Formation and Characterization of DNA Microarrays atSilicon 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, previouslydeposited by chemical vapor deposition at silicon wafer substrates, is reported. Each microarray fabricationprocess step, from silicon nitride substrate deposition, surface cleaning, amino-silanation, and attachmentof 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-targethybridization performance. The developed microarray fabrication methodology provides excellent (highsignal-to-background ratio) and reproducible responsivity to target oligonucleotide hybridization with arugged chemical stability that permits exposure of arrays to stringent pre- and posthybridization washconditions through many sustained cycles of reuse. Overall, the achieved performance features comparevery favorably with those of more mature glass based microarrays. It is proposed that this DNA microarrayfabrication strategy has the potential to provide a viable route toward the successful realization of futureintegrated DNA biochips.
IntroductionIt is widely acknowledged that, to accelerate the uptake
and exploitation of many microarray based geneticanalysis technologies, substantial increases in throughputmust be achieved while minimizing the cost per assay.Since the future demand for high-throughput geneticanalysis tools is expected to increase well beyond thecapabilities of current technologies, new approaches toDNA microarray fabrication such as the integration ofmicroarrays into electronically addressable “intelligent”substrates are required. Development of microelectronicsenabled functionally integrated biochips that permit on-chip biological assays, data acquisition, and even dataprocessing will therefore be a key enabler of thismicroarray revolution.
However, to successfully converge the materials andtools of semiconductor microfabrication with the reagentsand protocols of molecular biology, new approaches tosurface modification and interface engineering using apotentially diverse range of physical and chemical tech-niques must be explored. Integrated biochips are com-prised of two parts: a biologically active surface layerpatterned at an array of discrete sensing elements.1,2 Forbiomolecule detection, the former may be a layer ofantibodies, probe oligonucleotides, or cDNA strands, forexample. The latter is typically an array of microelectronicdevices capable of detecting molecular recognition andbinding events by transducing this information intomeasurable physical quantities such as changes inmass,3-5 temperature,6 conductance,7,8 or impedance.9-12
Critical to the overall performance of biochips is theavailability of methods for the deposition and immobiliza-tion of probe molecules onto sensor arrays in a mannerthat enables reproducible, sensitive operation.13-18 Siliconnitride (and silicon oxide) layers are key materialsemployed for the fabrication of integrated microelectronic,optoelectronic, and microelectromechanical devices andwill, in the future, assume increasing importance aspassivation layers, protecting integrated circuits frommechanical damage or other sources of degradation (Na+
ions or moisture),19 and as signal transduction layers, atthe interface between miniaturized, solid-state sensortechnologies and the adjacent analyte-bearing ambient.For DNA biochips, demands on passivation layers andassociated probe immobilization methods will be stringent,since the finishedmicroarray layersmustprotect theactivesensorelementsexposed toharshambient conditionswhilealso serving as DNA hybridization substrates.
To address some of these challenges, we report on thedevelopment of a versatile, microfabrication compatibleDNA deposition and immobilization method that suc-
* 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.; Nanofab-
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Souteyrand, E.; Polychronakos, C.; Lawrence, M. F. Biosens. Bioelectron.2002, 17, 405-412.
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cessfully permits direct, covalent attachment of probeoligonucleotides at the surface of silicon nitride layerspreviously deposited by chemical vapor deposition (CVD)at silicon wafer substrates. In this regard, a key achieve-ment has been the successful demonstration of reliable,direct covalent probe attachment combined with sensitivetarget hybridization at CVD Si3N4 layers while preservingpassivation layer and probe layer stability during reuse.
Experimental SectionMaterials. All chemicals and solvents were of reagent grade
quality or higher and were purchased from Fluka Chemie, GmbH,Switzerland, or Aldrich Chemie, GmbH, Germany. Deionizedwater (Millipore Q; >18 MΩ/cm) was used in all experiments.3-Aminopropyltrimethoxysilane (APTMS) was purchased fromABCR Gelest, Inc., U.K. Oligonucleotides both unlabeled andfluorescent-labeled (Cy3) oligonucleotides were purchased fromMWG Biotech, AG, Germany. Three general types were em-ployed: 3′-amino-modified probes, TGA AGG CTT ACC GTCATA GGT T (oligo A) and GGT ACT CTA TTT GTA GGT TCTTAC GT (oligo B). 5′-Cy3-modified targets, ACC TAT GAC GGTAAG CCT TGA (oligo A′) and CGT AAG AAC CTA CAA ATAGAG TAC C (oligo B′), A 5′-Cy3-, 3′-amino-modified probe oligoTCA AGG CTT ACC GTC ATA GGT (control modified) and a5′-Cy3-, nonmodified probe oligo CGT GGG CTC AAT ATG TTTAGA TTC CT (control nonmodified) as array controls. Siliconwafers [100]werepurchased fromWackerSiltronicAG,Germany.Glass microscope slides (25 mm × 75 mm) were purchased fromJ. Melvin Freed, Inc., U.S.A., and BDH Laboratory Supplies,U.K.
SiliconNitrideSubstrateLayerDeposition.N-typesingle-crystal silicon wafers of [100] orientation with a resistivity of2-4 Ω cm were cleaned in concentrated H2SO4 for 10 min.Following a H2O rinse, amorphous silicon nitride (Si3N4) layerswere deposited on these wafers using either of two depositiontechniques, plasma enhanced chemical vapor deposition (PECVD)or low pressure chemical vapor deposition (LPCVD). PECVDnitride films were deposited using a Trikon Delta 201 systemusing 300 sccm SiH4 and 500 sccm NH3 in the presence of 3500sccm N2 at 350 °C (2000 Å target layer thickness; 1985.0 Å actuallayer thickness measured following deposition using a Nano-metrics NanoSpec thin film measurement system). LPCVDnitride layers were deposited using a LPCVD nitride tube,employing 60 sccm NH3 and 20 sccm SiH2Cl2 at 800 °C (2000 Åtarget layer thickness; 1974.0 Å actual layer thickness measuredas explained above).
Deposition and Covalent Immobilization of Probe DNAMicroarrays. To reduce the risk of contamination, all substrateswere cleaned and silanated immediately prior to probe microarraydeposition. PECVD and LPCVD nitride layers deposited on 100mm silicon wafers were diced into 1.5 cm2 pieces prior to surfacecleaning. All substrates, nitride layers on silicon chips and bareglass microscope slides, were cleaned by immersion in 1:1methanol/HCl for 30 min (total volume 40 mL), H2O rinse,immersion in concentrated H2SO4 for 30 min, H2O rinse, andimmersion in boiling water for 30 min (to activate hydroxyl groupspresent on the surfaces).
Liquid phase silanation was performed under ambient condi-tions by sonication (Decon FS100B, U.K.) in Coplin jars usingfreshly 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 withalcohol and H2O, dried under N2, and cured in a fan-assistedoven at 120 °C for 15 min. Amino-silanated substrates wereactivated for the covalent attachment of amino-terminated probeoligonucleotides using a homobifunctional cross-linker, 1,4-phenylene diisothiocyante (PDITC). To this end, substrates wereimmersed in a 40 mL solution of 1 mM PDITC in 10% anhydrouspyridine/DMF (v/v) (pH 7.5) for 2 h followed by rinsing withdimethylformamide (DMF) and dichloroethane and dryingunder N2.
Deposition and immobilization of 3′-amino-modified probe oligomicroarrays at each of the substrates was then undertaken. Probeoligo solutions, varying in concentration from 10 to 0.001 µM,were prepared in one of three deposition and attachment
solutions: (a) 1 M Tris-HCl (pH 7.0) with added 1% N,N-diisopropylethylamine (hereafter referred to as Tris-HCl pH7.5), (b) 3xSSC (pH 7.0), or (c) dimethyl sulfoxide (DMSO) (pH8.0). Aliquots (10 µL) of these solutions were dispensed into a394 well-plate, and discrete 1 nL spots were deposited onto thesubstrates using an ArrayIt SpotBot microrobotic spotting toolequipped with Stealth SMP4 microspotting pins (TeleChemInternational, Inc., U.S.A.). Microarrays of up to 2800 individualspots were typically deposited in one cycle.
Following probe array deposition, substrates were incubatedin a humid environment at 37 °C overnight to facilitate covalentattachment of the amino-modified probes to the surface boundPDITC cross-linker molecules, rinsed with H2O and methanol,and dried under N2. Remaining unreacted, surface boundisothiocyanate groups were deactivated by immersion in asolution of 50 mM 6-amino-1-hexanol and 150 mM N,N-diisopropylethylamine in DMF (pH 7.5) for 2 h followed by rinsingin DMF, acetone, and H2O and drying under N2. Any nonco-valently bound probe oligos possibly remaining following arraydeposition and attachment were then removed by immersion ofeach array in 1xSSC and 0.1% sodium dodecyl sulfate (SDS) at95 °C (pH 7.0) for 15 min.
Target DNA Hybridization and Stripping. For hybridiza-tion, microarrays were placed on filter paper in a clean Petridish, covered with 10 µL of target oligonucleotide solution(concentrations from 10 µM to 100 pM) in SSARC hybridizationbuffer (600 mM NaCl, 60 mM Na-citrate, and 7.2% (v/v)Na-sarcosyl) at pH 7.0, and protected by a Hybri-slip (GraceBio-Labs, Inc., U.K.). A 200 µL portion of H2O was then appliedto the filter paper edges in the Petri dish. The dish, sealed withParafilm, was incubated at 42 °C overnight. Following incubation,the slides were rinsed with hybridization buffer and H2O anddried under N2. To strip (denature) the hybridized target oligosfor array reuse, the substrates were immersed in H2O at 95 °Cfor 2 min.
Substrate and Microarray Analysis. To provide routinereal time process monitoring of substrate cleaning, silanation,and PDITC activation, as mentioned above, static contact anglemeasurements were obtained using a home-built system. Toensure reproducibility, all measurements were undertakenimmediately following application of a specific process step.
Atomic force microscopy measurements, used to quantifysubstrate surface root-mean-square (rms) roughness after clean-ing, were performed using a JEOL JSPM-4200 system operatingin alternating current (ac) (tapping) mode. All measurementswere taken using probes from the same batch (Nanosensors NCHtapping mode probes). All scans consisted of 512 × 512 pixelsmeasured over a 1 × 1 µm2 area at 6 Hz under identical feedbackconditions. No processing was applied to the data apart from theusual background plane subtraction. The rms roughness valuefor each sample represented an average of multiple scansmeasured at different locations across each substrate surface.
Fluorescence images of the microarrays were acquired usinga Zeiss Axioskop II Plus epifluorescence microscope equippedwith an Optronics DEI-750 CCD camera and appropriate filtersets. The images were analyzed using Image Pro Express software(Media Cybernetics, Inc., U.S.A.). To quantify a given microarrayspot fluorescence intensity, pixel intensity values in an area of70×95 ((5) µm within a single spot were measured and averaged.For microarray performance analysis, single spot fluorescenceintensities acquired in this manner were typically averaged over10 printed spots. Unless otherwise stated, all averaged spotfluorescence intensity data reported herein were corrected byfluorescence background subtraction prior to data averaging. (Asimilar procedure for quantification of fluorescence backgroundwas employed using an average measurement area of 35 × 35((5) µm.) Fluorescence intensity line scans were plotted usingMATLAB software (The MathWorks, Inc., U.S.A.). Generally,no evidence for concentration quenching of fluorescence withinmicroarray spots was detected.
Results and Discussion
Formation of Probe DNA Microarrays. As describedin the Introduction, the objective of the present paper isto demonstrate the feasibility of DNA microarray forma-
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tion on Si3N4 layers using robust probe immobilizationmethods. The approach adopted was to develop methodsfor covalent attachment of amino-terminated probe oli-gonucleotide microarrays at amino-silanated substratesurfaces using a homobifunctional cross-linker, 1,4-phenylenediisothiocyanate (PDITC).Covalentattachmentof oligonucleotides to functionalized glass substrates in asimilar manner has previously been demonstrated to yieldstable DNA layers that exhibited high quality andreproducibility in terms of probe spot morphology andhomogeneity as well as hybridization efficiency andsensitivity.20-22
To monitor the progress of the substrate derivitizationprocess, contact angle measurements were routinelyemployed. In this manner, the increasing hydrophobicityof the newly cleaned substrate surfaces could be measuredfollowing silanation and PDITC based amino-silaneactivation; see Table 1. The magnitude, trend, and lowvariability of each of the measured values are consistentwith successful homogeneous stepwise molecule layerattachment.23
The relative performance of the subsequent amino-terminated probe oligonucleotide to 1,4-phenylene di-isothiocyanate covalent attachment step was then as-sessed by identifying an appropriate probe oligonucleotidedeposition and immobilization solution. Three differentsolution types, namely, Tris-HCl with added 1% di-iospropylethylamine (Tris-HCl) (pH 7.5), 3xSSC (pH 7.0),and DMSO (pH 8.0), were examined for their relativeeffectiveness with respect to deposition and linkage of20-mer, 5′-Cy3-, 3′-amino-modified probe oligonucleotidesat PDITC activated, amino-silane functionalized glass,LPCVD nitride, and PECVD nitride substrates. Followingdeposition and attachment of probe DNA (4 µM probeconcentration) and washing and drying of the substrates,fluorescence micrographs of each fabricated microarraywere acquired under 100 W mercury lamp illuminationusing a 1 s exposure time. To analyze the effectiveness ofprobe spot immobilization as a function of solution type,average spot fluorescence intensities and correspondingfluorescence backgrounds were measured for 10 spots eachper microarray, as described in the Experimental Section,and the data were then plotted with respect to substratetype; see Figure 1a.
It is clear from the presented data that both LPCVDand PECVD nitrides provided consistently high averagedimmobilized probe spot fluorescence intensities in com-bination with each of the deposition and attachmentsolutions employed. In particular, for both substrate types,a comparatively low background fluorescence, that is, agood signal-to-background ratio, was obtained while using
either 3xSSC or Tris-HCl solutions. By contrast, theDMSO solution yielded a much higher backgroundfluorescence. Also, consistent with previously reporteddata, probe deposition and attachment on glass substratesyielded low coupling efficiencies for both 3xSSC and DMSOsolutions (data not shown; see below) compared with thatachieved using the Tris-HCl solution.21,24
Concerning the influence of solution type on immobilizedspot 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 ofDMSO compared to water, whereby mechanical agitationof incompletely evaporated droplets during post-probedeposition slide handling causes streaking. For thissolution, probe spot quality was particularly poor whileusing glass as the microarray substrate. Nonuniform andpoorly defined probe oligo spots were observed to “spread”across the array substrate (since DMSO has a much lowersurface tension than water), rendering fluorescence dataacquisition very troublesome. For this reason, DMSO wasconsidered unsuitable as a probe spot deposition andattachment solution and omitted from further study. Forthe 3xSSC solution, a similarly poor spot quality wasobserved while using glass as the microarray substrate,while, interestingly, the solution afforded intense, rea-sonably well-defined fluorescent probe spots at both theLPCVD and PECVD nitride substrates. However, similarto previous findings, the Tris-HCl solution with added1% N,N-diisopropylethylamine (pH 7.5) provided the mostreproducible, well-defined, homogeneous spot morphol-ogies with high areal fluorescence and very low levels ofbackground fluorescence for all three substrates examined;see Figure 1b.20,21,25 The Tris-HCl solution works well
(20) Beier, M.; Hoheisel, J. D. Nucleic Acids Res. 1999, 27, 1970-1977.
(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.
(24) Diehl, F.; Grahlmann, S.; Beier, M.; Hoheisel, J. D. Nucleic AcidsRes. 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.
Figure 1. (a) Comparison of the averaged spot fluorescenceintensities of immobilized 5′-Cy3-, 3′-amino-modified probeoligos measured with respect to both probe deposition andimmobilization solution type and microarray substrate type.(b) Typical immobilized probe spot fluorescence images acquiredfor the probe deposition solutions and substrates used in parta above. Scale bar: 50 µm.
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because its slightly basic pH, afforded by the addeddiisopropylethylamine, optimizes the reactivity of thePDITC cross-linker by deprotonating the terminal aminemodification of the probe oligonucelotides, thereby facili-tating nucleophilic attack at the isothiocyanate carbonatom of PDITC and efficient thiourea cross-link formation.An additional advantage is that, because of its non-nucleophilic character, the diisopropylethylamine does notcompete for the reactive sites on the chip.
To investigate possible reasons for the quite impressiveperformance of nitride layers as substrates for theimmobilization of probe DNA microarrays, tapping modeatomic force microscopy (AFM) measurements were madeon each of the substrate types; see Figure 2. From theAFM images, distinct morphological differences wereobserved for each of the three substrates. rms roughnessmeasurements performed on multiple 1 µm2 portions ofthe surfaces showed that LPCVD nitride was the leastrough surface at 0.33 ( 0.007 nm/µm2 while PECVDnitride was the roughest at 1.76 ( 0.049 nm/µm2. Theroughness of the glass surface was intermediate betweenthe two at 0.38 ( 0.040 nm/µm2. Previously, a closecorrelation between surface roughness, effective surfacearea, and probe binding capacity has been observed forfused silica and glass substrates.26
However, the results presented herein indicate that,while the rms value of PECVD nitride was more than 4times that of glass, the averaged immobilized probe spotfluorescence intensities shown in Figure 1a for Tris-HClwere only increased by ∼20%. Similarly, while theroughness value of glass was close to that of LPCVDnitride, the averaged probe spot fluorescence intensitiesafforded by the latter substrate were also greater thanthose measured on glass by ∼30%. These results indicatethat, although surface roughness may play an importantrole in comparing the performance of substrates composedof similar materials, factors such as matching solutionconditions with surface chemistry (e.g., oxide/nitridestoichiometry, hydroxyl group density, and extent ofsurface hydration) and even optical effects, for example,modification of Cy3 dye fluorescence emission due toreflection of the excitation light by the Si substrate beneaththe Si3N4 layers, also contribute to the comparativeperformance of substrates composed of differing materials,such as those employed in this study.27,28
The relative performance of the covalent attachmentmethod for the formation of probe DNA microarrays atnitride substrates was also assessed by characterizingthe effect of deposited probe concentration on microarrayproperties. A series of 20-mer, 5′-Cy3-, 3′-amino-modifiedprobe oligonucleotide microarrays (with probe concentra-tions ranging from 0.001 to 10 µM) were fabricated atPDITC activated, amino-silane functionalized substratesusing Tris-HCl as the deposition solution. Following this,fluorescence micrographs of each microarray were ac-quired under the usual conditions. To analyze the ef-fectiveness of probe spot immobilization as a function ofdeposited probe concentration, spot fluorescence intensi-ties were measured for 10 spots each per microarray,background corrected, and averaged; see Figure 3a.
(26) Henke, L.; Nagy, N.; Krull, U. J. Biosens. Bioelectron. 2002, 17,547-555.
(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.
Figure 3. (a) Comparison of the averaged spot fluorescenceintensities of immobilized 5′-Cy3-, 3′-amino-modified probeoligonucleotides measured with respect to the deposited probeconcentration for each substrate type. (b) Typical immobilizedprobe spot fluorescence images acquired at each deposited probeconcentration for each substrate type. Scale bar: 50 µm.
398 Langmuir, Vol. 21, No. 1, 2005 Manning and Redmond
The averaged fluorescence intensities plotted in Figure3a with respect to substrate type suggest that the apparentrelative areal density of immobilized probe oligonucle-otides varied considerably with the concentration of probesdeposited onto each substrate prior to covalent im-mobilization. The apparent yield of immobilized probesat PECVD nitride substrates was lower than that achievedat LPCVD nitride or glass substrates. While for allsubstrates the yield of immobilized probes was compara-tively low for deposited concentrations of <0.4 µM, atconcentrations >1 µM, the yield of surface attached probesat LPCVD nitride and glass substrates was proportionalto the deposition concentration until it appeared to plateauslightly at the highest concentrations employed. Concern-ing the influence of deposited probe concentration on themorphology of subsequently immobilized spots, well-defined spots, ∼120 µm in diameter and without streakingor comet tails, were observed for all substrates at almostall concentrations examined; see Figure 3b. Fluorescenceintensity and homogeneity across the immobilized spotsdepended on the deposited probe concentration withdistinct patterns evident in spot fluorescence micrographsacquired at the lower probe concentrations due to, forexample, aggregation of probe at the spot edges duringdrying.24,25 However, LPCVD nitride substrates performedslightly better than glass and noticeably better thanPECVD nitride substrates in yielding reproducibly ho-mogeneous spots that were detectable even at the lowestdeposited probe concentrations and that displayed thegreatest measured fluorescence signals at higher depositedprobe concentrations (>1 µM), presumably correspondingto relatively high apparent areal densities of immobilizedprobe oligonucleotides.
Taken together, the data of Figures 2 and 3 indicatedthat probe oligonucleotide deposition and attachmentsolution type and deposited probe concentration wereindeed critical in determining the apparent relative arealdensity of probe molecules within immobilized spots, thequality and uniformity of spot size and morphology, andthe general reproducibility of the DNA microarray fab-rication process. In general, effective probe attachmentand high quality well-defined probe spots of consistentmorphology were observed while using 1 M Tris-HCl withadded 1% N,N-diisopropylethylamine (Tris-HCl) as theprobe oligo deposition and attachment solution.20,21,25 Onthe basis of these observations, Tris-HCl was selected asthe probe deposition and attachment solution for furtheruse.
Target DNA Hybridization. Although the covalentattachment strategy presented above was capable ofimmobilizing probes at all of the substrates investigated,it was imperative to demonstrate that probe oligonucle-otides were attached at each substrate in a manner suchthat efficient hybridization with complementary targetsequences in solution was possible. While it was shownabove that the apparent relative areal density of im-mobilized probe oligos varied with deposited probe con-centration for each substrate type, it was equally impor-tant to clarify how the effectiveness of target hybridizationmight also vary with probe concentration. In this regard,for example, previous research has indicated that lowdensities of immobilized probes resulted in poor hybrid-ization signals while high densities inhibited hybridiza-tion.29 To address this issue, the fabrication of a series of20-mer, 3′-amino-modified probe oligo microarrays (withprobe concentrations ranging from 0.001 to 10 µM) at
PDITC activated, amino-silane functionalized substratesusing Tris-HCl as the deposition solution was under-taken. Complementary dye-modified target oligos (of 4µM fixed concentration) were hybridized at each array, asdescribed in the Experimental Section. Posthybridizationfluorescence micrographs of each microarray were thenacquired in the usual manner. For comparison, thefluorescence intensity associated with 10 spots each permicroarray was measured, background corrected, andaveraged; see Figure 4a.
The data shown in Figure 4a indicated that targethybridization, specifically at LPCVD nitride and glasssubstrates, was excellent with detectability of hybridiza-tion across a 50-fold variation in initial spotted probeoligonucleotide concentration. Comparatively low fluo-rescence background levels were measured at both of thesesubstrates (the measured signal-to-background ratioswere typically 1:0.1 at nitride and 1:0.15 at glass). Whilethe averaged hybridization-related spot fluorescenceintensitiesdroppedmoresteeplywithdecreasingdepositedprobe concentration for LPCVD nitride than for glasssubstrates, the fluorescence intensities measured for bothof these substrates tracked the stepwise variation indeposited probe concentration over a wide dynamic rangewithout signal saturation, indicating no limitation intarget hybridization efficiency due to, for example, sterichindrance arising from excessive probe oligonucleotideareal surface density.30
Concerning the influence of deposited probe concentra-tion on the morphology of subsequently hybridized spots,high quality well-defined spots, ∼120 µm in diameter,with an excellent signal-to-background ratio and withminimal streaking were observed at LPCVD nitride andglass 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.
Figure 4. (a) Comparison of the averaged spot fluorescenceintensities of 5′-Cy3-modified target oligos hybridized at 3′-amino-modified probe oligo microarrays measured versus thedeposited probe for each substrate type. (b) Typical target spotfluorescence images acquired at each deposited probe concen-tration for each substrate type. Scale bar: 50 µm.
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intense for deposited probe concentrations exceeding 5µM with intensity decreasing with concentration. Higherdeposited probe concentrations yielded reproducibly uni-form and homogeneous spots with the highest measuredfluorescence signals, that is, relatively high apparent arealdensity of 5′-Cy3-modified target oligonucleotides hybrid-ized at the immobilized 3′-amino-modified probe oligospots. At intermediate and lower probe concentrations,the aggregation of probe oligos at spot edges duringpostdeposition drying caused the formation of ringlikeprobe deposits which were replicated in the fluorescenceimages acquired following target hybridization.25,31 Bycomparison, the hybridization-related fluorescence re-sponse of the PECVD nitride substrates was poorer in allrespects.
Finally, to investigate the effect of target oligonucleotideconcentration on hybridization, a further series of 20-mer, 3′-amino-modified probe oligo arrays was formed atPDITC activated, amino-silane functionalized substratesusing the standard fabrication methods. Following this,complementary dye-modified target oligos (at variousconcentrations between 10 µM and 100 pM) were hybrid-ized at each array and fluorescence image analysis wasundertaken in the manner already described. The result-ingdata, shownin Figure5a, indicated that LPCVDnitrideand glass arrays demonstrated a measurable averagefluorescence intensity associated with hybridization of 5′-Cy3-modified target oligos at 3′-amino-modifiedprobeoligospots with excellent fluorescent signal response overalmost the entire concentration range used. The highsensitivityof thesemicroarrays isavery important feature,especially when considering the application of DNAmicroarrays to assays for which available genetic materialmay be in limited supply. The generically poorer responseof the PECVD nitride layers in this regard is in agreementwith the probe concentration dependence of both probeimmobilization and target hybridization effectiveness
reported for this substrate above. For all microarraysubstrates employed, the hybridization-related averagefluorescence intensity apparently began to plateau attarget concentrations above 1 nM; see Figure 5a. Thiseffect might be attributable to the saturation of allavailable immobilized probes by the hybridized targetoligos at the substrate surface.
Regarding the morphology of the hybridized fluorescentspots, LPCVD nitride and glass based microarrays alsoexhibited very high spot quality and reproducibility; seeFigure 5b for representative fluorescence micrographsmeasured across the range of target concentrationsemployed. Target spots were typically well-defined almostcircular structures of ∼120 µm diameter, with uniformand homogeneous fluorescence within each spot and withan excellent signal-to-background ratio (measured signal-to-background ratio was typically 1:0.08). For both LPCVDnitride and glass substrates, spot fluorescence was mostintense for target concentrations exceeding 1 nM withintensity slightly increasing with target concentration.
Contrary to reports made by other researchers, the useof high target concentrations did not contribute toincreased background in the fluorescence images.29 Fur-ther, increased fluorescence background signals as re-ported by some researchers for hybridization timesexceeding 3 h were not observed during hybridization ofshort oligonucleotides on any of the three substratesexamined.15 In fact, low fluorescence background levelswere observed for hybridization reactions up to andexceeding 12 h, reflecting minimal nonspecific targetadsorption due to the effectiveness of the 6-amino-1-hexanol based substrate passivation/deactivation process.
Microarray Selectivity and Stability. Experimentswere undertaken to demonstrate that covalent probeimmobilization at nitride substrates also permitted thesuccessful discrimination of complementary and non-complementary target oligonucleotide sequences duringmultiple sequential hybridizations. For this purpose,microarrays comprising three different 20-mer probeoligonucleotides in three discrete columns (1-3) wereformed at PDITC activated, amino-silane functionalizedsubstrates using the protocols described above; see Figure6. Column 1 was an experimental control containing spotsof 20-mer, 5′-Cy3-, 3′-amino-modified probe oligonucle-otides covalently immobilized at each substrate. Columns2 and 3 contained spots of covalently immobilized 20-mer, 3′-amino-modified probe oligonucleotides with twoentirely different sequences. The fluorescence micrographsof Figure 6a show the microarrays following removal ofany nonspecifically bound probe oligos from the glasssurface and prior to hybridizationsfluorescence wasdetected only from the columns of dye-modified controloligos.
Following the hybridization of a target oligonucleotidecomplementary in sequence to the probe oligos of eachcolumn 2 using the standard protocols, fluorescence wasalso detected from these columns; see Figure 6b. Nofluorescence was detected from columns 3, demonstratingsuccessful discrimination of the target sequence by theprobe spots of these columns. Following the applicationof a stripping cycle to remove hybridized targets from thearrays and hybridization of target oligonucleotides comple-mentary in sequence to the probe oligos of columns 3,fluorescence was then detected from those columns; seeFigure 6c. No fluorescence was detected from any ofcolumns 2, demonstrating successful discrimination of thetarget sequence by the probe spots of these columns.
Note also that, following this second hybridization cycle,the fluorescence intensities measured from each of(31) Blossey, R.; Bosio, A. Langmuir 2002, 18, 2952-2954.
Figure 5. (a) Comparison of the averaged spot fluorescenceintensities of 5′-Cy3-modified target oligos hybridized at 3′-amino-modified probe oligo microarrays measured versus thetarget concentration for each substrate type. (b) Typical targetspot fluorescence images acquired at each target concentrationfor each substrate type. Scale bar: 50 µm.
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columns 1 on the LPCVD nitride and the glass substrateswere largely undiminished, consistent with the robustnessof the covalent probe immobilization method. In contrast,the fluorescence intensities measured from column 1 onthe PECVD nitride substrate appeared to be somewhatdiminished, suggesting that the stabilities either of theprobe oligos immobilized at this nitride surface or of theactual deposited nitride layers were lower than thoseachieved by the LPCVD nitride substrate.
To test the response of probe microarrays over cyclesof sustained reuse, multiple sequential hybridization andstripping (denaturation) cycles were performed on mi-croarrays comprising spots of 20-mer, 3′-amino-modifiedprobe oligonucleotides covalently attached at LPCVDnitride, PECVD nitride, and glass substrates usingstandard procedures; see Figure 7.
In agreement with the results presented above, thehybridization-related fluorescence response of PECVDnitride substrates was found to decrease rapidly after onehybridization and strip cycle, indicating a significantlylower stability of this substrate. In fact, optical inspectionof the PECVD nitride substrates following hybridizationand stripping revealed that progressive degradation ofthe nitride (by surface layer removal or thinning) hadoccurred as a result of application of the various processsteps. In contrast, for LPCVD nitride and glass substrates,the average hybridization-related spot fluorescence in-tensity was observed to remain practically constant overfive consecutive hybridization/strip/rehybridization cyclesof complementary 5′-Cy3-modified target oligos, irrespec-tiveof theharshnessof thestrippingconditions (immersionin H2O at 100 °C for 15 min). (Stripping resulted in theinterim removal of essentially all fluorescence signals;data not shown.) The particular performance of the nitride
substrate in this respect compares very favorably withdata reported from previous stability studies of siliconoxide, glass, and hydrogen-terminated silicon microarraysubstrates and is superior to that of DNA-modified goldsubstrates, which are known to degrade rapidly due tothiol group hydrolysis under basic conditions.18,25,32-36
Concerning the expected stability of response over morethan five cycles, an eventual decrease of microarrayperformance is likely to occur, since the degradation ofchemically modified surfaces incorporating Si-O linkages,especially under basic conditions or in the presence ofamines, is a known phenomenon.35,37,38 In this regard,recent work has demonstrated that substantial improve-ments in microarray stability may be achieved usingnanocrystalline diamond thin film substrates.36 However,the results presented herein confirm that our immobiliza-tion process results in the formation of stable covalentbonds between the terminal amino modification of theprobe oligos and the PDITC activated, amino-silanefunctionalized LPCVD nitride and glass substrates,respectively. The demonstrated selectivity, stability, andreusability of these probe microarrays suggest that thiscombination of microfabrication compatible Si3N4 sub-strates and novel probe immobilization protocols has thepotential to provide fundamental benefits in terms of bothease of fabrication and subsequent hybridization assayperformance for microelectronics enabled integrated DNAbiochips.
Conclusion
The goal of this work was to establish the feasibility ofDNA microarray formation on Si3N4 layers using, as abasis, covalent probe immobilization methods. Eachmicroarray fabrication process step, from silicon nitridesubstrate deposition, surface cleaning, amino-silanation,and attachment of homobifunctional cross-linking mol-ecule to the direct covalent immobilization of probeoligonucleotides, 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.
Figure 6. Micrographs showing (a) the fluorescence of co-valently immobilized dye-modified probe controls at columns1 (the probes covalently immobilized at columns 2 and 3 arenonmodified), (b) the fluorescence of covalently immobilizedcontrols at columns 1 (columns 2 show fluorescence due toselective hybridization of dye-modified targets), and (c) thefluorescence of covalently immobilized controls at columns 1(columns 3 now show target fluorescence due to selectivehybridization). Scale bar: 100 µm.
Figure 7. Comparison of averaged spot fluorescence intensitiesof5′-Cy3-modified targetshybridizedat3′-amino-modifiedprobemicroarrays measured with respect to hybridization cycle forfivesequentialhybridizationsateachmicroarraysubstrate type.Following each data acquisition, hybridized targets werestripped and the arrays were then reused.
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optimized to yield consistent probe microarray quality,homogeneity, and probe-target hybridization perfor-mance. With respect to the latter, the developed microar-ray fabrication methodology has been shown to provideexcellent (high signal-to-background ratio) and reproduc-ible responsivity to target oligonucleotide hybridizationwith a rugged chemical stability that permitted exposureof each array to stringent pre- and posthybridization washconditions through many sustained cycles of reuse.
Therefore, for this novel microarray substrate material,these features compare very favorably with the perfor-mance attributes of more mature glass microarray basedtechnologies. Consequently, it is proposed that this DNAmicroarray fabrication strategy has the potential toprovide a viable route toward successful realization offuture integrated DNA biochips.
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