Characterization of DNA Hybridization on Partially Aminated Diamond

The immobilized DNA on diamond is free from biological interface degradation ...... Elena V. Basiuk , Adriana Santamaría-Bonfil , Victor Meza-Laguna ...
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Langmuir 2006, 22, 11245-11250

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Characterization of DNA Hybridization on Partially Aminated Diamond by Aromatic Compounds Jung-Hoon Yang,†,‡ Kwang-Soup Song,†,‡ Guo-Jun Zhang,‡ Munenori Degawa,†,‡ Yoshinori Sasaki,†,‡ Iwao Ohdomari,†,‡ and Hiroshi Kawarada*,†,‡ School of Science and Engineering, Waseda UniVersity, 3-4-1 Okubo, Shinjuku-ku, Tokyo 169-8555, Japan, and Nanotechnology Research Center, Waseda UniVersity, 513 Waseda Tsurumaki-cho, Shinjuku-ku, Tokyo 162-0041, Japan ReceiVed March 12, 2006. In Final Form: July 18, 2006 Here, we report a novel method of micropatterning oligonucleotides via aromatic groups as linkers on partially amino-terminated diamond and the inherence on subsequent hybridization. The covalent immobilization of probe oligonucleotides and characterization of immobilized probe oligonucleotides with carboxylic compounds were investigated by X-ray photoelectron spectroscopy (XPS). To confirm the effects of linker flexibility in a low amino group on diamond for probe oligonucleotides, three kinds of dicarboxylic compoundsadipic acid, terephthalic acid, and trimesic acidswere used for immobilization of probe oligonucleotides, like linkers; and these oligonucleotides were hybridized with target oligonucleotides labeled with Cy 5 on the micropatterned diamond surface. The hybridization intensities determined by epifluorescence microscopy were compared and analyzed.

Introduction Diamond has attractive characteristics for some biological1 applications, such as its wide potential window,2 chemicalphysical stability,3 and biocompatibility.4 Biological materials, such as neurons5 and DNA,6 have been immobilized on the surface of diamond, because it can be modified with several types of surface termination, e.g., C-NH2, C-OH, and C-COOH. Moreover, an advanced application of diamond for the fabrication of transistor biosensors has been reported using an electrolytesolution-gate diamond field-effect transistor (SGFET) on the enzyme7 or anti-IgG antibody8 immobilized diamond surface. Two methods have been used for the immobilization of DNA on the surface of diamond: electrostatic adsorption9 and covalent binding.10 Of these methods, covalent binding onto several types of termination has mainly been used, because these surfaces provide sites for amide or disulfide bonds or Schiff bases according to the linkers used. The immobilization of DNA has been reported on polycrystalline,11 nanocrystalline,12 and single-crystalline13 * Corresponding author. E-mail: [email protected]. † School of Science and Engineering. ‡ Nanotechnology Research Center. (1) Knicker, T.; Strother, T.; Schwartz, M. P.; Russell, J. N., Jr.; Butler, J.; Smith, L. M.; Hamers, R. J. Langmuir 2003, 19, 1938. (2) Ferro, S.; Battisti, A. D. Anal. Chem. 2003, 75, 7040. (3) Lora Huang, L. C.; Chang, H. C. Langmuir 2004, 20, 5879. (4) Yang, W.; Auciello, O.; Butter, J. E.; Cai, W.; Carlisle, J. A.; Gruen, J. E.; Knickerbocker, T.; Lasseter, T. L.; Russell, J. N., Jr.; Smith, L. M.; Hamers, R. J. Nat. Mater. 2002, 1, 253. (5) Specht, C. G.; Williams, O. A.; Jackman, R. B.; Schoepfer, R. Biomaterial 2004, 25, 4073. (6) Takahashi, K.; Tanga, M.; Takai, O.; Okamura, H. Diamond Relat. Mater. 2003, 12, 572. (7) Song, K. S.; Degawa, M.; Nakamura, Y.; Kanazawa, H.; Umezawa, H.; Kawarada, H. Jpn. J. Appl. Phys. 2004, 43, 814. (8) Yang, W.; Hamers, R. J. Appl. Phys. Lett. 2004, 85, 3626. (9) Thompson, L. A.; Kowalik, J.; Josowicz, M.; Janata, J. J. Am. Chem. Soc. 2003, 125, 324. (10) Lenigk, R.; Carles, M.; Ip, N. Y.; Sucher, N. J. Langmuir 2001, 17, 2497. (11) Takahashi, K.; Tanga, M.; Takai, O.; Okamura, H. Diamond Relat. Mater. 2003, 12, 572. (12) Hamers, R. J.; Butler, J. E.; Lasseter, T.; Nichols, B. M.; Russell, J. N., Jr.; Tse, K. Y.; Yang, W. Diamond Relat. Mater. 2005, 14, 661. (13) Zhang, G. J.; Umezawa, H.; Hata, H.; Zako, T.; Funatsu, T.; Ohdomari, I.; Kawarada, H. Jpn. J. Appl. Phys. 2005, 44, 295.

diamond surfaces. The immobilized DNA on diamond is free from biological interface degradation over time to a greater extent than on other substrates, such as activated solid support, glassy carbon, amine-SiO2, silicon, and gold surfaces, because the interface is composed of strong surface chemical bonds.4 Moreover, modification of the diamond surface is an important objective in electrochemistry for material science to improve its mechanical properties.14 Aromatic groups are expected to act as mediators of electron transfer from oligonucleotides to electrodes if they are present independently between the diamond electrode and the oligonucleotides. Further, amide bonds formed by the combination of the aminated diamond surface and a carboxylic function on the DNA probe are more stable because the carboxylic group is an oxidized aldehyde group. Most conventional DNA chips consist of probe oligonucleotides immobilized on functionalized substrates.11-13 The primary problem in developing a selective DNA chip is retaining high hybridization efficiency in the probe oligonucleotides, because the linker size,15 the control density of probe oligonucleotides,16 and the strong chemical bonds between the surface and the probe oligonucleotides are important parameters for a high efficiency of DNA hybridization. In addition, various linker-based methods provide important information about the hybridization of DNA on the surface.17 However, it is still difficult, and often impossible, to obtain an interrelation between the flexibility of the linker and hybridization, because hybridization must be accompanied with a density of probe oligonucleotides on the substrate.18 We investigated the characteristics of diamond partially aminated by direct amination and strongly immobilized probe oligonucleotides to confirm the relation between the flexibility (14) Allongue, P.; Delamar, M.; Desbat, B.; Fagebaume, O.; Hitmi, R.; Pinson, J.; Saveant, J. J. Am. Chem. Soc. 1997, 119, 201. (15) Uslu, F.; Ingebrandt, S.; Mayer, D.; Bocker, S.; Odenthal, M.; Offenhausser, A. Biosens. Bioelectron. 2004, 19, 1723. (16) Peterson, A. W.; Heaton, R. J.; Georgiadis, R. M. Nucleic Acids Res. 2001, 29, 5163. (17) Taft, B. J.; O’Keefe, M.; Fourkas, J. T.; Kelley, S. O. Anal. Chim. Acta 2003, 496, 81. (18) Peterson, A. W.; Wolf, L. K.; Georgiadis, R. M. J. Am. Chem. Soc. 2002, 124, 14601.

10.1021/la060677l CCC: $33.50 © 2006 American Chemical Society Published on Web 11/04/2006

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Figure 1. The chemical structures of the linkers used. These experiments were performed with adipic acid, terephthalic acid, and trimesic acid as linkers to compare the effects of linker flexibility on hybridization. Moreover, dicarboxylic compounds were used to obtain information about hybridization according to carboxylic function, which represent the binding sites for probe oligonucleotides.

of the linker and the density of the carboxylic groups from dicarboxylic compounds for probe oligonucleotides with regard to hybridization. X-ray photoelectron spectroscopy (XPS) was used to determine the existence of probe oligonucleotides and amino groups on the diamond surface for probe oligonucleotides. The fluorescence intensities of complementary and noncomplementary DNA were compared with adipic acid, terephthalic acid, and trimesic acid by epifluorescence microscopy. Experimental Methods Fabrication of Polycrystalline Diamond and Partially Aminated Surface. A polycrystalline diamond was deposited by the microwave plasma-assisted chemical vapor deposition method (MPCVD) on a silicon substrate (100) in 0.1% methane gas diluted with hydrogen gas. The thickness of the diamond was about 8 µm. After deposition for 14 h, the diamond films were exposed to hydrogen plasma to realize hydrogen termination with a chamber pressure of 50 Torr and a substrate temperature of 800 °C. Prior to diamond surface functionalization, all diamond surfaces were rinsed with DI water, then dried under a stream of nitrogen gas. The partial amination on H-terminated diamond was performed in ammonia gas (99.9%) by ultraviolet (UV) irradiation at a wavelength of 253.7 nm. All processes for partial amination were carried out at room temperature. First, nitrogen gas was introduced before UV irradiation for 5 min to remove oxygen and other activated gases from the UV chamber. Then, the chamber was irradiated with UV in ammonia gas for 4 h. During the amination treatment, the gas was injected into the reaction chamber. Covalent Immobilization of DNA Oligonucleotides. Figure 1 shows adipic acid, terephthalic acid, and trimesic acid used as linkers for immobilization of probe oligonucleotides. All chemicals and solvents were purchased from Kanto Chemical Co., Inc. (Tokyo, Japan). The oligonucleotides used in this study were purchased from

Yang et al. Sigma Genosys Japan (Hokkaido, Japan). After washing three times with DI water for 5 min each time by ultrasonication, the partially aminated diamond surface was treated using a carboxylic compound with a 1:1 mixture of EDC (0.4 M) and NHS (0.1 M) for 1 h to activate the carboxylic function. The substrates were then washed three times with H2O for 5 min and dried under a stream of nitrogen gas prior to probe DNA attachment. The sequence of the complementary probe oligonucleotide with an amino group was as follows: 21-mer, H2N-5′-CCACGGACTACTTCAAAACTA-3′; the noncomplementary probe oligonucleotide had the sequence H2N-5′-ATCGATCGATCGATCGATCGA-3′; and the fluorescence-labeled target oligonucleotide had the sequence 3′-GGTGCCTGATGAAGTTTTGAT-5′-Cy 5. Ultrapure water was obtained from a Millipore system. Probe oligonucleotides were diluted with 3 × SCC to a final concentration of 20 µM. Small droplets of the solution were deposited manually on the surface. The substrate was incubated at 38 °C for 2 h in a humidified hybridization chamber to prevent the droplets drying out, washed once with washing buffer (PBS, 0.1% Tween-20) and three times with H2O, and then dried under a stream of nitrogen gas. XPS Analysis of Immobilized Oligonucleotides. All samples for XPS analysis were prepared for an amination time of 4 h, treatment with 50 mM linker solution, and immobilization of 20 µM aminoterminated probe oligonucleotides. All XPS spectra were subjected to chemical analysis by electron spectrometry using an Ulvac Φ 3300 (Ulvac-Phi, Kanagawa, Japan) with an anode source providing Al KR radiation. The electron takeoff angle (TOA, the angle between the analyzer and the sample surface) was 45 ( 3° relative to the substrate surface after focusing of monochromator radiation on the sample. In a separate test of the effects of the X-ray beam, irradiation of a representative sample for over 1 h using an X-ray source with a power rating of 125 W produced less than 5% variation in the N and P peak intensities.19 The N 1s, P 2p, and C 1s spectra and peak separation were acquired with Multipac spectrum software. The slit width (1.1 mm) and the TOA were kept constant for each of the samples measured to allow each to be probed at the same depth. The C 1s peak was chosen as the reference binding energy (285 eV). Fabrication of Micropatterned Diamond. Au masks for micropatterns were formed on aminated diamond by photolithography. The micropatterns on the partially aminated diamond surface were fabricated using the following steps: (1) deposition of the Au mask by chemical vapor deposition (CVD), (2) prebaking at 80 °C for 10 min to remove adsorbed water molecules, (3) coating with photoresist by spin coating, (4) prebaking at 80 °C for 20 min, (5) patterning by aligner, (6) immersion in drying solution for 20 s, and (7) etching at the outside of micropatterns by KI solution. For the cleaning of KI solution and Au, the diamond surface of the etched Au was sonicated three times with H2O for 5 min. The amino group after etching the Au mask was not destroyed in KI solution, because probe oligonucleotides can be immobilized with the existing amino group. To improve the signal-to-background ratio, the area beside the micropatterns masked by Au was fluorinated by C3F8 plasma treatment (RIE-101iPH; Samco International, Inc., Kyoto, Japan), because the nonspecific adsorption of probe and target oligonucleotides can be minimized by the negative charge of the F-terminated diamond.20 Furthermore, a polyfluorinated cyanine dye was reported to improve performance to a greater extent than cyanine dye in fluorescence imaging, because it reduces aggregation.21 Aminoterminated micropatterns on diamond were fabricated by etching Au patterns. Hybridization on fabricated micropatterns on diamond surfaces is shown in Figure 2. The micropatterns were finally imaged by epifluorescence microscopy (Olympus IX71; Olympus, Tokyo, Japan). (19) Petrovykh, D. Y.; Suda, H. K.; Tarlov, M. J.; Whitman, L. J. Langmuir 2004, 20, 429. (20) Zhang, G. J.; Umezawa, H.; Hata, H.; Zako, T.; Funatsu, T.; Ohdomari, I.; Kawarada, H. Jpn. J. Appl. Phys. 2005, 44, 295. (21) Renikuntla, B. R.; Rose, H. C.; Eldo, J.; Waggoner, A. S.; Armitage, B. A. Org. Lett. 2004, 6, 909.

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Figure 2. A schematic of the fabrication process for micropatterned DNA on the diamond surface: probe nucleotides were immobilized with dicarboxylic compounds on the micropatterned diamond surface.

Results and Discussion XPS Analysis. The elemental composition of the surface was determined by XPS. Quantitative analysis by XPS can be extremely useful for analysis of a large number of substrate surfaces or when monitoring the effectiveness of a particular surface reaction. The average coverage on a functionalized diamond surface was calculated using four samples for high accuracy, and the density of carbon atoms on C(111) is defined as one monolayer (2 × 1015/cm2). The XPS spectra of the diamond surfaces before and after partial amination are shown in Figure 3a. The spectra clearly showed that a new peak of nitrogen (N 1s) at 399.5 eV appeared and increased continuously after irradiation, accompanied with a peak of oxygen (O 1s) at 531.6 eV. Thus, the density of an amino group on the diamond surface increases with ammonia gas as a function of UV irradiation time. Amino groups cannot exist in bulk by amination processes due to the high density of carbon lattices. In addition, ammonia gas is formed like C-NH2 on dangling bonded hydrogen, because C-H bonds react easily with ammonia gas and undergo photochemical reaction on exposure to UV irradiation. Amino groups are present in less than one monolayer on the H-terminated diamond surface. The polycrystalline diamond films were synthesized by the MPCVD method using purified hydrogen and methane gas, and the deposited diamond surface was treated with hydrogen termination in this state; oxidation on the diamond surface could not occur during the synthetic process of diamond with oxygen. In the amination process, some portion of the H-terminated surface was also replaced by NH2 in the ammonia gas chamber by UV irradiation after purging of the air by introducing nitrogen gas for 5 min. In addition, oxygen (O 1s) was also increased as a function of UV irradiation time22 due to surface oxidation and water molecules adsorbed on the oxidized surface. (22) Zhang, G.; Song, K. S.; Nakamura, Y.; Kawarada, H. DNA microarray on directly aminated diamond surface. Langmuir, in press 2005.

We found that the quantity of amino groups on the diamond surface increases as a function of amination time. We characterize the nitrogen (N 1s) and phosphate (P 2p) to quantity the coverage on the modified diamond surface based on the results of XPS, a method commonly used in analysis of XPS spectra.23,24 Figure 3b shows the results of quantitative XPS for polycrystalline diamond substrates subjected to UV irradiation in the presence of ammonia gas. The results indicated that nitrogen (N 1s) on the diamond surface increased the 0.04 ML (monolayer) as a function of amination time. Moreover, the oxygen (O 1s) was also increased with nitrogen by the amination process. The oxygen peak was markedly increased in the 0.13 ML with amination time until 4 h, after which time the amount of oxygen deposited by oxidation or adsorption did not change. The results of XPS analyses confirmed that nitrogen and oxygen levels were increased as a function of the amination process and the amino groups can be created directly on the diamond surface. In addition, the amination rate on the diamond surface can be controlled by UV irradiation time in the presence of ammonia gas, and XPS measurement confirmed the existence of nitrogen in 0.15 ML (3 × 1014/cm2) in four samples, forming aminated micropatterns on diamond. The dicarboxylic compounds, adipic acid, terephthalic acid, and trimesic acid, were used to immobilize amino-modified oligonucleotides on aminated micropatterns as linkers. Table 1 shows the results of XPS analysis after immobilization of different linkers under the same conditions of amination time and concentration. As the three types of linker did not include nitrogen, the results indicated that the amount of nitrogen on the aminated diamond surface was almost the same. The amount of oxygen atoms is the sum of those on the oxidized diamond, adsorbed water molecules, and the linkers. In addition, the coverage of amide binding was comparable among the three types of linker. (23) Petrovykh, D. Y.; Suda, H. K.; Whitman, L. J.; Tarlov, M. J. J. Am. Chem. Soc. 2003, 125, 5219. (24) Ratner, B. D.; Castner, D. G. In Surface AnalysissThe Principal Techniques. Wiley: New York, 1997; p 43.

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Figure 3. XPS spectra of H-terminated and partially aminated diamond surface (A) and nitrogen and oxygen coverage as a function of amination time (B). In graph (A), the formation of partial amino groups on the diamond surface was confirmed by the presence of the N 1s peak and O 1s peak on UV irradiation. XPS spectra from probe oligonucleotides immobilized with trimesic acid on aminated diamond are also shown: N 1s spectrum (C), P 2p spectrum (D). The graphs (C) show combined trimesic acid on amino groups by NHS/EDC (C-1) and immobilized probe oligonucleotides on trimesic acid (C-2) on the diamond surface. The P 2p spectrum indicates the presence of probe oligonucleotides (D). Table 1. Surface Coverage of the Different Linkers on Partially Aminated Diamond surface O 1s N 1s CONH in N 1s

adipic acid

terephthalic acid

trimesic acid

1 × 1015 2 × 1014 4 × 1013

8 × 1014 3 × 1014 5 × 1013

1 × 1015 3 × 1014 5 × 1013

We estimated that there are almost the same number of binding sites for probe oligonucleotide immobilization for all three types of linker, because the results were within the margin of error of 1% for a monolayer of carbon (2 × 1015/cm2). We evaluated the specificity of binding sites for probe oligonucleotide immobilization. Figure 3c,d shows the XPS results for N 1s and P 2p from trimesic acid reacted with amino-modified probe oligonucleotides on partially aminated diamond surfaces, indicating the existence of these elements on the probe oligonucleotide/diamond surface. When trimesic acid combines with the amino group on aminated diamond by amide bonding, three

components at 398.5, 399.5, and 400.8 eV fit the N 1s signal from the activated carboxylic function by intracyclic nitrogen of EDC/NHS, amino group (-NH2), and amide bonding (CONH), as shown in Figure 3c(1). Examination of the trimesic acid combined surface from the partially aminated diamond confirmed that the amount of amide bonding between the amino function on diamond and the carboxyl group on trimesic acid was about 0.013 ML (3 × 1013/cm2) from the separated peaks, because trimesic acid is not included in amide bonding. These observations indicated that there were about 0.013 ML of binding sites for amino-terminated probe oligonucleotides on the functionalized diamond surface, because amino-terminated probe oligonucleotides can be immobilized on a carboxylic group of trimesic acid. As the thickness of the oligonucleotide is about 2 nm, the functionalized diamond surface produced using dicarboxylic compounds had a low density of binding sites for probe oligonucleotides, although combined trimesic acid includes two carboxylic groups.

DNA Hybridization on Partially Aminated Diamond

Figure 4. Fluorescent images of Cy 5-labeled DNA complementary to the probe DNA immobilized within the microstructured patterns: adipic acid (A), terephthalic acid (B), and trimesic acid (C) 10 µm in diameter (1), denaturation (2), and noncomplementary oligonucleotides (3), respectively. The final concentrations of probe and target oligonucleotides were 20 µM/L and 100 nM/L, respectively.

When probe oligonucleotides were immobilized on the activated carboxyl group of trimesic acid, two N 1s components of the amino group and the carboxyl groups activated by EDC/ NHS were increased due to intracyclic and exocyclic nitrogen atoms of the immobilized probe oligonucleotides, as shown in Figure 3c(2). From this amide bonding between immobilized trimesic acid and amino-terminated probe oligonucleotides, the increase in amide bonding was calculated as 0.007 ML (2 × 1013/cm2). Further, the existence of probe oligonucleotides was confirmed by increased intracyclic nitrogen and the generation of phosphate. These observations indicated that trimesic acid can be used as a linker based on the immobilization of probe oligonucleotide at a rate of over 50% on double the number of carboxylic binding sites. Patrick et al. reported two N 1s components at 399.7 eV and 401.1 eV, although they used BM(PEO)4 as a linker.25 Moreover, Cavic et al. reported increases in two peaks of intracyclic and exocyclic nitrogen atoms on hybridization of oligonucleotides.26

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The increases in the two intracyclic and exocyclic nitrogen peaks when probe oligonucleotides were immobilized on the substrate were similar to those reported by Cavic et al. In addition, the existence of immobilized probe oligonucleotides on the diamond surface was confirmed by the presence of the P 2p peak (Figure 3d). The phosphate signal was a good indicator of the immobilized probe oligonucleotides. Characterization of Micropatterned Diamond. Figure 4 shows epifluorescence microscopy images of Cy 5-labeled target oligonucleotides coupled with complementary and noncomplementary probe oligonucleotides with circular micropatterns of carboxylic compound modified surfaces 10 µm in diameter under the same conditions of probe oligonucleotide (20 µL of 20 µM) and target oligonucleotide concentration (20 µL of 100 nM), and temperature (immobilization of probe oligonucleotides at 39 °C, hybridization with target oligonucleotides at 59 °C) in 150 mM phosphate-buffered saline (PBS). The probe oligonucleotides were immobilized to the carboxylic groups within micropatterns (10 µm in diameter; A-1, B-1, and C-1) and were accessible to specific hybridization with their complements (Figure 4). The fluorescent micropatterns were homogeneous in size and shape, corresponding to the circular micropatterns seen with adipic acid, terephthalic acid, and trimesic acid after 1 h of hybridization of Cy 5-labeled target oligonucleotides. Moreover, the fluorescence images confirmed the denaturation of hybridized oligonucleotides on diamond except for the nonspecific adsorption of target oligonucleotides (Figure 4A-2, B-2, and C-2). These results indicated that probe oligonucleotides immobilized by carboxylic compounds as linkers can be hybridized with target oligonucleotides, and nonspecific binding of the noncomplementary probe to the target oligonucleotides labeled with Cy 5 yielded no signals (Figure 4A-3, B-3, and C-3). Further, the nonspecific adsorption of oligonucleotides on the diamond surface was decreased by fluorination. Therefore, the micropatterns on diamond were covalently immobilized with the probe DNA, and hybridization occurred specifically as a result of DNA-DNA interactions. Analysis of Fluorescent Images. To compare hybridization efficiency according to the binding sites for probe oligonucleotides and the flexibility of linkers, the intensities of DNA hybridization were measured on partially aminated diamond using three different carboxylic compounds: adipic acid, terephthalic acid, and trimesic acid (Figure 5). Analysis of the surface with probe oligonucleotides immobilized using dicarboxylic compounds confirmed that an area of 0.007 ML (2 × 1013/cm2) of diamond

Figure 5. The fluorescence intensity profiles of complementary DNA with adipic acid (A), terephthalic acid (B), and trimesic acid (C) on the hybridized areas of the micropatterned surface using H-terminated diamond. All Cy 5-labeled target oligonucleotide hybridization reactions were performed in a humid chamber for 1 h at 59 °C after immobilization of probe oligonucleotides for 2 h at 39 °C.

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was available for hybridization with target oligonucleotides. This result indicated that the density of binding sites for oligonucleotides was lower than for other modified films if carboxylic compounds were used as linkers. Further, the density of immobilized oligonucleotides can be controlled by UV irradiation time to overcome the primary problem for the high efficiency of hybridization.16,18,27 The hybridization intensities on adipic acid and trimesic acid were compared to determine hybridization efficiency according to linker flexibility (Figure 5A, C). In contrast to the results of Taft et al., who concluded that linker flexibility and baseconjugated linkers on the film are key parameters for efficient hybridization, our observations indicated that the flexibility of linkers is not a key parameter at low linker density.17 This discrepancy is most likely because linker flexibility is an important parameter for high efficiency of hybridization at high linker density, but is not related to DNA hybridization at low probe oligonucleotide binding site density due to the decreased steric effects of DNA-DNA interactions. Furthermore, the hybridization intensity on trimesic acid was increased in comparison with that on terephthalic acid. As the number of probe molecules immobilized on trimesic acid cannot be doubled due to steric effects and the intrinsic thickness of the oligonucleotides, the physical adsorption of target oligonucleotides may be decreased by the residual carboxylic function on trimesic acid (Figure 5B,C). These observations indicated that the availability of a large number of negative charges enables a high degree of stability of hybridization at low linker density. (25) Johnson, P. A.; Levicky, R. Langmuir 2003, 19, 10288. (26) Cavic, B. A.; McGovern, M. E.; Nisman, R.; Thompson, M. Analyst 2001, 126, 485. (27) Taira, S.; Yokoyama, K. Anal. Sci. 2004, 20, 267.

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Conclusions In summary, DNA immobilization on a microstructured pattern can be achieved successfully using dicarboxylic compounds on H-terminated diamond. Amino groups, binding sites for carboxylic compounds, can be controlled directly as a function of UV-irradiation time on the H-terminated diamond surface. Furthermore, dicarboxylic compounds can be used as linkers on the partially aminated diamond surface for probe oligonucleotide immobilization, hybridization, and denaturation. As trimesic acid provides twice the number of carboxylic functions for probe oligonucleotides, the hybridization intensity with trimesic acid is similar to that with terephthalic acid. From the viewpoint of linker flexibility, the hybridization intensity with adipic acid is also similar to that with terephthalic acid as a linker. The results of the present study showed that the flexibility of linkers is not related to DNA hybridization under conditions of low probe oligonucleotide density. Therefore, hybridization requires consideration of the surface densities of both the linker and probe oligonucleotide. Thus, the ability to immobilize DNA on the diamond surface by dicarboxylic compounds presents new possibilities for the development and fabrication of specialized biosensor systems. Acknowledgment. This work was supported in part by a Grant-in-Aid for Center of Excellence (COE) Research from the Ministry of Education, Culture, Sports, Science and Technology. This work was also supported in part by the Advanced Research Institute for Science and Engineering, Waseda University. LA060677L