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Langmuir 2006, 22, 3728-3734
DNA Micropatterning on Polycrystalline Diamond via One-Step Direct Amination Guo-Jun Zhang,*,† Kwang-Soup Song,*,†,‡ Yusuke Nakamura,‡ Taro Ueno,§ Takashi Funatsu,§ Iwao Ohdomari,†,‡,| and Hiroshi Kawarada†,‡ Nanotechnology Research Center & Institute of Biomedical Engineering, Waseda UniVersity, Waseda Tsurumaki-cho 513, Shinjuku-ku, Tokyo 162-0041, Japan, Department of Electronical Engineering and Bioscience, School of Science and Engineering, Waseda UniVersity, 3-4-1 Okubo, Shinjuku-ku, Tokyo 169-8555, Japan, Laboratory of Bio-Analytical Chemistry, Graduate School of Pharmaceutical Sciences, The UniVersity of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan, and Kagami Memorial Laboratory for Materials Science and Technology, Waseda UniVersity, 2-8-26 Nishi-waseda, Shinjuku-ku, Tokyo 169-0051, Japan ReceiVed April 4, 2005. In Final Form: NoVember 11, 2005 We report a novel method of one-step direct amination on polycrystalline diamond to produce functionalized surfaces for DNA micropatterning by photolithography. Polycrystalline diamond was exposed to UV irradiation in ammonia gas to generate amine groups directly. After patterning, optical microscopy confirmed that micropatterns covered with an Au mask were regular in size and shape. The regions outside the micropatterns were passivated with fluorine termination by C3F8 plasma, and the chemical changes on the two different surfacessthe amine groups inside the patterned regions by one-step direct amination and fluorine termination outside the patterned regionsswere characterized by spatially resolved X-ray photoelectron spectroscopy (XPS). The patterned areas terminated with active amine groups were then immobilized with probe DNA via a bifunctional molecule. The sequence specificity was conducted by hybridizing fluorescently labeled target DNA to both complementary and noncomplementary probe DNA attached inside the micropatterns. The fluorescence micropatterns observed by epifluorescence microscopy corresponded to those imaged by optical microscopy. DNA hybridization and denaturation experiments on a DNAmodified diamond show that the diamond surfaces reveal superior stability. The influence of a different amination time on fluorescence intensity was compared. Different terminations as passivated layers were investigated, and as a result, fluorine termination points to the greatest signal-to-noise ratio.
Introduction With the development of surface chemistry, many efforts have been made to realize real-time sensing and long-term monitoring of biomolecule interaction.1-3 Future biosensors are expected to make the best of signal amplification, processing, and the strengths of microelectronics. To this end, the attachment of biomolecules to microelectronics-compatible materials such as glass,4,5 gold,6 and silicon7,8 has been well developed. However, the interfaces between these materials and biomolecules lack long-term stability that is critical to developing integrated biosensors. Diamond is a particular material with high chemical stability9 and high sensitivity in electrochemical reactions.10 Moreover, it can also * To whom correspondence should be addressed. E-mail: zhang@ ohdomari.comm.waseda.ac.jp;
[email protected]. Tel and Fax: 81-3-5286-3391. † Nanotechnology Research Center & Institute of Biomedical Engineering, Waseda University. ‡ Department of Electronical Engineering and Bioscience, Waseda University. § The University of Tokyo. | Kagami Memorial Laboratory for Materials Science and Technology, Waseda University. (1) Yang, W. S.; Hamers, R. J. Appl. Phys. Lett. 2004, 85, 3626. (2) Cui, Y.; Wei, Q.-Q.; Park, H.-K.; Lieber, C. M. Science 2001, 293, 1289. (3) Fritz, J.; Cooper, E. B.; Gaudet, S.; Sorger, P. K.; Manalis, S. R. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 14142. (4) Guo, Z.; Guilfoyle, R. A.; Thiel, A. J.; Wang, R.; Smith, L. M. Nucleic Acids Res. 1994, 22, 5456. (5) Zammatteo, N.; Jeanmart, L.; Hamels, S.; Courtois, S.; Louette, P.; Hevesi, L.; Remacle, J. Anal. Biochem. 2000, 280, 143. (6) Herne, T. M.; Tarlov, M. J.J. Am. Chem. Soc. 1997, 119, 8916. (7) Zhang, L.; Strother, T.; Cai, W.; Cao, X. P.; Smith, L. M.; Hamers, R. J. Langmuir 2002, 18, 788. (8) Zhang, G.-J.; Tanii, T.; Zako, T.; Funatsu, T.; Ohdomari, I. Sens. Actuators, B 2004, 97, 243.
be deposited as a robust thin film on silicon and other microelectronics-compatible substrates. The unique properties of diamond make it an attractive substrate for chemical and biochemical modifications used to develop biosensors. Biomolecules, in particular, DNA, have successfully been attached to a variety of diamond surfaces associated with several approaches to the chemical modification of diamond proposed previously.11-14 Chemical modification on diamond usually involves two steps to produce amine-terminated surfaces via H-terminated diamond surfaces, which are subsequently functionalized with biomolecules. Although the reaction mechanism has not yet been clearly elucidated, the number of amine groups deposited on diamond might be reduced if a two-step process of generating amine groups is adopted. It may subsequently impact the surface coverage of amine groups, which is crucial for the immobilization of biomolecules. Beyond a doubt, the surface properties of diamond influence the corresponding immobilization of probe DNA molecules. To improve the surface density of probe DNA for hybridization, it is important to understand the conditions affecting the interaction of DNA with diamond. (9) Pleskov, Y. V.; Sakharova, A. Y.; Krotova, M. D.; Bouilov, L. L.; Spitsyn, B. V. J. Electroanal. Chem. 1987, 228, 19. (10) Swain, G. M.; Ramesham, R. Anal. Chem. 1993, 65, 345. (11) Yang, W. S.; Auciello, O.; Butler, J. E.; Cai, W.; Carlisle, J. A.; Gerbi, J. E.; Gruen, D. M.; Knickerbocker, T.; Lasseter, T. L.; Russell, J. N.; Smith, L. M.; Hamers, R. J. Nat. Mater. 2002, 1, 253. (12) Knickerbocker, T.; Strother, T.; Schwartz, M. P.; Russell, J. N.; Smith, L. M.; Hamers, R. J. Langmuir 2003, 19, 1938. (13) Ushizawa, K.; Sato, Y.; Mitsumori, T.; Machinami, T.; Ueda, T.; Ando, T. Chem. Phys. Lett. 2002, 351, 105. (14) Takahashi, K.; Tanga, M.; Takai, O.; Okamura, H. Diamond Relat. Mater. 2003, 12, 572.
10.1021/la050883d CCC: $33.50 © 2006 American Chemical Society Published on Web 03/18/2006
DNA Micropatterning on Polycrystalline Diamond
Besides polycrystalline diamond films, nanocrystalline diamond films have also been confirmed to be robust, highly selective substrates for biological modification.11 When the preparation approaches of nanocrystalline and polycrystalline diamond films are considered, the conditions needed to produce nanograined diamond are seen to be no different in terms of severity from those required to produce microcrystalline diamond. Only a simple transition between the two morphologies is required and is accomplished by altering the rate of renucleation/rate of growth ratio. This can be done by gas-phase adjustment or by substrate pretreatment. The polycrystalline and nanocrystalline diamond films have both been employed to undergo chemical modification for the immobilization of DNA and hybridization. However, an indirect strategy was introduced to generate amine groups on polycrystalline and nanocrystalline diamond films for the functionalization of the surfaces with DNA as described.11,12 Single-crystal diamond, another diamond material, is too expensive to be applicable, although it is an extremely flat surface. Herein, we show a one-step chemical modification to produce amine groups directly on polycrystalline diamond for patterning oligonucleotides with a fluorine-terminated surface as a passivation layer. We undertook the present study to investigate direct amination on polycrystalline diamond for DNA micropatterning. We chose micropolycrystalline diamond instead of single-crystal diamond or nanocrystalline diamond films for these experiments because it is cheap and easy to use. Polycrystalline diamond films are therefore ideal surfaces to functionalize with biological molecules. We then employed surface-sensitive analytical methods, such as XPS and epifluorescence microscopy, to characterize amine-modified surfaces by one-step direct amination on polycrystalline diamond because we wanted to identify the influence of different amination times on signal intensities after the immobilization of probe DNA and hybridization and evaluate the influence of different passivation layers on DNA patterning. Of significant interest in this work is that extremely good control over the feature size and shape of the DNA micropatterns has been obtained. Furthermore, amine groups are produced on a hydrogen-terminated diamond surface in only one step, not two, and more importantly, the lithographic process is introduced to immobilize DNA preferentially on the designated regions by satisfactorily controlling the chemical functionalization inside patterned regions and allowing very little nonspecific adsorption outside the patterned regions. This work is expected to benefit the chemical modification of diamond for biosensing applications. Experimental Section Materials. All of the chemicals and solvents used in this experiment were purchased from Kanto Chemical Co. Inc. (Tokyo, Japan). The DNA oligonucleotides used were purchased from Sigma Genosys Japan (Hokkaido, Japan). The complementary and noncomplementary oligonucleotides used for immobilization were amine modified at the 5′ end, and the oligonucleotides employed for hybridization to the surface were Cy 5 labeled at the 5′ end. Each of these oligonucleotides was 21-mers long. The three 21-mer sequences employed were H2N-5′-CCACGGACTACTTCAAAACTA-3′ (complementary), H2N-5′-ATCGATCGATCGATCGATCGA3′ (noncomplementary), and Cy 5-5′-TAGTTTTGAAGTAGTCCGTGG-3′ (target). H-Terminated Polycrystalline Diamond on Silicon. Polycrystalline diamond films were synthesized on a p-type silicon substrate (100) by a microwave-plasma-assisted chemical vapor deposition (MPCVD) method using purified hydrogen (396 sccm) and methane (4 sccm) at 45.5 Torr, 1.2 kW, and 840 °C for 12 h in a microwave plasma reactor (ASTEX 2115). The thickness of the deposited diamond film was approximately 8 µm. After deposition,
Langmuir, Vol. 22, No. 8, 2006 3729 the diamond surface was hydrogenated by plasma treatment in a hydrogen gas environment.15 Direct Amination on H-Terminated Polycrystalline Diamond. The hydrogen-terminated diamond surface was aminated by UV irradiation in an ammonia gas environment to produce amine groups directly. The wavelength of the UV light (halogen lamp) was 253.7 nm. Before UV irradiation, nitrogen gas was introduced for 6 min to remove oxygen and other activated gases in the UV chamber. Micropatterning by Photolithography. Au film approximately 100 nm thick was deposited on the polycrystalline diamond as a mask, and another resist film was then coated on the gold layer. The negative patterns were made by photolithography and obtained by etching off the gold with a solution containing KI and I2. After the removal of the resist film, the etched areas were exposed to C3F8 plasma, using inductively coupled plasma (ICP), for 20-30 s to generate a fluorine-terminated surface as a passivation layer outside micropatterned regions. The micropatterns, in which the aminemodified surface was still covered with the gold mask and showed no problems from the C3F8 plasma inside the micropatterned regions, finally occurred when the remaining gold mask on the surface was etched off. As a result, the amine groups were again exposed inside the micropatterned regions for the immobilization of DNA after the gold mask was etched. Immobilization of Probe DNA on Micropatterned Regions. The micropatterned polycrystalline diamond was treated with a solution of 1% glutaraldehyde in water for 1 h and washed three times with water. Twenty-one-mer 5′ amine-modified oligonucleotide probes were diluted with 3 × SSC to a final concentration of 20 µM. Twenty microliters of the solution was deposited manually on the surface, which was covered with a slip. The substrates were incubated at 38 °C for 2 h in a humidified chamber and then washed once with a washing buffer (PBS, 0.1% Tween-20), once with PBS, and once with H2O and then blow dried. Hybridization and Detection. The DNA-modified polycrystalline diamond was exposed to 2 µL of 1 µM 5′ Cy 5-labeled target DNA in 2 × SSC containing 0.2% SDS, covered with a slip, and hybridized at 59 °C for 1 h in a humidity chamber. The hybridized micropatterns were then rinsed once for 5 min in 2 × SSC containing 0.2% SDS, once for 5 min in 2 × SSC, and once for 5 min in 0.2 × SSC to remove any unhybridized complement. The DNA micropatterns were finally observed by epifluorescence microscopy.
Results and Discussion The schematic diagram of micropatterning DNA on polycrystalline diamond via one-step direct amination is illustrated in Figure 1. The amine groups are directly generated on polycrystalline diamond by one-step amination. Photolithography is used to fabricate micropatterns in which amine groups still remain inside patterned areas and fluorine termination as a passivation layer is deposited outside patterned regions. DNA is then patterned on the designated areas and undergoes a hybridization with a target labeled with Cy 5. To observe the shape of micropatterns fabricated by photolithography, we imaged the micropatterns with the gold mask by optical microscopy. Before it could be immobilized with DNA on patterned areas, the gold mask had to be etched off, which led to difficulties in imaging and evaluating the shape of the micropatterns. After the removal of the resist, the patterned regions were covered with the gold mask. Therefore, the shape of the gold micropatterns has a decisive impact on fluorescence DNA patterns after DNA immobilization and hybridization. Shown in Figure 2 are gold micropatterns on polycrystalline diamond fabricated by photography. Four types of micropatterns were fabricated: 20-µm size with 20-µm spacing (Figure 2a), 15-µm size with 15-µm spacing (Figure 2b), 10-µm size with 10-µm (15) Song, K. S.; Sakai, T.; Kanazawa, H.; Araki, Y.; Umezawa, H.; Tachiki, M.; Kawarada, H. Biosens. Bioelectron. 2003, 19, 137.
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Figure 1. Schematic diagram of micropatterning DNA on polycrystalline diamond via one-step direct amination by photolithography.
Figure 2. Optical images of micropatterns covered with Au as a mask prior to being etched off: (a) 20-µm size with 20-µm spacing, (b) 15-µm size with 15-µm spacing, (c) 10-µm size with 10-µm spacing, and (d) 5-µm size with 5-µm spacing. The scale bar represents 20 µm.
spacing (Figure 2c), and 5-µm size with 5-µm spacing (Figure 2d). As shown, the shape of each pattern is very regular and well proportioned. Meanwhile, the corresponding microarrays are homogeneous. These uniform micropatterns reveal that their shapes are well controlled by the surface-patterning technique. However, polycrystalline diamond films have a somewhat rough surface with approximately 8-µm thickness, which leads to difficulties in imaging small patterns optically. Consequently, minimal gold patterns down to a size of 5 µm can be fabricated on polycrystalline diamond in this work. To investigate the amine groups generated within the patterned regions by one-step direct amination and fluorine termination outside these regions, a spatially resolved X-ray photoelectron spectroscopy (XPS) was used to characterize the chemical changes on the two different surfaces. However, because of the limitation of resolution of a spatially resolved XPS, it is impossible to use a conventional XPS to characterize the amine-modified surface
inside a 20-µm micropattern. Thus, larger patterns of 500 µm were made using the same fabrication process as for 20-µm patterns fabricated by photolithography. The amine-modified surface in the patterned area of 500 µm and the fluorine-terminated layer between the patterns were then characterized by XPS with a monochromatic Al KR X-ray (1486.7 eV) source. Figure 3 shows the XPS spectra for the C 1s, O 1s, N 1s, and F 1s areas on the two different surfaces. In the amine-modified areas, the main carbon C 1s from bulk diamond was detected at 284.8 eV after modification with ammonia gas under UV for 2.5 h. The N 1s spectrum shows a significant peak centered at 402.0 eV, indicating that all the nitrogen was present as amine. Noticeably, steric limitations will certainly limit the amine group coverage on the surface. In other words, not every terminal H group will be replaced with an NH2 group. The theoretical coverage (maximum) of amine groups on the surface calculated from XPS data is 12%. The O 1s spectrum shows a peak at 531.1 eV because an aminated polycrystalline diamond surface is highly hydrophilic, leading to the adsorption of water molecules in the air on the surface. However, the hydrogenated diamond surface is somewhat oxidized by ozone in the UV chamber. The O 1s signal seen on the aminated surface is actually attributed to both adsorbed water and surface-bound oxygen. Therefore, the O 1s signal occurs under any conditions. Generally, one would expect the NH2 groups to be air oxidized to NO2 groups. Therefore, another experiment was conducted by storing the directly aminated diamond surface in air for 6 weeks and measuring the XPS of the surface once again. The XPS data show that N 1s peak did not change, indicating that the NH2 groups generated on the diamond surface are very stable and do not change to NO2 groups via a long-time exposure to air. However, the solution containing KI and I2 is able to etch a Au mask, but it is a strong oxidizing agent that can also damage the aminated diamond surface under long-time incubation of the substrates with the solution. After 3 min of incubation, the XPS spectrum shows that a very weak N 1s peak appears, which indicates that almost all amine groups on the surface have been oxidized. To etch the Au mask completely and stabilize the functional amine groups on the surface, 1-min incubation was employed in this experiment. The theoretical coverage of amine groups on the surface calculated from XPS data is 6%, showing that 50% of amine groups are oxidized after
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Figure 3. X-ray photoelectron spectra of an aminated polycrystalline diamond surface.
the removal of the Au mask. Usually, DNA hybridization does not require dense probe DNA because of steric hindrance. Such a loss of amine coverage of the surface caused by oxidization of the solution of KI and I2 does not affect the subsequent hybridization process. The resulting amine coverage is sufficient for DNA immobilization and hybridization, thereby resulting in the very strong fluorescence signals shown in Figure 4a-d. The number densities of carbon, nitrogen, and oxygen groups calculated on the basis of XPS data are 1 × 1015, 1.2 × 1014, and 2.9 × 1014 atoms/cm2, respectively. The corresponding ratios of O/C and N/C are 0.29 and 0.12, respectively. As described above, the N/C signal does not change as a function of time during exposure to air. However, in the fluorine-protected areas only C 1s and F 1s peaks were detected at 284.8 and 685.9 eV, respectively. However, no O 1s or N 1s peaks were observed on this surface. The number density of fluorine groups calculated on the basis of XPS data is 7.7 × 1014 atoms/cm2, and the ratio of F/C is 0.77. These XPS results show that the diamond surfaces have successfully been terminated with amine groups inside the patterned regions and with fluorine outside them. After being passivated with fluorine termination outside the patterned regions, the amine-patterned diamond surfaces were reacted with a cross-linker molecule, glutaraldehyde. The aldehyde group at one end in this molecule reacts specifically with the amine groups of the surface to form an imide bond. The other aldehyde moiety was then reacted with amine-modified DNA. To demonstrate sequence specificity, two types of sequences, complementary and noncomplementary, were employed. After patterning amine-modified DNA on diamond surfaces, the sequence specificity was investigated using surfaceimmobilized sequences, complementary and noncomplementary, to hybridize with the applied fluorescently labeled DNA. The Cy 5-labeled target DNA was hybridized to the micropatterned surface immobilized with complementary and noncomplementary sequences. Figure 4a-e shows the fluorescence images of a polycrystalline diamond surface patterned with two sequences via a hybridization with Cy 5-labeled target DNA. Figure 4a-d shows that hybridization occurs as expected. Strong intensity in the micropatterned areas has been observed, indicating that the
specific binding of the probe complementary to the target had occurred. Moreover, the shape of these fluorescence micropatterns corresponded to those observed by optical microscopy. Quantitatively, the fluorescence intensity of each spot on each scale analyzed in Figure 4a-d reveals uniformity and little difference. However, Figure 4e shows that no patterns are distinguishable from the background, which indicates that no hybridization occurred between a noncomplementary probe and the target. Furthermore, all of the images show a low background, revealing that little nonspecific adsorption of DNA was observed on the fluorinated surface. Consequently, the complementray and noncomplementary sequences are both patterned on the surface and undergo hybridization with a target, and hybridization occurs only between a pair of complementary sequences, as expected. Diamond has been demonstrated to be unique in its ability to achieve very high stability by the comparison of DNA-modified ultrananocrystalline diamond films with other commonly used surfaces for biological modification, such as gold, silicon, glass, and glassy carbon.11 To investigate the stability of the surfacebound oligonucleotides on the directly aminated polycrystalline diamond, we conducted repetitive cycles of hybridization and denaturation on patterned polycrystalline diamond using fluorescence imaging. In each cycle, the complementary oligonucleotides that were patterned on the surface were hybridized with their fluorescently labeled complementary targets for 1 h at 59 °C in a humidity chamber. The DNA microarrays were imaged, and the mean intensity of fluorescence was measued. The substrate was then denatured twice with an aqueous solution of 0.2 M NaOH containing 0.1% SDS at 37 °C for 15 min and rinsed with 2 × SSC and distilled water, and the fluorescence intensity was measured again after each denaturation with epifluorescence microscopy to confirm that it was zero. This hybridization/ denaturation process was repeated 20 times. As shown in Figure 4f, the mean fluorescence intensity measured in relative fluorescence units exhibits almost no loss of DNA over the 20 hybridization/denaturation cycles. This experiment further demonstrates that the DNA immobilized preferentially inside micropatterns has ultrahigh stability on the directly aminated diamond surface.
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Figure 4. Fluorescence images and fluorescence intensity profile of the hybridization of a Cy 5-labeled target DNA with complementary probe (a-d) and noncomplementary probe (e) micropatterned on polycrystalline diamond with fluorine termination as a passivated layer outside the patterns: (a) 20-µm size with 20-µm spacing, (b) 15-µm size with 15-µm spacing, (c) 10-µm size with 10-µm spacing, and (d) 5-µm size with 5-µm spacing. The scale bar represents 20 µm. (f) Stability of DNA-modified polycrystalline diamond during 20 successive cycles of hybridization and denaturation.
To determine surface coverage of the attached DNA molecules, we used a method similar to the one reported by Knickerbocker et al.12 Complementary probe DNAs were immobilized on aminated diamond surfaces with no pattern using glutaraldehyde as a linker, and fluorescently labeled target DNA was hybridized and washed thoroughly with buffer. The substrate was then denatured with 5 mL of an aqueous solution of 0.2 M NaOH containing 0.1% SDS. The surfaces were observed with the epifluorescence microscopy to ensure that the denaturation of the duplex was complete. The fluorescence yielded from the solution was measured on a fluorimeter. The concentration of DNA strands present in the sample was determined by reference to a standardization curve prepared from a series of fluorescently labeled DNA samples of known concentrations. These experiments yielded a concentration of 2.5 × 1012 DNA molecules/ cm2, which is in good agreement with the result observed on diamond.12
Many factors may affect the direct amination reaction on polycrystalline diamond, among which the amination time is most likely to be the key issue. To demonstrate the influence of amination time on the resulting fluorescence signals, various durations of amination were used to generate amine groups for DNA immobilization and subsequent hybridization, resulting in the corresponding DNA fluorescence micropatterns. However, the fluorescence images observed are micropatterned arrays on a different scale. To compare the influence of different amination times on the resulting signal intensity, we cropped the spots from one fluorescence image (patterning with a 20-µm size) at each different amination time and arranged them in an array corresponding to the different times. An obvious correlation of amination times and signals is shown in Figure 5. Clearly, at a short amination time of less than 30 min the hydrogenated diamond surface was not completely aminated, resulting in less probe DNA being immobilized on patterned areas and weaker
DNA Micropatterning on Polycrystalline Diamond
Figure 5. Influence of direct amination time on the resulting fluorescence intensity via hybridization. The scale bar represents 20 µm.
fluorescence signals being observed by fluorescence microscopy via hybridization. The fluorescence intensity increases along with an enhancement of the amination time, but it does not change obviously after 2.5 h. In this work, 2.5 h is the optimal amination time. Because the hydrophobicity and electronegativity of fluorine termination have excess electron density at the surface, the fluorinated diamond surface is capable of minimizing the adsorption of unspecific DNA. However, the use of ICP for fluorine termination involves special gas and expensive equipment, which is a limitation of DNA microarray fabrication. To this end, an oxygen-terminated surface deposited by an easy method as another kind of passivation layer was investigated. Moreover, to confirm the necessity of a passivation layer for DNA patterning, we conducted a control experiment that required that no treatment be conducted outside the patterned areas after the removal of the resist. Figure 6 shows fluorescence images (patterning with a 20-µm size) using three different passivated layers. If the surface outside the patterned areas was not treated by any molecules, it is still terminated with amine groups, subsequently immobilized with probe DNA, and hybridized with
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fluorescently labeled target DNA, thereby resulting in barely visible fluorescence patterns because there is no contrast between the patterned regions and the background (Figure 6c). In another case, the polycrystalline diamond surface outside patterned regions was oxidized by UV and ozone to produce oxygen termination, instead of fluorine termination, as another passivation layer.15 Oxygen termination is negatively charged. However, because of incomplete oxidization by UV and ozone on diamond, this surface is probably terminated not only with ether but also with hydroxyl. A small quantity of hydroxyl termination on polycrystalline diamond makes the surface slightly hydrophilic, thereby reducing the signal-to-noise ratio (Figure 6b). The fluorescence signals in Figure 6b are much weaker than those in Figure 6a (fluorine termination as a passivation layer). These results demonstrate that oxygen termination may be used as a passivation layer but that fluorine termination is the best candidate.
Conclusions The functionalization of diamond surface is of great importance to the attachment of biomolecules. One-step direct amination is able to modify polycrystalline diamond with amine groups, although the exact mechanism of this reaction on the surface has not yet been determined. The functionalized diamond surface can be used to micropattern DNA by introducing a passivated layer outside the patterned areas using photolithography. The above results show that DNA fluorescence micropatterns fabricated on polycrystalline diamond by direct amination are selective, stable, and significantly homogeneous in shape and size. The direct amination method on polycrystalline diamond
Figure 6. Comparison of fluorescence images using three different passivated layers involving fluorine (a), oxygen (b), and amine termination (c), respectively. The scale bar represents 20 µm.
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is easy and effective and is expected to be a new method of chemical modification for integration with biomolecules. Acknowledgment. This work is supported in part by the Japan Society for the Promotion of Science (JSPS) and by the Core Research for Evolutional Science and Technology (CREST)
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of the Japan Science and Technology Corporation (JST) and by a Grant-in-Aid for Center of Excellence (COE) Research and the Establishment of Consolidated Research Institute for Advanced Science and Medical Care from the Ministry of Education, Culture, Sports, Science and Technology, Japan. LA050883D
