BE) of ≈162.0 eV. The decrease of the intermediate amidebonds due to the addition of the oligonucleotides was calculatedby 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 UsingSSOP Method. The oligonucleotides, Primer 1 and AntisenseProbe, with a concentration of 5 µM were first verified by 100µM SSOP diluted to five different concentrations (Figure 2a).The indications revealed a good relationship with the dilutedconcentrations. The SSOP diluted to 0.1 µM was still distin-guishable (mark 4 in Figure 2a). In Figure 2b, the quality ofthe 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 buffersolution and the reaction time did not cause structural damageor hydrolysis in the oligonucleotides.

3.2. Chemical Structures Examined by FTIR with 75°Grazing Incident Angle Reflectance. An analogous FTIR curveof the pristine surface was taken as the reference and is shownin the bottom curves of Figures 3–6. The characteristic IR peaksfor 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 createan amide bond with the NH2-modified Primer 1 using EDC andNHS as the coupling agents (35, 40). In the case of the acidicenvironment at pH 4.5 and 37 °C (Figure 3), the addition of 1mM EDC and NHS buffered in MES solution (Figure 3b)mainly created ester compounds (1265.5 cm-1 and 1108.4cm-1). The NHS-ester (1744.3 cm-1) was formed transitionally,whereas the coupling of the NH2-modified Primer 1 was notfound. As the buffered concentration of EDC and NHS wasincreased to 10 mM (Figure 3c), IR absorptions of C-O indeoxyribose 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 associationwith Primer 1, were detected (40). Nevertheless, IR absorptionsin the range of 1500–1650 cm-1, correlating with the presence

Figure 3. IR absorption spectra of Primer 1/MHDA/Au prepared indifferent 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 theconcentrations of EDC and NHS at (b) 1 mM, (c) 10 mM, and (d) 100mM.

Figure 4. IR absorption spectra of Primer 1/MHDA/Au prepared indifferent concentrations of EDC and NHS at a pH value of 4.5 at 4 °C.The as-measured surfaces were (a) MHDA/Au and (a) with theconcentrations of EDC and NHS at (b) 1 mM, (c) 10 mM, and (d) 100mM.

Figure 5. IR absorption spectra of Primer 1/MHDA/Au prepared indifferent concentrations of EDC and NHS at a pH value of 7.4 at 37°C. The as-measured surfaces were (a) MHDA/Au and (a) with theconcentrations of EDC and NHS at (b) 1 mM, (c) 10 mM, and (d) 100mM.

Figure 6. IR absorption spectra of Primer 1/MHDA/Au prepared indifferent concentrations of EDC and NHS at a pH value of 4.5 at 4 °C.The as-measured surfaces were (a) MHDA/Au and (a) with the concentra-tions of EDC and NHS at (b) 1 mM, (c) 10 mM, and (d) 100 mM.

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of amide bonds, were considerably weak. It is likely that thesensitivity of the measurement is insufficient to interpret a minorquantity of the intermediate amide bonds. Furthermore, withthe 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 speciesassociated with Primer 1 were simultaneously found.

Analogous results were obtained in the case of the acidicenvironment at pH 4.5 and 4 °C (Figure 4) and the alkalineenvironment at 37 °C (Figure 5) and 4 °C (Figure 6). Bycomparison, the NH2-modified Primer 1 was well coupled withMHDA/Au in the case of 100 mM EDC and NHS buffered inMES solution at pH 4.5 and 37 °C, while IR intensity of thecharacteristic amide group and most of the analogous speciesassociated with Primer 1 were clearly found.

From the above measurements, as the coupling agents werebuffered to a concentration of 10 mM, the covalently bondedPrimer 1 could be identified, whereas the IR intensity correlated

with the amide group, the intermediate structure, was relativelyinsignificant.

3.3. Chemical Bonds Examined by HRXPS. For thepristine MHDA/Au, the characteristic structures of MHDA weredetermined by HRXPS and presented in the bottom curves ofFigures 7 and 8. After carrying out the decompositions of theC 1s, O 1s, and N 1s spectra illustrated in Figure 9, the C 1sspectrum exhibited a major emission peak at a BE of ≈284.8eV (i.e., C*-C or C*-H) with a fwhms of ≈0.9 eV and chemicalshifts 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 witha BE of ≈162.0 eV for the S 2p3/2 component. This doublet iscommonly related to the thiolate species bonded to Ausurface (36, 38, 41, 42). The O 1s spectrum exhibited theemission peaks at BEs of 532.1 eV (C-O) and 533.4 eV (C)O),resulting from the presence of MHDA. No traces of nitrogenor phosphorus were found.

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 illustratedin 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 illustratedin Figure 11.

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Interfacing of surface characterization with analytical data isnot well-developed. Particularly, chemical species of covalentattachment presenting in the multiple linkages are difficult tobe defined by configurations. In this section, the respectivesamples with the coupling agents of 10 mM are purposelystudied using HRXPS to gain information of the aptamers uponMHDA/Au under different pH values and coupling or annealingtemperatures.

The HRXPS spectra for Primer 1/MHDA/Au, prepared atdifferent pH values or temperatures, are shown in Figures 7band d and 8b and d, which exhibit analogous BEs. Followedby hybridization with Antisense Probe, their C and O intensitiesand BEs exhibited variable data (shown in Figures 7c and 7eand 8c and e). Among them, the HRXPS spectra for Primer

1/MHDA/Au prepared in 10 mM EDC and NHS at 37 °C werecurve-fitted and studied. In Figure 9, the decompositions of theC 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*-OHor from the oligonucleotide C. The decompositions of the O 1sspectra were assigned: (1) 532.1 eV, C-O*-C from the oligo-nucleotide G or C, (2) 532.8 eV, amide group or (N)2C)O*from the oligonucleotide G, (3) 533.4 eV, C)O* from theoligonucleotide, (4) 534.4 eV, O*)C-OH or C-O-P from theoligonucleotide and (5) 535.6 eV, NHS-ester. The decomposi-tions 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 fromthe amide group, (3) 400.5 eV, -N*-, -N*H-, -N*H2 from the

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. Thedecomposition of the respective C 1s, O 1s, and N 1s spectra is described in section 3.4.

Oligonucleotides with 16-Mercaptohexadecanoic Acid Bioconjugate Chem., Vol. 18, No. 6, 2007 1901

oligonucleotide G or C, and (4) 402.5 eV, -N*-O from NHS-ester. For the P 2p spectra shown in Figures 7 and 8, theemission peak at a BE of 133.2 eV was associated with thepresence of oligonucleotides.

In Figure 9, the ratio of peak 1 over peak 3 at b of N1s spectrawas 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 1sspectra was close to 3/5 (39), which is approximately the productof Primer 1 in hybridization with Antisense Probe. On the basisof these qualitative measurements, the hybridization betweenPrimer 1 and Antisense Probe noticeably occurred as a resultof the particular treatments.

3.4. Semiquantitative Measurements for S–Au andAmide-Coupled Bonds. The addition of Antisense Probe inhybridization with Primer 1 also resulted in decreasing theHRXPS intensity of the amide group, which was associated withthe assignments of peak 3 of C 1s, peak 2 of O 1s, and peak 2of N 1s (Figure 9). In the case of b of Primer 1 coupled onMHDA/Au at pH 4.5 and c of surface b in hybridization withAntisense Probe, the relative intensities of peak 2 over peak 1in 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 ≈0.57to that of ≈0.22 (or -61.4%) due to the hybridization withAntisense Probe that increased the packing density of theoligonucleotide 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 terminatedthiols with analogous chain length on Au, whereas a relativelydisordered carboxyl terminated SAM on Au is anticipated (43).In our previous calculations, the intermolecular spacing of theheadgroup S–S for MHDA/Au is presumably larger than thatfor CH3 terminated thiols on Au (≈4.97 Å) (44) due to thefunctions and/or the interactions of hydrogen bonds betweenthe tail groups (43). As a result, the headgroup (i.e., S adsorbedon Au) of MHDA/Au is not as closely packed as that of CH3-terminated thiols on Au. As illustrated in Figures 7 and 8, theS–Au bonds kept nearly intact during the coupling andsubsequent hybridization processes. It implied that the MHDAuultrathin film on Au was suitable as a molecular support. Fora stable coupling process, the bond angle for O)C*-O (≈120°)and that for O)C*-N (≈109°) is changed, which will alterthe molecular arrangement as well as the packing density ofthe oligonucleotide on MHDA/Au. With these calculations, the

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 surfaceswere (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-measuredsurfaces 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.

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photoelectron intensity of the intermediate amide bonds continu-ously decreases because of the completion of hybridizationnaturally increases the packing density of the oligonucleotideson MHDA/Au.

The element’s ratios calculated from Figures 7 and 8 withrespect to the pristine MHDA/Au surface are shown in Figures10 and 11, respectively. Note that an equal concentration (i.e.,10 mM) of the coupling agent was used. The intensity ratios ofthe C 1s, O 1s, N 1s, and P 2p spectra increased with theaddition of Primer 1 and subsequent to the hybridization ofAntisense Probe. Those of the S 2p spectra decreased with theaddition of the oligonucleotides because of the increase ofmolecular density on MHDA/Au. No significant traces of othersulfur-derived species (45, 46) were found during the processes.The coupling process in the acid environment (i.e., pH 4.5) eitherat 37 or 4 °C steadily decreased the S 2p intensities, that is, cor e with respect to b or d of S 2p spectra in Figures 10 and 11.In combination with the calculation of molecular density onMHDA/Au, an optimized coupling condition was first suggestedat pH 7.4 and 37 °C. However, the increased packing densityof the oligonucleotides on MHDA/Au resulted in increasing theelements’ 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’ ratioof the oligonucleotide G, C, A, or T, an optimized couplingcondition was suggested at pH 4.5 and 37 °C.

Comparing the case of the coupling temperature at 37 °Cwith that at 4 °C, the former exhibited proficiency in increasingthe quantity of the oligonucleotides on MHDA/Au, particularlyon the basis of the increased intensity ratios of N 1s and P 2pspectra and the decreased intensity ratios of S 2p spectra.However, comparing the pH value for amide coupling at 4.5with that at 7.4, the former exhibited values in correlation withthe elements’ ratio of the oligonucleotide. From these measure-ments, the optimized coupling conditions in this study werepresumably placed at pH 4.5 and 37 °C.

CONCLUSIONS

The use of tailored SAMs chemically adsorbed upon Au isincreasingly important for recent techniques in microelectronics.In combination with specific short-chain oligonucleotides alongwith rich SNP profiles on the microelectronic device, theinvention meets the demands for rapid screening with reducedsample amounts. The coupling of the carboxyl tailed MHDA(≈2 nm) with a 5′-modified amino group oligonucleotide 15-mers in length forms an ultra-thin layer. Such a coupledoligonucleotide is relevant to determine the presence of nucleicacids and to act as a diagnostic probe for a target sequence. Aperceptive detection for the specific molecular binding on amicrostructured chip surface has been highly improved. There-fore, this study particularly emphasizes the stability of molecularinteractions under treatment conditions such as pH values andtemperatures for the coupling or annealing process. On the basisof the high-resolution spectroscopic studies and the definiteconcentration of coupling agents (i.e., 10 mM), the optimizedconditions for the coupling process have been proposed at pH4.5 and 37 °C. At the same time, the S–Au bonds and theintermediate amide-coupled structure are considerably resistantto subsequent treatments. Thus, this work provides a methodto intensify the joining of short-chain DNA strands with thetailored SAMs/Au prior to completing a hybridization process.It is also promising in creating nanopatterned SAMs/Au foradvanced DNA chips, in particular for the detection of SNPs.

ACKNOWLEDGMENT

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

and Technology and the Sustainable Environment ResearchCenter, National Cheng Kung University, and National Syn-chrotron Radiation Research Center, Hsinchu, Taiwan for accessto equipment, technical support, and partial financial support.

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