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Surface Characterization of a Silicon-Chip-Based DNA Microarray Ralf Lenigk,† Maria Carles,† Nancy Y. Ip, and Nikolaus J. Sucher* Biotechnology Research Institute and Department of Biology, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong SAR, China Received September 22, 2000. In Final Form: January 22, 2001 The immobilization of DNA (deoxyribonucleic acid) on solid supports is a crucial step for any application in the field of DNA microarrays. It determines the efficacy of the hybridization and influences the signal strength for the detection. We used solid supports made from silicon wafers as an alternative substrate to the commonly used microscope glass slides. The covalent immobilization of thiol-terminated DNA oligonucleotides on self-assembled layers of (3-mercaptopropyl)trimethoxysilane (MPTS) by disulfide bond formation was investigated. Contact angle measurement, variable angle spectral ellipsometry (VASE), X-ray photoelectron spectroscopy (XPS), and atomic force microscopy (AFM) were used to characterize the changing properties of the surface during the DNA array fabrication. During wafer processing the contact angle changed from 3° for the hydroxylated surface to 48.5° after deposition of MPTS. XPS data demonstrated that all sulfur in the MPTS layer was present in the form of reduced SH or S-S groups. VASE measurements indicated a layer thickness of 57.8 Å for the immobilized 16 base oligonucleotides including a 18 carbon atom spacer located between the disulfide bond and the oligomer. AFM was used to characterize the DNA layer before and after hybridization to a complementary target. The data recorded after hybridization revealed a sharp increase in particle size from 89 nm2 to a mean value of 363 nm2. Fluorescence microscopy was used to monitor the hybridization of a fluorescently labeled DNA target to the immobilized probe. The heat stable disulfide-linkage formed during the oligonucleotide immobilization allowed the stripping of complementary DNA targets as well as rehybridization. These data show the advantages and applicability of silicon wafers that have been processed with CMOS (complementary metal oxide semiconductor) compatible processes as solid support in DNA technology. This approach offers the possibility of integration with other silicon-based components such as PCR microreactors and capillary electrophoresis units into a “lab-on-a-chip”.
Introduction A quarter century ago Edwin Southern first demonstrated that DNA fragments that had been size separated by gel electrophoresis could be transferred to a membrane and subsequently be probed for the presence of specific sequences.1 The “Southern” blot became rapidly a standard technique in molecular biology laboratories extending and superseding earlier solution-based hybridization methods that were developed by Paul Doty and colleagues.2 More recently, the original design of the “Southern” blot has been extended to nonporous solid supports and the development of methods allowing high-density deposition of oligonucleotides or DNA fragments resulting in the creation of DNA microarrays.3 A common denominator of these DNA microarray-based analytical methods is the immobilization of DNA probes onto a solid support allowing their hybridization to previously labeled complementary sequences (targets). The recent surge in sequence data from an increasing number of genome projects has spurred the interest in the use of this DNA microarray technology and prompted the automation of array fabrication, data acquisition, and data analysis. Automation is facilitated by the use of rigid supports, including glassslides, nitrocellulose-coated microscope slides, or siliconbased DNA arrays rather than the traditional membranebased blots. * To whom correspondence should be addressed. Department of Biology. Telephone: (852) 2358 7306. Fax: (852) 2358 1559. E-mail address:
[email protected]. † These authors contributed equally to the work. (1) Southern, E. M. J. Mol. Biol. 1975, 98, 503. (2) Schildkraut, C. L.; Marmur, J.; Doty, P. J. Mol. Biol. 1961, 3, 595. (3) Lander, E. S. Nat. Genet. 1999, 21 (1 Suppl.), 3.
The use of silicon wafers as solid support for DNA arrays has several additional advantages compared to the commonly employed microscope slides. First, silicon wafers have less surface roughness. This allows for increased uniformity of DNA deposition with a higher density and smaller size of DNA spots that is made possible by the development of new printing technology.4 Second, the flat surface allows high-density DNA arrays to be analyzed with confocal laser-based scanners. Third, silicon surfaces provide a better signal-to-noise ratio because silicon wafers show less background fluorescence and the dark, nontransparent surface absorbs excitation light. Finally, silicon chip-based DNA microarrays will facilitate the fabrication of the so-called “lab-on-a-chip” devices. The immobilization methods used to attach DNA molecules to surfaces include (a) cross-linking to poly-Llysine-coated microscope slides,5 (b) attraction of the negatively charged DNA to a positively polarized electrode that is coated with a gel containing functional groups for subsequent cross-linking,6 (c) synthesis of DNA on glass wafers making use of photolithographic techniques,7,8 (d) covalent attachment of DNA to self-assembled monolayers (SAMs) of aminosilanes using heterobifunctional crosslinkers,9 (e) binding of thiol-modified oligonucleotides to (4) Harris, T. M.; Massimi, A.; Childs, G. Nat. Biotechnol. 2000, 18, 384. (5) Schena, M.; Shalon, D.; Davis, R. W.; Brown, P. O. Science 1995, 270, 467. (6) Edman, C.; Raymond, D.; Wu, D.; Tu, E.; Sosnowski, R.; Butler, W.; Nerenberg, M.; Heller, M. Nucleic Acids Res. 1998, 25, 4907. (7) Anderson, R. C.; McGall, G.; Lipshutz, R. J. Microsystem Technology in Chemistry and Life Sciences; Springer-Verlag: Berlin, 1998; p 117. (8) Pease, L. A.; Solas, D.; Sullivan, E. J.; Cronin, M. T.; Holmes, C. P.; Fodor, S. P. A. Proc. Natl. Acad. Sci. 1994, 91, 5022.
10.1021/la001355z CCC: $20.00 © 2001 American Chemical Society Published on Web 03/23/2001
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thiol-functionalized silicon wafers using bifunctional alkyltrichlorosilanes,10 (f) 5′-end amino-modified oligonucleotides bound to (3-glycidoxypropyl)trimethoxysilanecoated planar waveguides,11 (g) tethering of DNA to glass slides via an epoxysilane-amine covalent linkage,12 and (h) chemoselective coupling of oligonucleotides to thiol SAMs on gold.13 Which method is chosen depends on the intended application (i.e. the sequencing of DNA, the detection of mutations in certain genes, or the identification of organisms), leading to different necessities regarding the density of DNA and the orientation of immobilized DNA. Although the self-assembly of alkylsiloxane derivative monolayers on silicon dioxide is well established and has been investigated previously by various groups,14 little is known to date about the deposition and characterization of such layers on silicon wafers. The present study was undertaken in order to investigate the covalent immobilization of thiol-terminated DNA oligonucleotides on self-assembled layers of (3-mercaptopropyl)trimethoxysilane (MPTS) by disulfide bond formation. Surface sensitive analytical methods (contact angle measurement, variable angle spectral ellipsometry (VASE), X-ray photoelectron spectroscopy (XPS), and atomic force microscopy (AFM)) were used to characterize the fabrication steps necessary to produce a silicon-based DNA microarray. We demonstrated the hybridization of oligonucleotides labeled with Texas Red (TX) fluorescent dye molecules to complementary oligonucleotide probes that were immobilized on MPTS-treated silicon chips. Using stringent buffer conditions, it was possible to separate the annealed strands and rehybridize again, thereby allowing repeated use of the same chip. Experimental Section Materials. All chemicals and solvents were purchased from Fluka and Sigma-Aldrich (Milwaukee, WI). Fresh MPTS was used since it is subject to oxidation of the SH groups and crosslinking of the molecules due the hydrolysis of silanol groups when humidity condenses inside the bottle. Oligonucleotides were purchased from Synthetic Genetics (San Diego, CA). The DNA oligonucleotides that were immobilized onto the MPTS layer had the sequence 5′-HS-carbon18-CAC AAA ACG GGG GCG G -3′. The complementary labeled oligo sequence was 5′-TX-CCG CCC CCG TTT TGT G-3′. Texas Red is a registered trademark of Molecular Probes (Eugene, OR). The wafers used for the experiments were n-type Si(100) wafers. Thermal Oxidation of Silicon. All wafers were subjected to a thorough cleaning procedure before use. After immersion in “piranha” etch solution (90% H2SO4, 10% H2O2; 125 °C) for 10 min, the wafers were treated with a 2% solution of hydrofluoric acid (HF) for 1 min to remove the native oxide layer. After each step the wafers were rinsed four times with copious amounts of double-distilled (dd) water and then blow-dried. A 1000 Å layer of dry silicon dioxide was grown in an oxidation furnace at 1100 °C, followed by 4000 Å of wet oxide. The steps above were performed in a class 1000 clean room using CMOS (complementary metal-oxide semiconductor) compatible processing. Finally, the wafers were cut into squares of 2.0 cm × 2.0 cm using a diamond scribe. Cleaning and Surface Hydroxylation of the Chips. Organic contaminants on the surface of the chips were removed (9) Chrisey, L. A.; Lee, G. U.; O’Ferrall, C. E. Nucleic Acids Res. 1996, 24, 3031. (10) McGovern, M. E.; Thompson, M. Can. J. Chem. 1999, 77, 1678. (11) Budach, W.; Abel, A. P.; Bruno, A. E.; Neuschafer, D. Anal. Chem. 1999, 71, 3347. (12) Beattie, W. G.; Meng, L.; Turner, S. L.; Varma, R. S.; Dao, D. D.; Beattie, K. L. Mol. Biotechnol. 1995, 4, 213. (13) Boncheva, M.; Scheibler, L.; Lincoln, P.; Vogel, H.; Akerman, B. Langmuir 1999, 15, 4317. (14) Ulman, A. Ultrathin Organic Films; Academic Press: San Diego, CA, 1991.
Lenigk et al. by immersion of the 4 cm2 silicon chips into an etchant solution (1 g of potassium permanganate dissolved in 30 mL of concentrated sulfuric acid) for 90 s followed by several washes with dd H2O. Boiling for 1 h in a 1% ammonium hydroxide solution was used to hydroxylate the surface of the chips. Formation of Self-Assembled (3-Mercaptopropyl)trimethoxysilane (MPTS) Layer. Glassware used in the selfassembly process was sonicated with a 5% Decan 90 solution and then rinsed with copious amounts of water. The chip was immersed into a solution consisting of 10 mL of 2-propanol p.a., 250 µL of MPTS, and 100 µL of H2O and boiled under reflux for 30 min. After the solution cooled, the chips were removed using stainless steel tweezers, rinsed first with copious amounts of 2-propanol and then with water and subsequently blow-dried with nitrogen gas. The layer was annealed in an oven at 115 °C for 30 min, as previously described only for alkanethiol monolayers on gold;15 the heat treatment yielded surfaces of superior quality. Immobilization of Oligonucleotides. Sixteen base oligonucleotide probes were diluted with sodium chloride/sodium citrate buffer (SSC; 3 M NaCl, 0.3 M Na citrate - 2 H2O, pH 4.5) to a final concentration of 20 µM (modified from ref 16). Small drops of the solution were deposited manually on the surface of freshly prepared chips using a pipet tip. The dots had a diameter of about 500 µm and a volume of about 50 nL (mean value). The chips were incubated overnight at room temperature in a humidified chamber to prevent the drying of the drops (when drops dry, substances are transported to the rim, leading to uneven distribution of the concentration) and then washed three times with washing buffer (10 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.05% Tween 20) and three times with water. They were blow-dried with nitrogen gas to ensure that the surface was dry and free of dust particles. Hybridization Conditions and Stripping of Oligonucleotides. Sixteen base oligonucleotide targets labeled with a fluorophore (Texas Red) were diluted in hybridization buffer (consisting of 6x SSC pH 7, 5x Denhardt’s solution (1% BSA (bovine serum albumin), 2% Ficoll 400, and 2% polyvinylpyrollidone (PVP)), 0.5% sodium dodecyl sulfate (SDS), and sheared salmon sperm DNA (55 µg/mL)) to a final concentration of 20 µM. Two small strips of adhesive tape were affixed along the rim of a chip with the immobilized oligonucleotide capture probes and then covered with a cleaned microscope slide cover glass. The cavity formed between the chip and the cover glass was filled by slowly loading 10 µL of the hybridization solution by capillary force. The chip was incubated in a custom-made hybridization chamber. The humidified chamber (100 µL of water was placed next to the chip) was immersed in a 55 °C water bath for 2 h. After the cover glass was removed, the chip was washed three times with washing buffer and then with copious amounts of water and blow-dried with nitrogen gas. The stripping of the bound oligonucleotides of the chip was investigated by placing it into a boiling solution of (a) 200 mM TrisCl pH 7.0, 0.1x SSC, and 0.1% (w/v) SDS for 10 min or (b) 0.1% (w/v) SDS for 10 min in order to remove the labeled oligonucleotides. Contact Angle Measurements. A Rame´-Hart goniometer model # 100-00 was used for the contact angle measurement. A mass of approximately 20 mg of dd H2O was deposited onto the surface using a microsyringe, and the reading was taken 2 min after deposition. The temperature was 25 °C, and the relative humidity was 60%. Characterization of the MPTS Layer by XPS. All XPS spectra were collected using a Physical Electronics, Model PHI 5600, multitechnique system with an anode source providing Al KR (1486.6 eV) radiation. After monochromation the radiation was focused on the sample, at an electron takeoff angle (TOA: angle between the analyzer and the sample surface) of 45 ( 3° relative to the substrate surface. The S(2p), O(1s), and C(1s) spectra were acquired using Physical Electronics software. The slit width (0.8 mm) and the TOA were kept constant for each of the samples measured in order to probe each sample at the same (15) Delamarche, E.; Michel, B.; Kang, H.; Gerber, Ch. Langmuir 1994, 10, 4103. (16) Rogers, Y. H.; Jiang-Baucom, P.; Huang, Z. J.; Bodganov, V.; Anderson, S.; Boyce-Jacino, M. T. Anal. Biochem. 1999, 266, 23.
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depth. A flood gun was used to compensate for sample charging, necessitating adjustment of the binding energies of the spectra. The Si(2p) peak was chosen as the reference binding energy (103.3 eV). Thickness Measurement Using VASE. The instrument used was a variable angle ellipsometry system from J. A. Woollam Co. For the measurements the optical constant of the MPTS layer was set to 1.4420 (25 °C), as found in the literature.17 Fluorescence Microscopy. A Zeiss Axioskope microscope fitted with a CCD camera (Photometrics Sensys) and a Dell personal computer running “V” for Windows software (Photometrics Ltd.) was used for image acquisition. Atomic Force Microscopy (AFM). Nanoscope 3a from Digital Instruments was used for the experiments. Data were acquired in the tapping mode using a resolution of 512 pixels per line and analyzed using Digital Instrument software.
Results and Discussion Pretreatment and Analysis of Oxidized Silicon Wafers. Initial experiments aiming to form an MPTS layer on oxidized silicon wafers indicated the need for a thorough pretreatment and the hydroxylation of the silicon surface. An oxide layer was grown on the silicon wafers by dry oxidation at high temperature followed by subsequent rehydroxylation because at temperatures above 800 °C a loss of all hydroxyl groups occurred.18 Adequate substrate rehydration has been proven to be crucial for the selfassembly of alkylsiloxane monolayers.19;20 Accordingly, the cleaning and pretreatment of the oxidized wafer assured the reproducibility of the results. Organic contaminants on the surface of the chip were removed, and the surface of the chip was hydroxylated. The change of the surface wetting behavior was monitored by goniometry.21 A contact angle of 3° after the treatment of the chips in NH4OH indicated the almost complete hydroxylation of the surface. The analysis of the XPS spectrum taken immediately after cleaning indicated a low carbon content of 4.1% on the surface and demonstrated that a certain amount of contamination could not be avoided under the conditions used. The amount of carbon recorded on the surface was likely due to the deposition of contaminants present in the ambient air. We therefore concluded that it was important to deposit the MPTS layer by self-assembly immediately after cleaning. To investigate the surface changes that occur during the processing of the chips by AFM, 1 cm2 silicon chips were cut to fit onto the probe stage of the microscope and prepared as described above. The AFM data are presented in Figure 1. During the cleaning and rehydroxylation process, some material appeared to be removed (desorption of Si(OH)4 into the solution), leading to an increase in roughness and of the surface area (Figure 1a). VASE was used for the determination of the layer thickness using the same samples as prepared for the AFM experiments. On the basis of the topography of the cleaned chips that was observed with AFM, a “roughness layer” (50% voidness was assumed) was included into the layer model. Using the Bruggemann and Maxwell-Garnett effective medium approximation (EMA), a thickness of 16.8 Å resulted in the best correspondence between the calculated and measured spectra. The inclusion of this (17) In Handbook of data on organic compounds; CRC Press: Boca Raton, FL, 1989. (18) Iler, R. K. The Chemistry of Silica; John Wiley and Sons: New York, 1979. (19) Allara, D. L.; Parikh, A. N.; Rondelez, F. Langmuir 1995, 11, 2357. (20) Legrange, J. D.; Markham, J. L. Langmuir 1993, 9, 1749. (21) Bauer, J.; Drescher, G.; Illig, M. J. Vac. Sci. Technol., B 1996, 14, 2485.
Figure 1. Atomic force microscopy of silicon chip surfaces: (a) cleaned, rehydroxylated silicon surface; (b) self-assembled MPTS layer on silicon; (c) immobilized oligonucleotide probe; (d) surface after hybridization to a complementary target.
Figure 2. High-resolution XPS spectra of a (C2H5O)3SiOC3H7SH layer on oxidized silicon prepared by self-assembly.
roughness layer significantly improved the quality of the model, resulting in an increase of 2 Å in the overall thickness of the MPTS layer. Investigation of Self-Assembled MPTS Layer. After the bonding of MPTS to the surface, the contact angle increased from 3° to 48.5° ( 1.5°. The hydrophobic MPTS layer facilitated the deposition of the oligonucleotide (aqueous solution) because it prevented the spreading of the drop over the surface. The elemental composition of the surface under study was determined by XPS. In the XPS spectra of the MPTS layer (Figure 2) the sulfur peak (S 2p) was detected at 163.7 eV, which indicated that all of the sulfur was present as thiol or disulfide.22 XPS was also used to study the composition of mixed layers of silanes (MPTS and methyltriethoxysilane (MeTS)) resulting from the selfassembly process on the silicon dioxide surface at different (22) Moulder, J. F.; Stickle, W. F.; Sobol, P. E.; Bomben, K. D. Handbook of X-ray Photoelectron Spectroscopy; Perkin-Elmer Corporation: Eden Prairie, MN, 1992.
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Table 1. Sulfur Content Analysis by XPS of Mixed Silane Layers Using the S 2p Peak sulfur MPTS
pure MPTS
MeTS/MPTS 1:1
MeTS/MPTS 5:1
9.1% 100%
5.4% 59%
0.85% 9%
ratios (Table 1). The application of mixtures of silanes was used to control the density of free thiol groups and thus the density of immobilized oligonucleotides on silicon dioxide surfaces. A similar approach was reported previously to influence the extent of immobilization of protein probes on modified gold surfaces.23 The carbon content of the MPTS layer (11.1%) was found to be 2.3 times greater than the carbon content of MeTS with 4.9%. Taking into account some surface contamination, the measured values matched the 3:1 ratio predicted from the carbon content of the molecules immobilized on the chip. When a 1:1 ratio of the compounds was used in the immobilization mixture, the percentage of MPTS in the layer dropped from 100% to 59% (as monitored by the relative S 2p peak height normalized to the S 2p signal for pure MPTS). When a 5-fold excess of MeTS was present in the mixture, the MPTS content dropped to 9% rather than the expected 20%, indicating nonlinear behavior of mixed solutions of MeTS and MPTS. The contact angles were found to increase from 52.5° ( 1.5° for the1:1 mixture to 55.5° ( 1.5° for the 1:5 mixture while the value for pure MeTS was determined to be 57° ( 1.5°. Nonetheless and as expected, these experiments indicate that control of the immobilization density can be achieved by using mixtures of alkyl- and mercaptoalkoxysilanes in the self-assembly process. However, the surface density of the groups of interest has to be determined experimentally. The AFM data (Figure 1b) indicated that immobilization of MPTS led to an increase in the grain size from 44 to 89 nm2 and a small change in the topography. A thickness of 38.8 ( 7 Å was calculated for the MPTS layer by the modeling software. This value was several times larger than expected for a monolayer. One possible reason for this discrepancy might be the formation of multiple layers due to the polymerization of MPTS molecules under the chosen reaction conditions (presence of water for bond hydrolysis). Immobilization and Analysis of DNA Oligonucleotides on (3-Mercaptopropyl)trimethoxysilane. Mercaptosilanes were used for the immobilization of 5′-(thiolmodified) oligonucleotides. As reported previously, the dithiol bond formed was heat stable at least to 95 °C.24 The 5′-(thiol-modified) oligonucleotide probes were covalently bound to the MPTS layer by direct disulfide bond formation. The oxygen in the air catalyzes the oxidation of mercaptanes to disulfides, if a small amount of base is present. The mechanism involves loss of a proton, oxidation to a free radical, and radical coupling.25 Besides the heterodimer formation (DNA-S-S-MPTS), homodimerization (DNA-S-S-DNA and MPTS-S-S-MPTS) may also occur in the solution. The AFM image of the immobilized oligonucleotide layer (Figure 1c) showed a pattern that suggested a certain order in the arrangement of the molecules on the surface. Surface coverage is influenced by a number of factors including the concentration of the oligonucleotides, the pH of the buffer, and the incubation time. Very dense (23) Cotton, C.; Glidle, A.; Beamson, A.; Cooper, J. M. Langmuir 1998, 14, 5139. (24) National Research Development Corporation, Immobilised Polynucleotides, Patent WO 91/00868, 1991. (25) March, J. Advanced Organic Chemistry; John Wiley and Sons: New York, 1985; p 1092.
Figure 3. Fluorescence microscope images: (a) hybridization of target labeled with Texas Red (20 µM concentration) to the complementary oligonucleotide; (b) hybridization of a noncomplementary target labeled with Texas Red (20 µM concentration) to the immobilized probe.
coverage is not desirable because steric hindrance can interfere with the hybridization of the target to the probe.26 VASE measurements of the thickness proved to be difficult because the AFM data indicated no dense layer was present. The recorded vase data were put into a mathematical model for the determination of the refractive index, and the resulting value was used in the calculation of the layer thickness. The problem of finding a mathematical model with a high accuracy implied that the surface coverage was very low. Along these lines, it was reported previously that the accessibility of surfaceattached thiol groups may have been reduced, due to their reaction with available hydroxyls either on the hydrolyzed silane or on the wafer27 leading to a decrease in the density of oligonucleotide groups immobilized on the surface. Analysis of Hybridization with Labeled Complementary Oligonucleotides. Fluorescence micrographs were taken to illustrate the hybridization of labeled oligonucleotide targets to complementary probes. No binding was observed to noncomplementary probes (Figure 3a and b, respectively). (26) Shchepinov, M. S.; Case-Green, S. C.; Southern, E. M. Nucleic Acids Res. 1997, 25, 1155. (27) Yee, J. K.; Parry, D. B.; Caldwell, K. D.; Harris, J. M. Langmuir 1991, 7, 307.
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Table 2. VASE Measurement Results for the Cauchy Fit Model of the Monolayer Optical Parameters layer
thickness
underlying silicon SiO2 roughness layer (50% void) MPTS Cauchy (hybridization layer)
1 mm 4972.1 Å 16.8 Å 38.8 Å 29.0 Å
AFM data obtained following hybridization (Figure 1d) revealed that big clusters of hybridized oligonucleotides were distributed over the surface. Fluorescence microscope images of the surface also confirmed the existence of areas with higher fluorescence (not illustrated). The mean grain size of these spots as calculated from AFM data increased from 250 nm2 (immobilized oligonucleotide layer) to 1500 nm2 (hybridized layer). In the VASE measurement, the “cauchy” layer (Table 2) corresponds to the oligonucleotide layer hybridized with the complementary, labeled DNA strand. The index of refraction was determined to be 1.509 at 530 nm, and the thickness was 29.0 ( 5 Å. Because that layer was not uniform, as indicated by the AFM data, it was estimated that the maximal thickness was probably twice that of the mean value. To improve the accuracy of these measurements, either the uniformity of the layer would have to be improved (very difficult to achieve considering that it takes the deposition of three layers onto each other) or a roughness value would have to be calculated and integrated into the thickness calculation by the VASE software. Stripping of Labeled Target and Rehybridization. The potential reusability of the chip was investigated by using a low stringency (Figure 4a) or high stringency wash (Figure 4c), as described in the method section, in order to remove the labeled oligonucleotides. As confirmed by fluorescence microscopy, this procedure resulted in the removal of the target oligonucleotides. When the chip was used for another set of hybridization experiments, the fluorescent dot was detected with a small decrease in intensity compared to that for the first hybridization (Figure 4b and d). The chip could be reused at least five times without significant loss of immobilized oligonucleotide probes (not illustrated). Concluding Remarks Self-assembled layers of oligonucleotides on silicon chips were prepared using (3-mercaptopropyl)trimethoxysilane as a bifunctional linker molecule. XPS, VASE, AFM, and contact-angle measurements were taken to obtain information concerning n-type Si(100) surfaces on which multiple layers were deposited. XPS spectra revealed that
Figure 4. Fluorescence microscope images of stripping/ rehybridization experiments: (a) after stripping of Texas Redlabeled oligonucleotide with a medium stringency solution; (b) after first rehybridization (20 µM concentration); (c) after second stripping using high stringency conditions; (d) after second rehybridization (20 µM concentration).
thorough cleaning procedures of the wafer surface greatly reduced but did not preclude surface contamination by hydrocarbons present in ambient air. Photoelectron spectroscopy was also used to analyze the composition of mixed monolayers. VASE measurements yielded thickness values for the deposited layers on the chip surface. The hybridization to complementary probes labeled with a fluorescent marker was demonstrated by fluorescence microscopy. The stability of the film formed by immobilization of the oligonucleotide probes via disulfide bonds allowed for repeated hybridization and stripping of the same chip. The described procedure may find increased use in the manufacturing of nucleic acid microarrays based on silicon surfaces. Acknowledgment. This work was supported by grants from the Industry Department of the Hong Kong SAR (AF/150/99) and the Hong Kong Jockey Club. The authors would like to thank Dr. K. L. Yeung of the Department of Chemical Engineering and Mr. J. Squire of the Materials Characterization and Preparation Center, HKUST, for helpful discussions and their experimental support. LA001355Z
