Formation and Characterization of DNA Microarrays at Silicon Nitride

Dec 2, 2004 - A versatile method for direct, covalent attachment of DNA microarrays at silicon nitride layers, previously deposited by chemical vapor ...
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Langmuir 2005, 21, 395-402

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Formation and Characterization of DNA Microarrays at Silicon Nitride Substrates Mary Manning and Gareth Redmond* Nanotechnology Group, NMRC, Lee Maltings, Prospect Row, Cork, Ireland Received August 9, 2004. In Final Form: October 18, 2004 A versatile method for direct, covalent attachment of DNA microarrays at silicon nitride layers, previously deposited by chemical vapor deposition at silicon wafer substrates, is reported. Each microarray fabrication process step, from silicon nitride substrate deposition, surface cleaning, amino-silanation, and attachment of a homobifunctional cross-linking molecule to covalent immobilization of probe oligonucleotides, is defined, characterized, and optimized to yield consistent probe microarray quality, homogeneity, and probe-target hybridization performance. The developed microarray fabrication methodology provides excellent (high signal-to-background ratio) and reproducible responsivity to target oligonucleotide hybridization with a rugged chemical stability that permits exposure of arrays to stringent pre- and posthybridization wash conditions through many sustained cycles of reuse. Overall, the achieved performance features compare very favorably with those of more mature glass based microarrays. It is proposed that this DNA microarray fabrication strategy has the potential to provide a viable route toward the successful realization of future integrated DNA biochips.

Introduction It is widely acknowledged that, to accelerate the uptake and exploitation of many microarray based genetic analysis technologies, substantial increases in throughput must be achieved while minimizing the cost per assay. Since the future demand for high-throughput genetic analysis tools is expected to increase well beyond the capabilities of current technologies, new approaches to DNA microarray fabrication such as the integration of microarrays into electronically addressable “intelligent” substrates are required. Development of microelectronics enabled functionally integrated biochips that permit onchip biological assays, data acquisition, and even data processing will therefore be a key enabler of this microarray revolution. However, to successfully converge the materials and tools of semiconductor microfabrication with the reagents and protocols of molecular biology, new approaches to surface modification and interface engineering using a potentially diverse range of physical and chemical techniques must be explored. Integrated biochips are comprised of two parts: a biologically active surface layer patterned at an array of discrete sensing elements.1,2 For biomolecule detection, the former may be a layer of antibodies, probe oligonucleotides, or cDNA strands, for example. The latter is typically an array of microelectronic devices capable of detecting molecular recognition and binding events by transducing this information into measurable physical quantities such as changes in mass,3-5 temperature,6 conductance,7,8 or impedance.9-12 * Corresponding author. E-mail: [email protected]. (1) Jain, K. K. Pharmogenomics 2000, 1, 289-298. (2) Hoch, C. H., Jelinski, L. W., Craighead, H. G., Eds.; Nanofabrication and Biosystems: Integrating Materials Science, Engineering, and Biology; Cambridge University Press: Cambridge, U.K., 1996. (3) Liu, Y.; Yu, X.; Zhao, R.; Shangguan, D.; Bo, Z.; Liu, G. Biosens. Bioelectron. 2003, 18, 1419-1427. (4) Zhang, J.; O’ Shea, S. Sens. Actuators, B 2003, 94, 65-72. (5) Hang, T. C.; Guiseppi-Elie, A. Biosens. Bioelectron. 2004, 19, 1537-1548. (6) Ramamathan, K.; Jo¨nsson, B. R.; Danielsson, R. Anal. Chim. Acta 2001, 427, 1-10. (7) Yao, S.; Liu, D.; Ge, K.; Chen, K.; Nie, L. Enzyme Microb. Technol. 1995, 17, 413-417.

Critical to the overall performance of biochips is the availability of methods for the deposition and immobilization of probe molecules onto sensor arrays in a manner that enables reproducible, sensitive operation.13-18 Silicon nitride (and silicon oxide) layers are key materials employed for the fabrication of integrated microelectronic, optoelectronic, and microelectromechanical devices and will, in the future, assume increasing importance as passivation layers, protecting integrated circuits from mechanical damage or other sources of degradation (Na+ ions or moisture),19 and as signal transduction layers, at the interface between miniaturized, solid-state sensor technologies and the adjacent analyte-bearing ambient. For DNA biochips, demands on passivation layers and associated probe immobilization methods will be stringent, since the finished microarray layers must protect the active sensor elements exposed to harsh ambient conditions while also serving as DNA hybridization substrates. To address some of these challenges, we report on the development of a versatile, microfabrication compatible DNA deposition and immobilization method that suc(8) Xu, B. Q.; Zhang, P. M.; Li, X. L.; Tao, N. J. Nano Lett. 2004, 4, 1105-1108. (9) He, F.; Zhao, J.; Zhang, L.; Su, X. Talanta 2003, 59, 935-941. (10) Lasseter, L. T.; Cai, W.; Hamers, R. J. Analyst 2004, 129, 3-8. (11) Cai, W.; Peck, J. R.; van der Weide, D. W.; Hamers, R. J. Biosens. Bioelectron. 2004, 19, 1013-1019. (12) Cloarec, J. P.; Deligianis, N.; Martin, J. R.; Lawrence, I.; Souteyrand, E.; Polychronakos, C.; Lawrence, M. F. Biosens. Bioelectron. 2002, 17, 405-412. (13) Proudnikov, D.; Timofeev, E.; Mirzabekov A. Anal. Biochem. 1998, 259, 34-41. (14) Bruckbauer, A.; Zhou, D.; Kang, D. J.; Korchev, Y. E.; Abell, C.; Klenerman, D. J. Am. Chem. Soc. 2004, 126, 6508-6509. (15) Bruckbauer, A.; Ying, L.; Rothery, A. M.; Zhou, D. Shevchuk, A. I.; Abell, C.; Korchev, Y. E.; Klenerman, D. J. Am. Chem. Soc. 2002, 124, 8810-8811. (16) Stamou, D.; Musil, C.; Ulrich, W. P.; Leufgen, K.; Padeste, C.; David, C.; Gobrecht, J.; Duschl, C.; Vogel, H. Langmuir 2004, 20, 34953497. (17) Yin, H. B.; Brown, T.; Greef, R.; Wilkinson, J. S.; Melvin, T. Microelectron. Eng. 2004, 73, 830-836. (18) Dugas, V.; Depret, G.; Chevalier, Y.; Nesme, X.; Souteyrand, E. Sens. Actuators, B 2004, 101, 112-121. (19) Plummer, J. D.; Deal, M. D.; Griffin, P. B. Silicon VLSI Technology: Fundamentals, Practice and Modeling; Prentice Hall, NJ, 2000.

10.1021/la0480033 CCC: $30.25 © 2005 American Chemical Society Published on Web 12/02/2004

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cessfully permits direct, covalent attachment of probe oligonucleotides at the surface of silicon nitride layers previously deposited by chemical vapor deposition (CVD) at silicon wafer substrates. In this regard, a key achievement has been the successful demonstration of reliable, direct covalent probe attachment combined with sensitive target hybridization at CVD Si3N4 layers while preserving passivation layer and probe layer stability during reuse. Experimental Section Materials. All chemicals and solvents were of reagent grade quality or higher and were purchased from Fluka Chemie, GmbH, Switzerland, or Aldrich Chemie, GmbH, Germany. Deionized water (Millipore Q; >18 MΩ/cm) was used in all experiments. 3-Aminopropyltrimethoxysilane (APTMS) was purchased from ABCR Gelest, Inc., U.K. Oligonucleotides both unlabeled and fluorescent-labeled (Cy3) oligonucleotides were purchased from MWG Biotech, AG, Germany. Three general types were employed: 3′-amino-modified probes, TGA AGG CTT ACC GTC ATA GGT T (oligo A) and GGT ACT CTA TTT GTA GGT TCT TAC GT (oligo B). 5′-Cy3-modified targets, ACC TAT GAC GGT AAG CCT TGA (oligo A′) and CGT AAG AAC CTA CAA ATA GAG TAC C (oligo B′), A 5′-Cy3-, 3′-amino-modified probe oligo TCA AGG CTT ACC GTC ATA GGT (control modified) and a 5′-Cy3-, nonmodified probe oligo CGT GGG CTC AAT ATG TTT AGA TTC CT (control nonmodified) as array controls. Silicon wafers [100] were purchased from Wacker Siltronic AG, Germany. Glass microscope slides (25 mm × 75 mm) were purchased from J. Melvin Freed, Inc., U.S.A., and BDH Laboratory Supplies, U.K. Silicon Nitride Substrate Layer Deposition. N-type singlecrystal silicon wafers of [100] orientation with a resistivity of 2-4 Ω cm were cleaned in concentrated H2SO4 for 10 min. Following a H2O rinse, amorphous silicon nitride (Si3N4) layers were deposited on these wafers using either of two deposition techniques, plasma enhanced chemical vapor deposition (PECVD) or low pressure chemical vapor deposition (LPCVD). PECVD nitride films were deposited using a Trikon Delta 201 system using 300 sccm SiH4 and 500 sccm NH3 in the presence of 3500 sccm N2 at 350 °C (2000 Å target layer thickness; 1985.0 Å actual layer thickness measured following deposition using a Nanometrics NanoSpec thin film measurement system). LPCVD nitride layers were deposited using a LPCVD nitride tube, employing 60 sccm NH3 and 20 sccm SiH2Cl2 at 800 °C (2000 Å target layer thickness; 1974.0 Å actual layer thickness measured as explained above). Deposition and Covalent Immobilization of Probe DNA Microarrays. To reduce the risk of contamination, all substrates were cleaned and silanated immediately prior to probe microarray deposition. PECVD and LPCVD nitride layers deposited on 100 mm silicon wafers were diced into 1.5 cm2 pieces prior to surface cleaning. All substrates, nitride layers on silicon chips and bare glass microscope slides, were cleaned by immersion in 1:1 methanol/HCl for 30 min (total volume 40 mL), H2O rinse, immersion in concentrated H2SO4 for 30 min, H2O rinse, and immersion in boiling water for 30 min (to activate hydroxyl groups present on the surfaces). Liquid phase silanation was performed under ambient conditions by sonication (Decon FS100B, U.K.) in Coplin jars using freshly made amino-alkylsilane solutions at 3% concentration (v/v) in methanol/water solvent (95:5 v/v) at pH 7.0 for 30 min. Following silanation, substrates were sequentially rinsed with alcohol and H2O, dried under N2, and cured in a fan-assisted oven at 120 °C for 15 min. Amino-silanated substrates were activated for the covalent attachment of amino-terminated probe oligonucleotides using a homobifunctional cross-linker, 1,4phenylene diisothiocyante (PDITC). To this end, substrates were immersed in a 40 mL solution of 1 mM PDITC in 10% anhydrous pyridine/DMF (v/v) (pH 7.5) for 2 h followed by rinsing with dimethylformamide (DMF) and dichloroethane and drying under N2. Deposition and immobilization of 3′-amino-modified probe oligo microarrays at each of the substrates was then undertaken. Probe oligo solutions, varying in concentration from 10 to 0.001 µM, were prepared in one of three deposition and attachment

Manning and Redmond solutions: (a) 1 M Tris-HCl (pH 7.0) with added 1% N,Ndiisopropylethylamine (hereafter referred to as Tris-HCl pH 7.5), (b) 3xSSC (pH 7.0), or (c) dimethyl sulfoxide (DMSO) (pH 8.0). Aliquots (10 µL) of these solutions were dispensed into a 394 well-plate, and discrete 1 nL spots were deposited onto the substrates using an ArrayIt SpotBot microrobotic spotting tool equipped with Stealth SMP4 microspotting pins (TeleChem International, Inc., U.S.A.). Microarrays of up to 2800 individual spots were typically deposited in one cycle. Following probe array deposition, substrates were incubated in a humid environment at 37 °C overnight to facilitate covalent attachment of the amino-modified probes to the surface bound PDITC cross-linker molecules, rinsed with H2O and methanol, and dried under N2. Remaining unreacted, surface bound isothiocyanate groups were deactivated by immersion in a solution of 50 mM 6-amino-1-hexanol and 150 mM N,Ndiisopropylethylamine in DMF (pH 7.5) for 2 h followed by rinsing in DMF, acetone, and H2O and drying under N2. Any noncovalently bound probe oligos possibly remaining following array deposition and attachment were then removed by immersion of each array in 1xSSC and 0.1% sodium dodecyl sulfate (SDS) at 95 °C (pH 7.0) for 15 min. Target DNA Hybridization and Stripping. For hybridization, microarrays were placed on filter paper in a clean Petri dish, covered with 10 µL of target oligonucleotide solution (concentrations from 10 µM to 100 pM) in SSARC hybridization buffer (600 mM NaCl, 60 mM Na-citrate, and 7.2% (v/v) Na-sarcosyl) at pH 7.0, and protected by a Hybri-slip (Grace Bio-Labs, Inc., U.K.). A 200 µL portion of H2O was then applied to the filter paper edges in the Petri dish. The dish, sealed with Parafilm, was incubated at 42 °C overnight. Following incubation, the slides were rinsed with hybridization buffer and H2O and dried under N2. To strip (denature) the hybridized target oligos for array reuse, the substrates were immersed in H2O at 95 °C for 2 min. Substrate and Microarray Analysis. To provide routine real time process monitoring of substrate cleaning, silanation, and PDITC activation, as mentioned above, static contact angle measurements were obtained using a home-built system. To ensure reproducibility, all measurements were undertaken immediately following application of a specific process step. Atomic force microscopy measurements, used to quantify substrate surface root-mean-square (rms) roughness after cleaning, were performed using a JEOL JSPM-4200 system operating in alternating current (ac) (tapping) mode. All measurements were taken using probes from the same batch (Nanosensors NCH tapping mode probes). All scans consisted of 512 × 512 pixels measured over a 1 × 1 µm2 area at 6 Hz under identical feedback conditions. No processing was applied to the data apart from the usual background plane subtraction. The rms roughness value for each sample represented an average of multiple scans measured at different locations across each substrate surface. Fluorescence images of the microarrays were acquired using a Zeiss Axioskop II Plus epifluorescence microscope equipped with an Optronics DEI-750 CCD camera and appropriate filter sets. The images were analyzed using Image Pro Express software (Media Cybernetics, Inc., U.S.A.). To quantify a given microarray spot fluorescence intensity, pixel intensity values in an area of 70 × 95 ((5) µm within a single spot were measured and averaged. For microarray performance analysis, single spot fluorescence intensities acquired in this manner were typically averaged over 10 printed spots. Unless otherwise stated, all averaged spot fluorescence intensity data reported herein were corrected by fluorescence background subtraction prior to data averaging. (A similar procedure for quantification of fluorescence background was employed using an average measurement area of 35 × 35 ((5) µm.) Fluorescence intensity line scans were plotted using MATLAB software (The MathWorks, Inc., U.S.A.). Generally, no evidence for concentration quenching of fluorescence within microarray spots was detected.

Results and Discussion Formation of Probe DNA Microarrays. As described in the Introduction, the objective of the present paper is to demonstrate the feasibility of DNA microarray forma-

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Table 1. Contact Angles Measured Immediately Following Substrate Cleaning, Silanation, and PDITC Activation for Each of the Substrates Employed in This Study contact angle (deg) substrate type

thickness (nm)

clean

APTMS

PDITC

glass LPCVD nitride PECVD nitride

n/a 197.4 198.5

8(1 18 ( 2 6(1

42 ( 2 42 ( 2 38 ( 2

50 ( 1 60 ( 2 47 ( 1

tion on Si3N4 layers using robust probe immobilization methods. The approach adopted was to develop methods for covalent attachment of amino-terminated probe oligonucleotide microarrays at amino-silanated substrate surfaces using a homobifunctional cross-linker, 1,4phenylene diisothiocyanate (PDITC). Covalent attachment of oligonucleotides to functionalized glass substrates in a similar manner has previously been demonstrated to yield stable DNA layers that exhibited high quality and reproducibility in terms of probe spot morphology and homogeneity as well as hybridization efficiency and sensitivity.20-22 To monitor the progress of the substrate derivitization process, contact angle measurements were routinely employed. In this manner, the increasing hydrophobicity of the newly cleaned substrate surfaces could be measured following silanation and PDITC based amino-silane activation; see Table 1. The magnitude, trend, and low variability of each of the measured values are consistent with successful homogeneous stepwise molecule layer attachment.23 The relative performance of the subsequent aminoterminated probe oligonucleotide to 1,4-phenylene diisothiocyanate covalent attachment step was then assessed by identifying an appropriate probe oligonucleotide deposition and immobilization solution. Three different solution types, namely, Tris-HCl with added 1% diiospropylethylamine (Tris-HCl) (pH 7.5), 3xSSC (pH 7.0), and DMSO (pH 8.0), were examined for their relative effectiveness with respect to deposition and linkage of 20-mer, 5′-Cy3-, 3′-amino-modified probe oligonucleotides at PDITC activated, amino-silane functionalized glass, LPCVD nitride, and PECVD nitride substrates. Following deposition and attachment of probe DNA (4 µM probe concentration) and washing and drying of the substrates, fluorescence micrographs of each fabricated microarray were acquired under 100 W mercury lamp illumination using a 1 s exposure time. To analyze the effectiveness of probe spot immobilization as a function of solution type, average spot fluorescence intensities and corresponding fluorescence backgrounds were measured for 10 spots each per microarray, as described in the Experimental Section, and the data were then plotted with respect to substrate type; see Figure 1a. It is clear from the presented data that both LPCVD and PECVD nitrides provided consistently high averaged immobilized probe spot fluorescence intensities in combination with each of the deposition and attachment solutions employed. In particular, for both substrate types, a comparatively low background fluorescence, that is, a good signal-to-background ratio, was obtained while using (20) Beier, M.; Hoheisel, J. D. Nucleic Acids Res. 1999, 27, 19701977. (21) Manning, M.; Harvey, S.; Galvin, P.; Redmond, G. Mater. Sci. Eng., C 2003, 23, 347-351. (22) Manning, M.; Galvin, P.; Redmond, G. Am. Biotechnol. Lab. 2002, 7, 16-17. (23) Charles, P. T.; Vora, G. J.; Andreadis, J. D.; Fortney A. J.; Meador, C. E.; Dulcey, C. S.; Stenger, D. A. Langmuir 2003, 19, 1586-1591.

Figure 1. (a) Comparison of the averaged spot fluorescence intensities of immobilized 5′-Cy3-, 3′-amino-modified probe oligos measured with respect to both probe deposition and immobilization solution type and microarray substrate type. (b) Typical immobilized probe spot fluorescence images acquired for the probe deposition solutions and substrates used in part a above. Scale bar: 50 µm.

either 3xSSC or Tris-HCl solutions. By contrast, the DMSO solution yielded a much higher background fluorescence. Also, consistent with previously reported data, probe deposition and attachment on glass substrates yielded low coupling efficiencies for both 3xSSC and DMSO solutions (data not shown; see below) compared with that achieved using the Tris-HCl solution.21,24 Concerning the influence of solution type on immobilized spot morphology, when the DMSO solution was employed, streaking of spots was observed for all substrate types; see Figure 1b. This effect is due to the lower volatility of DMSO compared to water, whereby mechanical agitation of incompletely evaporated droplets during post-probe deposition slide handling causes streaking. For this solution, probe spot quality was particularly poor while using glass as the microarray substrate. Nonuniform and poorly defined probe oligo spots were observed to “spread” across the array substrate (since DMSO has a much lower surface tension than water), rendering fluorescence data acquisition very troublesome. For this reason, DMSO was considered unsuitable as a probe spot deposition and attachment solution and omitted from further study. For the 3xSSC solution, a similarly poor spot quality was observed while using glass as the microarray substrate, while, interestingly, the solution afforded intense, reasonably well-defined fluorescent probe spots at both the LPCVD and PECVD nitride substrates. However, similar to previous findings, the Tris-HCl solution with added 1% N,N-diisopropylethylamine (pH 7.5) provided the most reproducible, well-defined, homogeneous spot morphologies with high areal fluorescence and very low levels of background fluorescence for all three substrates examined; see Figure 1b.20,21,25 The Tris-HCl solution works well (24) Diehl, F.; Grahlmann, S.; Beier, M.; Hoheisel, J. D. Nucleic Acids Res. 2001, 29, e38. (25) Dolan, P.; Wu, Y.; Ista, L. K.; Metzenberg, R. L.; Nelson, M. A.; Lopez, G. P. Nucleic Acids Res. 2001, 29, e107.

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Figure 3. (a) Comparison of the averaged spot fluorescence intensities of immobilized 5′-Cy3-, 3′-amino-modified probe oligonucleotides measured with respect to the deposited probe concentration for each substrate type. (b) Typical immobilized probe spot fluorescence images acquired at each deposited probe concentration for each substrate type. Scale bar: 50 µm.

Figure 2. Typical 1 µm2 tapping mode AFM images and associated rms roughness values (per square micrometer) measured for cleaned (a) PECVD nitride, (b) LPCVD nitride, and (c) glass substrates.

because its slightly basic pH, afforded by the added diisopropylethylamine, optimizes the reactivity of the PDITC cross-linker by deprotonating the terminal amine modification of the probe oligonucelotides, thereby facilitating nucleophilic attack at the isothiocyanate carbon atom of PDITC and efficient thiourea cross-link formation. An additional advantage is that, because of its nonnucleophilic character, the diisopropylethylamine does not compete for the reactive sites on the chip. To investigate possible reasons for the quite impressive performance of nitride layers as substrates for the immobilization of probe DNA microarrays, tapping mode atomic force microscopy (AFM) measurements were made on each of the substrate types; see Figure 2. From the AFM images, distinct morphological differences were observed for each of the three substrates. rms roughness measurements performed on multiple 1 µm2 portions of the surfaces showed that LPCVD nitride was the least rough surface at 0.33 ( 0.007 nm/µm2 while PECVD nitride was the roughest at 1.76 ( 0.049 nm/µm2. The roughness of the glass surface was intermediate between the two at 0.38 ( 0.040 nm/µm2. Previously, a close correlation between surface roughness, effective surface area, and probe binding capacity has been observed for fused silica and glass substrates.26 (26) Henke, L.; Nagy, N.; Krull, U. J. Biosens. Bioelectron. 2002, 17, 547-555.

However, the results presented herein indicate that, while the rms value of PECVD nitride was more than 4 times that of glass, the averaged immobilized probe spot fluorescence intensities shown in Figure 1a for Tris-HCl were only increased by ∼20%. Similarly, while the roughness value of glass was close to that of LPCVD nitride, the averaged probe spot fluorescence intensities afforded by the latter substrate were also greater than those measured on glass by ∼30%. These results indicate that, although surface roughness may play an important role in comparing the performance of substrates composed of similar materials, factors such as matching solution conditions with surface chemistry (e.g., oxide/nitride stoichiometry, hydroxyl group density, and extent of surface hydration) and even optical effects, for example, modification of Cy3 dye fluorescence emission due to reflection of the excitation light by the Si substrate beneath the Si3N4 layers, also contribute to the comparative performance of substrates composed of differing materials, such as those employed in this study.27,28 The relative performance of the covalent attachment method for the formation of probe DNA microarrays at nitride substrates was also assessed by characterizing the effect of deposited probe concentration on microarray properties. A series of 20-mer, 5′-Cy3-, 3′-amino-modified probe oligonucleotide microarrays (with probe concentrations ranging from 0.001 to 10 µM) were fabricated at PDITC activated, amino-silane functionalized substrates using Tris-HCl as the deposition solution. Following this, fluorescence micrographs of each microarray were acquired under the usual conditions. To analyze the effectiveness of probe spot immobilization as a function of deposited probe concentration, spot fluorescence intensities were measured for 10 spots each per microarray, background corrected, and averaged; see Figure 3a. (27) Kain, C. R.; Marason, E. G.; Johnston R. F. U.S. Patent 6,008,892, 1999. (28) Opila, R. L.; Legrange, J. D.; Markham, G.; Heyer, G. Schroeder, C. M. J. Adhes. Sci. Technol. 1997, 11, 1-10.

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The averaged fluorescence intensities plotted in Figure 3a with respect to substrate type suggest that the apparent relative areal density of immobilized probe oligonucleotides varied considerably with the concentration of probes deposited onto each substrate prior to covalent immobilization. The apparent yield of immobilized probes at PECVD nitride substrates was lower than that achieved at LPCVD nitride or glass substrates. While for all substrates the yield of immobilized probes was comparatively low for deposited concentrations of 1 µM, the yield of surface attached probes at LPCVD nitride and glass substrates was proportional to the deposition concentration until it appeared to plateau slightly at the highest concentrations employed. Concerning the influence of deposited probe concentration on the morphology of subsequently immobilized spots, welldefined spots, ∼120 µm in diameter and without streaking or comet tails, were observed for all substrates at almost all concentrations examined; see Figure 3b. Fluorescence intensity and homogeneity across the immobilized spots depended on the deposited probe concentration with distinct patterns evident in spot fluorescence micrographs acquired at the lower probe concentrations due to, for example, aggregation of probe at the spot edges during drying.24,25 However, LPCVD nitride substrates performed slightly better than glass and noticeably better than PECVD nitride substrates in yielding reproducibly homogeneous spots that were detectable even at the lowest deposited probe concentrations and that displayed the greatest measured fluorescence signals at higher deposited probe concentrations (>1 µM), presumably corresponding to relatively high apparent areal densities of immobilized probe oligonucleotides. Taken together, the data of Figures 2 and 3 indicated that probe oligonucleotide deposition and attachment solution type and deposited probe concentration were indeed critical in determining the apparent relative areal density of probe molecules within immobilized spots, the quality and uniformity of spot size and morphology, and the general reproducibility of the DNA microarray fabrication process. In general, effective probe attachment and high quality well-defined probe spots of consistent morphology were observed while using 1 M Tris-HCl with added 1% N,N-diisopropylethylamine (Tris-HCl) as the probe oligo deposition and attachment solution.20,21,25 On the basis of these observations, Tris-HCl was selected as the probe deposition and attachment solution for further use. Target DNA Hybridization. Although the covalent attachment strategy presented above was capable of immobilizing probes at all of the substrates investigated, it was imperative to demonstrate that probe oligonucleotides were attached at each substrate in a manner such that efficient hybridization with complementary target sequences in solution was possible. While it was shown above that the apparent relative areal density of immobilized probe oligos varied with deposited probe concentration for each substrate type, it was equally important to clarify how the effectiveness of target hybridization might also vary with probe concentration. In this regard, for example, previous research has indicated that low densities of immobilized probes resulted in poor hybridization signals while high densities inhibited hybridization.29 To address this issue, the fabrication of a series of 20-mer, 3′-amino-modified probe oligo microarrays (with probe concentrations ranging from 0.001 to 10 µM) at

PDITC activated, amino-silane functionalized substrates using Tris-HCl as the deposition solution was undertaken. Complementary dye-modified target oligos (of 4 µM fixed concentration) were hybridized at each array, as described in the Experimental Section. Posthybridization fluorescence micrographs of each microarray were then acquired in the usual manner. For comparison, the fluorescence intensity associated with 10 spots each per microarray was measured, background corrected, and averaged; see Figure 4a. The data shown in Figure 4a indicated that target hybridization, specifically at LPCVD nitride and glass substrates, was excellent with detectability of hybridization across a 50-fold variation in initial spotted probe oligonucleotide concentration. Comparatively low fluorescence background levels were measured at both of these substrates (the measured signal-to-background ratios were typically 1:0.1 at nitride and 1:0.15 at glass). While the averaged hybridization-related spot fluorescence intensities dropped more steeply with decreasing deposited probe concentration for LPCVD nitride than for glass substrates, the fluorescence intensities measured for both of these substrates tracked the stepwise variation in deposited probe concentration over a wide dynamic range without signal saturation, indicating no limitation in target hybridization efficiency due to, for example, steric hindrance arising from excessive probe oligonucleotide areal surface density.30 Concerning the influence of deposited probe concentration on the morphology of subsequently hybridized spots, high quality well-defined spots, ∼120 µm in diameter, with an excellent signal-to-background ratio and with minimal streaking were observed at LPCVD nitride and glass substrates for all probe concentrations employed; see Figure 3b. Hybridized spot fluorescence was most

(29) Guo, Z.; Guilfoyle, R. A.; Theil, A. J.; Wang, R.; Smith, L. M. Nucleic Acids Res. 1994, 22, 5456-5465.

(30) Bordoni, R.; Consolandi, C.; Castiglioni, B.; Busti, E.; Bernardi, L. R.; Battaglia, C.; De Bellis, G. Nucleic Acids Res. 2002, 30, e34.

Figure 4. (a) Comparison of the averaged spot fluorescence intensities of 5′-Cy3-modified target oligos hybridized at 3′amino-modified probe oligo microarrays measured versus the deposited probe for each substrate type. (b) Typical target spot fluorescence images acquired at each deposited probe concentration for each substrate type. Scale bar: 50 µm.

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Figure 5. (a) Comparison of the averaged spot fluorescence intensities of 5′-Cy3-modified target oligos hybridized at 3′amino-modified probe oligo microarrays measured versus the target concentration for each substrate type. (b) Typical target spot fluorescence images acquired at each target concentration for each substrate type. Scale bar: 50 µm.

intense for deposited probe concentrations exceeding 5 µM with intensity decreasing with concentration. Higher deposited probe concentrations yielded reproducibly uniform and homogeneous spots with the highest measured fluorescence signals, that is, relatively high apparent areal density of 5′-Cy3-modified target oligonucleotides hybridized at the immobilized 3′-amino-modified probe oligo spots. At intermediate and lower probe concentrations, the aggregation of probe oligos at spot edges during postdeposition drying caused the formation of ringlike probe deposits which were replicated in the fluorescence images acquired following target hybridization.25,31 By comparison, the hybridization-related fluorescence response of the PECVD nitride substrates was poorer in all respects. Finally, to investigate the effect of target oligonucleotide concentration on hybridization, a further series of 20mer, 3′-amino-modified probe oligo arrays was formed at PDITC activated, amino-silane functionalized substrates using the standard fabrication methods. Following this, complementary dye-modified target oligos (at various concentrations between 10 µM and 100 pM) were hybridized at each array and fluorescence image analysis was undertaken in the manner already described. The resulting data, shown in Figure 5a, indicated that LPCVD nitride and glass arrays demonstrated a measurable average fluorescence intensity associated with hybridization of 5′Cy3-modified target oligos at 3′-amino-modified probe oligo spots with excellent fluorescent signal response over almost the entire concentration range used. The high sensitivity of these microarrays is a very important feature, especially when considering the application of DNA microarrays to assays for which available genetic material may be in limited supply. The generically poorer response of the PECVD nitride layers in this regard is in agreement with the probe concentration dependence of both probe immobilization and target hybridization effectiveness (31) Blossey, R.; Bosio, A. Langmuir 2002, 18, 2952-2954.

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reported for this substrate above. For all microarray substrates employed, the hybridization-related average fluorescence intensity apparently began to plateau at target concentrations above 1 nM; see Figure 5a. This effect might be attributable to the saturation of all available immobilized probes by the hybridized target oligos at the substrate surface. Regarding the morphology of the hybridized fluorescent spots, LPCVD nitride and glass based microarrays also exhibited very high spot quality and reproducibility; see Figure 5b for representative fluorescence micrographs measured across the range of target concentrations employed. Target spots were typically well-defined almost circular structures of ∼120 µm diameter, with uniform and homogeneous fluorescence within each spot and with an excellent signal-to-background ratio (measured signalto-background ratio was typically 1:0.08). For both LPCVD nitride and glass substrates, spot fluorescence was most intense for target concentrations exceeding 1 nM with intensity slightly increasing with target concentration. Contrary to reports made by other researchers, the use of high target concentrations did not contribute to increased background in the fluorescence images.29 Further, increased fluorescence background signals as reported by some researchers for hybridization times exceeding 3 h were not observed during hybridization of short oligonucleotides on any of the three substrates examined.15 In fact, low fluorescence background levels were observed for hybridization reactions up to and exceeding 12 h, reflecting minimal nonspecific target adsorption due to the effectiveness of the 6-amino-1hexanol based substrate passivation/deactivation process. Microarray Selectivity and Stability. Experiments were undertaken to demonstrate that covalent probe immobilization at nitride substrates also permitted the successful discrimination of complementary and noncomplementary target oligonucleotide sequences during multiple sequential hybridizations. For this purpose, microarrays comprising three different 20-mer probe oligonucleotides in three discrete columns (1-3) were formed at PDITC activated, amino-silane functionalized substrates using the protocols described above; see Figure 6. Column 1 was an experimental control containing spots of 20-mer, 5′-Cy3-, 3′-amino-modified probe oligonucleotides covalently immobilized at each substrate. Columns 2 and 3 contained spots of covalently immobilized 20mer, 3′-amino-modified probe oligonucleotides with two entirely different sequences. The fluorescence micrographs of Figure 6a show the microarrays following removal of any nonspecifically bound probe oligos from the glass surface and prior to hybridizationsfluorescence was detected only from the columns of dye-modified control oligos. Following the hybridization of a target oligonucleotide complementary in sequence to the probe oligos of each column 2 using the standard protocols, fluorescence was also detected from these columns; see Figure 6b. No fluorescence was detected from columns 3, demonstrating successful discrimination of the target sequence by the probe spots of these columns. Following the application of a stripping cycle to remove hybridized targets from the arrays and hybridization of target oligonucleotides complementary in sequence to the probe oligos of columns 3, fluorescence was then detected from those columns; see Figure 6c. No fluorescence was detected from any of columns 2, demonstrating successful discrimination of the target sequence by the probe spots of these columns. Note also that, following this second hybridization cycle, the fluorescence intensities measured from each of

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Figure 7. Comparison of averaged spot fluorescence intensities of 5′-Cy3-modified targets hybridized at 3′-amino-modified probe microarrays measured with respect to hybridization cycle for five sequential hybridizations at each microarray substrate type. Following each data acquisition, hybridized targets were stripped and the arrays were then reused.

Figure 6. Micrographs showing (a) the fluorescence of covalently immobilized dye-modified probe controls at columns 1 (the probes covalently immobilized at columns 2 and 3 are nonmodified), (b) the fluorescence of covalently immobilized controls at columns 1 (columns 2 show fluorescence due to selective hybridization of dye-modified targets), and (c) the fluorescence of covalently immobilized controls at columns 1 (columns 3 now show target fluorescence due to selective hybridization). Scale bar: 100 µm.

columns 1 on the LPCVD nitride and the glass substrates were largely undiminished, consistent with the robustness of the covalent probe immobilization method. In contrast, the fluorescence intensities measured from column 1 on the PECVD nitride substrate appeared to be somewhat diminished, suggesting that the stabilities either of the probe oligos immobilized at this nitride surface or of the actual deposited nitride layers were lower than those achieved by the LPCVD nitride substrate. To test the response of probe microarrays over cycles of sustained reuse, multiple sequential hybridization and stripping (denaturation) cycles were performed on microarrays comprising spots of 20-mer, 3′-amino-modified probe oligonucleotides covalently attached at LPCVD nitride, PECVD nitride, and glass substrates using standard procedures; see Figure 7. In agreement with the results presented above, the hybridization-related fluorescence response of PECVD nitride substrates was found to decrease rapidly after one hybridization and strip cycle, indicating a significantly lower stability of this substrate. In fact, optical inspection of the PECVD nitride substrates following hybridization and stripping revealed that progressive degradation of the nitride (by surface layer removal or thinning) had occurred as a result of application of the various process steps. In contrast, for LPCVD nitride and glass substrates, the average hybridization-related spot fluorescence intensity was observed to remain practically constant over five consecutive hybridization/strip/rehybridization cycles of complementary 5′-Cy3-modified target oligos, irrespective of the harshness of the stripping conditions (immersion in H2O at 100 °C for 15 min). (Stripping resulted in the interim removal of essentially all fluorescence signals; data not shown.) The particular performance of the nitride

substrate in this respect compares very favorably with data reported from previous stability studies of silicon oxide, glass, and hydrogen-terminated silicon microarray substrates and is superior to that of DNA-modified gold substrates, which are known to degrade rapidly due to thiol group hydrolysis under basic conditions.18,25,32-36 Concerning the expected stability of response over more than five cycles, an eventual decrease of microarray performance is likely to occur, since the degradation of chemically modified surfaces incorporating Si-O linkages, especially under basic conditions or in the presence of amines, is a known phenomenon.35,37,38 In this regard, recent work has demonstrated that substantial improvements in microarray stability may be achieved using nanocrystalline diamond thin film substrates.36 However, the results presented herein confirm that our immobilization process results in the formation of stable covalent bonds between the terminal amino modification of the probe oligos and the PDITC activated, amino-silane functionalized LPCVD nitride and glass substrates, respectively. The demonstrated selectivity, stability, and reusability of these probe microarrays suggest that this combination of microfabrication compatible Si3N4 substrates and novel probe immobilization protocols has the potential to provide fundamental benefits in terms of both ease of fabrication and subsequent hybridization assay performance for microelectronics enabled integrated DNA biochips. Conclusion The goal of this work was to establish the feasibility of DNA microarray formation on Si3N4 layers using, as a basis, covalent probe immobilization methods. Each microarray fabrication process step, from silicon nitride substrate deposition, surface cleaning, amino-silanation, and attachment of homobifunctional cross-linking molecule to the direct covalent immobilization of probe oligonucleotides, has been defined, characterized, and (32) Chrisey, L. A.; Lee, G. U.; O’Ferrall, C. E. Nucleic Acids Res. 1996, 24, 3031-3039. (33) Strother, T.; Cai, W.; Zhao, X.; Hamers, R. J.; Smith, L. M. J. Am. Chem. Soc. 2000, 122, 1205-1209. (34) Strother, T.; Hamers, R. J.; Smith, L. M. Nucleic Acids Res. 2000, 28, 3535-3541. (35) Lin, Z.; Strother, T.; Cai, W.; Cao, X.; Smith, L. M.; Hamers, R. J. Langmuir 2002, 18, 788-796. (36) Yang, W. S.; Auciello, O.; Butler, J. E.; Cai, W.; Carlisle, J. A.; Gerbi, J.; Gruen, D. M.; Knickerbocker, T.; Lasseter, T. L.; Russell, J. N.; Smith, L. M.; Hamers, R. J. Nat. Mater. 2002, 1, 253-257. (37) Gray, D. E.; Case-Green, S. C.; Fell, T. S.; Dobson, P. J.; Southern, E. M. Langmuir 1997, 13, 2833-2842. (38) Major, R. C.; Zhu, X.-Y. Langmuir 2001, 17, 5576-5580.

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optimized to yield consistent probe microarray quality, homogeneity, and probe-target hybridization performance. With respect to the latter, the developed microarray fabrication methodology has been shown to provide excellent (high signal-to-background ratio) and reproducible responsivity to target oligonucleotide hybridization with a rugged chemical stability that permitted exposure of each array to stringent pre- and posthybridization wash conditions through many sustained cycles of reuse.

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Therefore, for this novel microarray substrate material, these features compare very favorably with the performance attributes of more mature glass microarray based technologies. Consequently, it is proposed that this DNA microarray fabrication strategy has the potential to provide a viable route toward successful realization of future integrated DNA biochips. LA0480033