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Copyright © 2004 Oxford University Press Submicron patterning of DNA oligonucleotides on silicon *To whom correspondence should be addressed at School of Electronics and Computer Science, University of Southampton, Highfield, Southampton, SO17 1BJ, UK. Tel: +44 0 23 8059 6505; Fax: +44 0 23 8059 3092; Email: tm/at/ecs.soton.ac.uk Received June 9, 2004; Revised July 9, 2004; Accepted July 27, 2004. | |||||||||||||
Abstract The covalent attachment of DNA oligonucleotides onto crystalline silicon (100) surfaces, in patterns with submicron features, in a straightforward, two-step process is presented. UV light exposure of a hydrogen-terminated silicon (100) surface coated with alkenes functionalized with N-hydroxysuccinimide ester groups resulted in the covalent attachment of the alkene as a monolayer on the surface. Submicron-scale patterning of surfaces was achieved by illumination with an interference pattern obtained by the transmission of 248 nm excimer laser light through a phase mask. The N-hydroxysuccinimide ester surface acted as a template for the subsequent covalent attachment of aminohexyl-modified DNA oligonucleotides. Oligonucleotide patterns, with feature sizes of 500 nm, were reliably produced over large areas. The patterned surfaces were characterized with atomic force microscopy, scanning electron microscopy, epifluorescence microscopy and ellipsometry. Complementary oligonucleotides were hybridized to the surface-attached oligonucleotides with a density of 7 × 1012 DNA oligonucleotides per square centimetre. The method will offer much potential for the creation of nano- and micro-scale DNA biosensor devices in silicon. | |||||||||||||
INTRODUCTION Recent advances in the methodology used for the immobilization of biomolecules in patterns on surfaces have enabled the rapid development of biosensors for the analysis of DNA (1,2). Numerous DNA sensors with patterned biomolecular motifs, of feature sizes from tens to hundreds of microns, have been fabricated (1–6). An increasing need for miniaturized tools for pharmaceutical and diagnostic applications (7), as well as the newly emerging areas of DNA-based nanoassembly of electronic devices (8) has resulted in the need to develop methods for the high-resolution patterning of DNA on surfaces at the nanometre scale. Scanning probe lithography or dip-pen nanolithography offers attractive possibilities for precisely anchoring nanoscale patterns of DNA on gold (9,10). Feature sizes of the order of tens to a few hundred nanometres are achievable, although, unfortunately, with slow ‘write speeds’ and small patterning areas. In contrast, the advanced lithographic techniques used in the microelectronics industry achieve high-throughput fabrication of lateral dimensions in a range of a few nanometres to hundreds of microns (11) and would have much potential for the construction of miniaturized biosensors. There have already been a number of approaches derived from photolithographic methods for the attachment of biomolecules to surfaces, which can be categorized as follows.
Crystalline silicon has an excellent surface chemical purity and flatness, and it is ubiquitous in the semiconductor industry. Although largely unexplored to date, the use of bulk silicon as a substrate for biosensor construction is anticipated to enable further miniaturization of existing device technologies as well as to provide a platform for novel biosensor creation, such as practical devices that are actuated by DNA-mediated charge transfer (21) and nanowires (8). The possibility for the direct immobilization of DNA on crystalline silicon was facilitated by the discovery of the photo and thermal reaction of hydrogen-terminated silicon surfaces with alkenes (22). The first examples of the immobilization of DNA on silicon have recently been demonstrated (23–26). For all the methods presented so far, the whole silicon surface was modified with functionalized alkenes and the DNA oligonucleotide solutions spotted onto the modified silicon surface; oligonucleotide patterns on the millimetre scale have been achieved (25,26). Although nanometre-sized motifs have been fabricated on gold surfaces (9,10), gold is incompatible with microelectronics fabrication because of contamination issues (gold easily diffuses into silicon, thus affecting the electrical properties of the subsequent microelectronic devices). We were motivated to explore the potential of creating nanometre-sized motifs of covalently attached DNA oligonucleotides on surfaces for the creation of silicon-based biosensors. It was important to provide a highly reproducible method for the synthesis of motifs with high oligonucleotide density and accessibility towards selective hybridization with the complementary sequence. Chemical modification of the silicon surface has been tailored for optimization of the photoreaction and to facilitate the immobilization of the oligonucleotide (27). In this paper, we present the first report of the fabrication of DNA oligonucleotide patterns on silicon on a submicron scale. The attachment of the DNA to the surface is achieved in a straightforward two-step process. The immobilized oligonucleotide patterns show excellent hybridization properties and high attachment densities. The method will offer the potential for the creation of a range of novel silicon-based biosensing devices in the future. | |||||||||||||
MATERIALS AND METHODS Chemicals All chemicals were obtained from Aldrich Chemical Company and used without further purification. Deionized water (ELGA genetics system) was used for all the aqueous solutions. Undecylenic acid N-hydroxysuccinimide ester (UANHS) was synthesized inhouse as described elsewhere (27).Scanning electron microscopy Scanning electron microscopy (SEM) (LEO 430 SEM, Oxford Instruments) was used to image the alkene-patterned silicon surfaces, which were scanned at low voltage without gold coating. Alkene-patterned surfaces, once evaluated by SEM, were not used further; identically prepared samples were used for the attachment of DNA oligonucleotides.Epifluorescence microscopy The fluorescein-labelled oligonucleotide-patterned surfaces were visualized using a Zeiss Axiovert 200 inverted microscope, coupled with epifluorescence illumination and fitted with filter sets. For low-resolution imaging, all samples were placed face down on a drop of Tris–HCl buffer over a microscope slice towards an Epiplan-Neofluar 10× or 20× objective lenses. The fluorescence images were captured under the same imaging conditions for each experiment. The fluorescence signal-to-noise ratio (SNR) was determined by comparing the fluorescence intensity of single pixel points within the patterned area to the surrounding background using AxioVision Viewer software (Carl Zeiss). At least 20 random points within a single scan were determined. The average value of three to six scans for one wafer was given.The fluorescence images of submicron, oligonucleotide-patterned silicon surfaces were obtained using a confocal laser microscope (Carl Zeiss, LSM510 Meta) with an Epiplan-Neofluar 63× (water immersion) objective lens. The excitation laser wavelength was fixed at 488 nm and the emission filter was a 505 nm long pass. A laser power of 6 mW and a pixel dwell time of 3.2 μs were used. All samples were covered in Tris–HCl buffer (20 mM Tris–HCl, 100 mM MgCl2, pH 8.0) during the imaging process. Atomic force microscopy An Explorer™ atomic force microscopy (AFM) from Veeco Instruments was used in the tapping mode with high-resonance-frequency, non-contact tips for scanning the surface of the oligonucleotide patterned silicon. All measurements were carried out under air-ambient conditions (temperature of 20°C and relative humidity of 45%).Ellipsometry Ellipsometric measurements were carried out with an imaging ellipsometer (I-Elli2000, Nanofilm Technology) as described previously (27).Photopatterning of alkene layers A single-polished silicon wafer (100) was pre-cleaned as described in (27), etched in 2% aqueous hydrofluoric acid for 2 min, washed thoroughly with water, dried under pressurized nitrogen and immediately spin-coated with 40 μl of 10% UANHS solution in CH2Cl2 at 2000 r.p.m. for 20 s.The wafer was exposed using a KrF excimer laser (IPEX™-800, GSI Lumonics) at a wavelength of 248 nm through a photolithographic mask or a phase mask. The photolithography masks were prepared with chrome-coated UV grade fused silica (Suprasil) using the wet etching method. A +1/−1 phase mask, having a periodicity of 2 μm was fabricated by the Ibsen Photonics Company. The masks were placed above a silicon wafer. After photoreaction, the wafer was thoroughly washed with dichloromethane and rinsed with acetone. Immobilization of DNA oligonucleotides Oligonucleotides were synthesized in-house using conventional phosphoramidite chemistry and purified by reverse phase high-performance liquid chromatography (HPLC). Oligonucleotide concentrations were determined using extinction coefficients at a wavelength of 260 nm. The sequences used are 5′-FAM-TGC AGA TAG ATA GCA GT-3′–aminomodifier C7 CpG (Glen Research) (oligonucleotide A), 5′-TGC AGA TAG ATA GCA GT-3′–aminomodifier C7 CpG (oligonucleotide B) and 5′-TGC AGA TAG ATA GCA GTTTTTTTTTTTTTTTT-3′–aminomodifier C7 CpG (oligonucleotide BT). Sequence A was labelled with the fluorescein dye at the 5′ end, which allows for the direct visualization of the fluorescent pattern of the attached single strands of the oligonucleotide. The oligonucleotides were dissolved in a 0.1 M sodium carbonate solution to a concentration of 10 μM (pH = 8.4). An aliquot of 8 μl of the oligonucleotide solution was deposited on the alkene-patterned silicon wafer and covered with a cover slip (2 × 2 cm2). The wafer was incubated overnight at room temperature in a humid chamber. After incubation, the wafer was washed thoroughly by rinsing with 0.1 M MES [2-(N-Morpholino)ethanesulfonic acid] buffer (pH 6.4) and deionized water. Samples immobilized with the oligonucleotides were kept in deionized water at 4°C prior to characterization or further hybridization.Hybridization and denaturation experiments To explore the hybridization properties of the immobilized oligonucleotides (DNA oligonucleotides B and BT), three 5′-fluorescein-labelled oligonucleotides were used.Oligonucleotide b (5′-FAM-CTG CTA TCT ATC TGC A-3′) is complementary to oligonucleotide B and BT, whereas oligonucleotide b1 (5′-FAM-CTGCTTTCTATCTGCA-3′) has one thymine base mismatch, and oligonucleotide c (5′-FAM-CTTACCGATCGTATTC-3′) is not complementary. The hybridization solutions were prepared with the fluorescein-labelled oligonucleotides (10 μM) in Tris–HCl buffer (20 mM Tris–HCl, 100 mM MgCl2, pH 8.0). A wafer patterned with oligonucleotides B or BT was pre-soaked in the Tris–HCl buffer for 20 min and then excess buffer was removed. Immediately, an aliquot of 8 μl of the oligonucleotide hybridization solution was deposited on the wafer and covered with a cover slip (2 × 2 cm2). The wafer was incubated in a humid chamber for 3 h at room temperature. After incubation, the wafer was rinsed thoroughly with the Tris–HCl buffer and kept in a Tris–HCl buffer prior to characterization. Denaturation was accomplished by placing the samples in an 8.3 M urea solution for 15 min at 40°C followed by rinsing with water (25). The surfaces were then evaluated by using epifluorescence microscopy to make sure that the denaturation was complete. Subsequent hybridization was repeated using the same procedure. Density of hybridization with the surface-attached oligonucleotides A similar method to that reported by Strother et al. (25) was employed. Oligonucleotides B were patterned on silicon surfaces using a photolithographic mask as described. Complementary fluorescent oligonucleotides b were hybridized and washed thoroughly with Tris–HCl buffer. The fluorescent images were acquired to calculate the exact surface area of the oligonucleotide patterns. The wafer was then placed in 800 μl of water at 90°C for 15 min to allow for the denaturation of the duplex. The wafer was removed and rinsed twice with 200 μl of water; the denaturation solution 800 μl was mixed with the rinsing solutions (2 × 200 μl). The surfaces were then observed with the epifluorescence microscope to ensure that the denaturation of the duplex was complete. The water containing the denatured fluorescent DNA strands was freeze dried, and the resulting dried oligonucleotides were re-dissolved in 50 μl of 0.05 M Tris buffer (pH 9.0). The fluorescence yield from the sample was measured in a 50 μl cuvette in a fluorimeter (Cary Ellipse, Varian Inc., λexc = 488 nm, λem = 525 nm). The concentration of DNA strands present in the sample was determined by reference to a standardization curve prepared from a series of fluorescent-labelled DNA oligonucleotide samples of known concentration. | |||||||||||||
RESULTS AND DISCUSSION Surface attachment chemistry UANHS attachment onto hydride terminated silicon surfaces has been demonstrated (27). This method has been exploited, in the current study, for submicron patterning. Briefly, a hydride-terminated silicon surface was spin-coated with a dichloromethane solution of UANHS and irradiated with an excimer laser of wavelength 248 nm. Patterns of the surface-attached UANHS were achieved by illumination through either a photolithographic mask (Suprasil) or a phase mask. Typically, the laser pulse (20 ns) intensity was 15 kW, delivered with a repetition rate of 10 Hz, with illumination times of up to 2 min. Significantly, shorter illumination times were necessary than for previously reported photo-initiated alkene reactions with silicon (23,25,26,28), where long-time exposures (several hours or a day) and illumination under nitrogen gas was necessary. The N-hydroxysuccinimide ester group is a robust coupling agent for amino-modified biomolecules (29). Coupling the N-hydroxysuccinimide ester terminated silicon surface in an aqueous solution with an amino-modified oligonucleotide provides a method for the creation of DNA oligonucleotide surfaces without further processing (Figure 1).
Submicron patterning of oligonucleotides A reduction in the dimensions of oligonucleotide patterns below 1 μm is desirable for the creation of small-scale sensors. In practice, a creation of a motif by illumination of dimensions approaching 1 μm is difficult to achieve by standard photolithographic mask techniques, even with the mask in close contact (11). Interference lithography, which has been widely used to generate sub-100 nm features (30) or even sub-50 nm features when UV light has been used for other types of surface patterning (31,32), and appeared to be a promising approach for our aims.Figure 2 shows the scheme adopted for the submicron patterning of oligonucleotides used here. A +1/−1 phase mask (2 μm period) was placed above a silicon wafer coated with UANHS. On one of the flat surfaces of the phase mask, a one-dimensional periodic surface relief structure had been etched. The laser light, incident normal to the phase mask, was diffracted by the periodic corrugations of the phase mask diffract light into the +1 and −1 orders. Interference between these two orders created a light intensity profile of alternate maximum peak and minimum valleys of 1 μm period. After the exposure, the unreacted UANHS was washed off the silicon wafer and an amino-modified DNA oligonucleotide was added to the patterned surface to couple the oligonucleotide onto the UANHS motifs on the surface.
A wide range of exposure conditions were tested at a fixed repetition rate of 10 Hz, between 0.3 and 2 mJ per pulse per square centimetre, and exposure times from 20 s to 2 min. The resulting alkene motifs were evaluated by SEM and ellipsometry to enable the optimal exposure conditions to be determined. No reaction was detected when the sample was irradiated at 0.3 mJ per pulse per square centimetre for 30 s, but an increase in the exposure time to 60 s yielded a grating pattern that could be detected by SEM as shown in Figure 3, where a periodic pattern of ~1 μm is observable. Irradiation at higher intensity with a shorter exposure time yielded a very similar result (i.e. 1.5 mJ/cm2 for 15 s). Patterns of 1 μm periodicity could be produced using a range of light fluences (0.17–0.85 J/cm2). Ellipsometry was used to evaluate the film thickness. Because the ellipsometer could not be used to resolve nanometre-sized patterns, the UANHS films were irradiated without the phase mask (the phase mask only reduces the transmitted light by 5%). The thickness of the formed UANHS layers, determined by ellipsometry, obtained with the fluences of 0.17 J/cm2 were 0.85 J/cm2, are 8.6 and 11.2 Å, respectively, which is in the approximate range for the formation of a monolayer of the alkene containing less than 18 carbons (22). However, the difference between the thickness values is only 3 Å, almost at the margin of the experimental error (1–2 Å). We suggest that there may be a denser film of surface-attached UANHS molecules obtained with the higher fluences; as demonstrated previously by ellipsometry for other films (22).
Subsequent coupling with fluorescein-labelled oligonucleotides onto the patterned UANHS leads to an oligonucleotide pattern on the submicron scale and this was visualized by high resolution AFM as shown in Figure 4. The oligonucleotides (bright spots) are mainly located on the raised uniform parallel lines.
Hybridization of complementary and mismatched DNA oligonucleotides with the surface-patterned DNA surfaces To evaluate the submicron patterned DNA oligonucleotides with respect to hybridization with the complementary sequence, an unlabelled oligonucleotide B pattern was prepared on the silicon surface. A series of identical samples were tested towards hybridization to the fluorescein-labelled oligonucleotides b, b1 and c; these oligonucleotides are complementary (b), contain a one base mismatch (b1) and are non-complementary (c) to oligonucleotide B. After stringent washing, the epifluorescence images of the hybridized samples were obtained (Figure 5); only the sample hybridized with the complementary oligonucleotide b revealed a highly fluorescent pattern.
The fluorescence signal after treatment of the patterned surfaces with each of the oligonucleotides was quantified as the normalized fluorescent signal-to-noise ratio N-SNR (fluorescent SNR of the sample after hybridization experiment /SNR of the same sample before hybridization) in Figure 5. The N-SNR for samples hybridized with one-mismatch (sequence B–b1) was 1.25 ± 0.24 and non-complementary sequence was 1.09 ± 0.05 (sequence B–c), indicating the presence of a very small amount of non-specifically-bound oligonucleotide to the silicon attached oligonucleotides. The N-SNR for the complementary sequences was evaluated as 2.99 ± 0.45 (sequence B–b). As a test to prove that there was no variation between the hybridization efficiency between different DNA oligonucleotide patterned silicon surfaces, (i) a silicon sample with surface-attached oligonucleotides was treated under hybridization conditions with the complementary oligonucleotides b; (ii) following this the sample was evaluated and the hybridized oligonucleotides removed under denaturation conditions; and then (iii) the same surface patterned silicon sample was treated with the oligonucleotide b1, the mis-match sequence under hybridization conditions, [a sample where steps (i) and (iii) are exchanged was also tested]. In all cases, fluorescent patterns were obtained if the complementary oligonucleotide sequence (b) was used; the normalized fluorescent N-SNRs were comparable to those obtained for the freshly prepared oligonucleotide-patterned silicon samples. Thus, hybridization with the patterned DNA oligonucleotide surfaces is specific. The length of the alkane linking chain between the oligonucleotide and the silicon surface was estimated to be ~12 Å. It was therefore considered that there might be some surface effects that could influence the hybridization of the complementary sequence. In order to investigate this, oligonucleotide BT was attached to the silicon surface in patterns; the oligonucleotide BT consists of sequence B and 15 thymine bases to which the 3′ end is attached to the silicon surface. The hybridization tests, described above, were carried out and the N-SNR obtained, 3.20 ± 0.21 (sequence BT–b), was similar to that obtained previously for oligonucleotide B (without the spacer group) [2.99 ± 0.45 (sequence B–b)], suggesting that there was little detectable difference on the attachment distance of the surface-attached oligonucleotides towards specific hybridization, under the conditions used here. The density of fluorescein-labelled oligonucleotides that hybridized to the surface was determined using a modified version of a previously reported method (see Materials and Methods) (25). The surface density of complementary strands hybridized onto oligonucleotide-B-patterned surface was found to be 6.7 × 1012 oligonucleotides per square centimetre (one oligonucleotide per 15 square nanometre) which is slightly higher than that obtained previously for covalently attached DNA oligonucleotides on glass (2.3 × 1012 oligonucleotides per square centimetre) (26). Hydrolysis of the succinimide ester linked to the silicon surface during the coupling reaction with the amine-functionalized oligonucleotides might have been predicted. However, the density of surface hybridized fluorescent-labelled oligonucleotides is such as to indicate that the hydrolysis reaction is not a major problem. Thus, the method for the attachment of DNA in patterns to silicon provides excellent surface coverage of the attached DNA oligonucleotides that are accessible towards hybridization with complementary sequences. If oligonucleotide patterns are reduced towards the nanometre scale for the creation of biosensors, highly effective hybridization of the immobilized oligonucleotide with complementary sequences is vital. A silicon wafer with the non-fluorescent oligonucleotide B, attached onto the silicon surface with a submicron-scale periodic pattern, (fabricated as described above) was prepared. Fluorescent-labelled oligonucleotides b were hybridized with the patterned oligonucleotide surface and then evaluated with a laser confocal microscope. A fluorescence pattern of 500 nm lines separated by 500 nm spaces was observed over the whole region (Figure 6). The results demonstrated that the immobilized oligonucleotides, in patterns of submicron dimensions, were accessible towards specific hybridization.
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CONCLUSIONS DNA oligonucleotides have been successfully attached onto bulk silicon semiconductor with motifs with submicron feature sizes. This has been achieved by a two-step process by (i) photo-exposure using UV light in interference patterns onto films of UANHS on hydrogen-terminated silicon surfaces followed by (ii) treatment of the surface motifs with amine-functionalized DNA oligonucleotides. The immobilized oligonucleotide patterns on the silicon surfaces show excellent specific hybridization with the complementary sequence in high densities. The method is straightforward and should offer much potential for the creation of self-assembled nano-bioelectronic devices and for the miniaturization of emerging bio-analytical tools, such as integrated silicon BioMEMS devices. | |||||||||||||
ACKNOWLEDGEMENTS We are grateful for the financial support of the BBSRC (ref 51/16611 and 51/18926) and the Royal Society (Biomolecule Patterns on Silicon). | |||||||||||||
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