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Ellipsometric and Interferometric Characterization of DNA Probes Immobilized on a Combinatorial Array D. E. Gray,*,† S. C. Case-Green,‡ T. S. Fell,‡ P. J. Dobson,† and E. M. Southern‡ Department of Engineering Science, University of Oxford, Oxford OX1 3PJ, U.K., and Department of Biochemistry, University of Oxford, Oxford OX1 3QU, U.K. Received December 31, 1996. In Final Form: March 17, 1997X We have used ellipsometry to study oligonucleotides bound on an array by observing changes in the optical thickness of the organic material attached to the surface. Interferometry has been used as a complementary technique to confirm our ellipsometry measurements. We have used these optical methods to characterize the chemical steps involved in synthesizing oligonucleotides on a solid support. Large area arrays have been mapped by ellipsometry with a spatial resolution of ∼1 mm2 and a sub-nanometer optical thickness resolution. We have demonstrated that this method can differentiate between areas containing oligonucleotides of different lengths.
Introduction From the earliest years of its development as an analytical instrument, ellipsometry has been used to study biological films. One popular application has been the determination of antibody-antigen interactions where ellipsometry is used to measure changes in the thickness of an absorbed antigen layer following interaction with antibodies in solution.1-3 Alternatively, ellipsometry has been used to calibrate the enzyme-linked immunosorbant assay (ELISA) in kinetic studies of antibody reactions.4 Similar studies can be found on the kinetics of protein binding5 and many other biological systems.6 An early ellipsometric investigation of DNA molecules has also been undertaken with a view to developing a rapid detection method for filter hybridization assays.7 By using a combination of ellipsometry and pseudoBrewster angle reflectometry the authors were able to detect an increase in thickness of an immobilized layer of single-stranded DNA under hybridization conditions. The authors recognized the potential use of ellipsometry in this field, but it appears that the technique was not developed any further for this particular application. However, DNA sequencing by hybridization is a recently proposed methodology8,9 which is currently undergoing rapid development for use as a genetic diagnostic tool and in which the characterization of surface bound DNA molecules is of paramount importance. In sequencing by hybridization, the coupling between an oligonucleotide probe of known sequence and a DNA target of unknown sequence establishes that the probe’s complementary sequence exists in the target. Therefore, if the unknown target is hybridized against a library of different oligo* To whom correspondence should be addressed. † Department of Engineering Science. ‡ Department of Biochemistry. X Abstract published in Advance ACS Abstracts, May 1, 1997. (1) Rothen, A. Int. Rev. Cytol. 1982, 80, 267-304. (2) Ruzgas, T. A.; Razumas, V. J.; Kulys J. J. Biosens. Bioelectron. 1992, 7, 305-308. (3) Jin, G.; Jansson, R.; Arwin, H. Rev. Sci. Instrum. 1996, 67, 29302936. (4) Werthe´n, M.; Nygren, H. Biochim. Biophys. Acta 1993, 1162, 326332. (5) Giesen, P. L. A.; Willems, G. M.; Hemker, H. C.; Stuart, M. C. A.; Hermens, W. T. Biochim. Biophys. Acta 1993, 1147, 125-131. (6) Azzam, R. M. A.; Bashara, N. M. Ellipsometry and polarised light; North-Holland: Amsterdam, 1977 (ISBN 0-7204-0694-3). (7) Mandenius, C. F.; Chollet, A.; Mecklenburg, M.; Lundstro¨m, I.; Mosbach, K. Anal. Lett. 1989, 22, 2961-2973. (8) Bains, W.; Smith, G. C. J. Theor. Biol. 1988, 135, 303-307. (9) Drmanac, R.; Labat, I.; Brukner, I.; Crkvenjakov, R. Genomics 1989, 4, 114-128.
S0743-7463(96)02139-7 CCC: $14.00
nucleotide probes, it is in principle possible to determine all the subsequences present in the target and hence from these elements the whole target sequence can be assembled. The common manifestation of this technology involves attaching the library of oligonucleotide probes to a solid support in a well-defined matrix. This arrangement enables the identification of the oligonucleotides by their spatial position on the support, which is commonly a sheet of functionalized glass, polymer, or as in this work an oxidized silicon wafer. Three main strategies for fabricating matrices of oligonucleotides have been reported. The first involves the spotting of microdroplets of presynthesized oligonucleotides onto the support,10-12 the second performs oligonucleotide synthesis directly onto the support where reagents are spatially confined by use of a mechanical mask,13,14 and the last involves localized deprotection of the solid support by photolithographic15,16 or electrochemical methods17,18 prior to either direct oligonucleotide synthesis or attachment of presynthesized probes. However, what these methods all share is a primary concern for the final quality of the oligonucleotide array. An unsuccessful support functionalization or oligonucleotide synthesis step can easily go undetected and has obvious catastrophic consequences for the integrity of the final device. While those methods that utilize oligonucleotide synthesis directly onto the support have the advantage that a combinatorial approach19 may be used to generate a far larger library than is practicable (10) Khrapko, K. R.; Lysov, Y. P.; Khorlin, A. A.; Shick, V. V.; Florentiev, V. L.; Mirzabekov, A. D. FEBS Lett. 1989, 256, 118-122. (11) Khrapko, K. R.; Lysov, Y. P.; Khorlin, A. A.; Ivanov, I. B.; Yershov, G. M.; Vasilenko, S. K.; Florentiev, V. L.; Mirzabekov, A. D. J. DNA Seq. 1991, 1, 375-388. (12) Beattie, K.; Eggers, M.; Shumaker, J.; Hogan, M.; Varma, R.; Lanture, J.; Hollis, M.; Ehrlich, D.; Rathman, D. Clin. Chem. 1993, 39, 719-722. (13) Maskos, U.; Southern, E. M. Nucleic Acids Res. 1992, 20, 16751678. (14) Maskos, U.; Southern, E. M. Nucleic Acids Res. 1992, 21, 46634669. (15) Fodor, S. P. A.; Read. J. L.; Pirrung, M.; Stryer, L.; Lu, R. T.; Solas, D. Science 1991, 251, 767-773. (16) Sheldon, E. L.; Briggs, J.; Bryan, R.; Cronin, M.; Oval, M.; McGall, G.; Gentalen, E.; Miyada, C. G.; Masino, R.; Modlin, D.; Pease, A.; Solas, D.; Fodor, S. P. A. Clin. Chem. 1993, 39, 718-719. (17) Southern E. M. Electrochemical treatment of surfaces; US Patent Application Serial No 08/325,337. (18) Livache, T; Roget, A.; Dejean, E.; Barthet C.; Bidan G.; Te´oule R. Nucleic Acids Res. 1994, 22, 2915-2921. (19) Lowe G. Chem. Soc. Rev. 1995, 309, 311-317.
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using presynthesized probes, these methods are the most vulnerable to such fabrication failures. The aim of this work is to assess the suitability of ellipsometry and interferometry to characterize oligonucleotide arrays at different stages during their fabrication. Their perceived advantages were that they were noninvasive to the oligonucleotide array, fast, easy to use, and relatively inexpensive while having the sensitivity to detect transparent organic layers at the molecular level. If successful for nucleic acids, it is anticipated that these optical methods will have similar applications to other array chemistries which measure interactions between specific ligands and test substances, e.g., enzymes with their substrates, antibodies with antigens, and drugs with their target receptors. Experimental Procedure (i) Preparation of the Solid Support. Three inch silicon wafers with a highly uniform thermal oxide (from the Microelectronics Industrial Unit, Southampton University, U.K.) were used as the solid support material. The thickness of the silicon dioxide layer was measured by ellipsometry and found to be 124 ( 0.4 nm across each entire wafer. The highly reflective silicon and wellcharacterized oxide provided an ideal substrate for the optical measurements especially with regard to our HeNe laser ellipsometer, which is particularly sensitive to this specific thickness of oxide covering on silicon.20 Each wafer was cleaned thoroughly to minimize any contamination that may disrupt the even deposition of the organic layers or generate artifacts during their optical characterization. The wafers were immersed in piranha solution (a 7:3 (v/v) ratio mix of 98% H2SO4 and 30% (w/v) H2O2) for 30 min at 90 °C twice. They were rinsed in deionized water, ethanol, and methanol and dried under nitrogen. The wafers were then baked at 100 °C for 5 min to minimize surface water species. The cleaning processes and wafer handling were carried out in a class 1000 clean room environment. (ii) Support Functionalization with Linker Molecules. If the oligonucleotide probes are anchored directly onto the solid support the probability of them hybridizing with the unknown target is reduced due to steric hindrance effects with the surface.21,22 Therefore, the support is functionalized with linker molecules which tether the probes while providing them with a greater freedom of movement. The linker layer was attached to the substrate in two steps: silanation of the wafer with (3-glycidoxypropyl)trimethoxysilane followed by opening of the epoxide group with polyethylene glycol which produced primary hydroxyl groups available for oligonucleotide synthesis.23 Wafers which were to be used as supports for oligonucleotide synthesis were typically silanated by immersion in a 7.7% (v/v) solution of (3-glycidoxypropyl)trimethoxysilane in xylene with a trace of diisopropylethylamine for 6 h at 85 °C. The wafers were washed in acetone and dried under nitrogen before and after performing ellipsometry readings on them at this stage. In addition, a series of wafer samples were also silanated at room temperature using four different concentrations (0.03, 3.8, 7.7, and 19.4% (v/v)) of (3-glycidoxypropyl)(20) Tompkins, H. G. A user’s guide to ellipsometry; Academic Press: San Diego, CA, 1993; Chapter 3. (21) Guo, Z; Guilfoyle, R. A.; Thiel, A. J.; Wang, R.; Smith, L. M. Nucleic Acids Res. 1994, 22, 5456-5465. (22) Shchepinov, M. S.; Case-Green, S. C.; Southern, E. M. Nucleic Acids Res. 1997, 25, 1155-1161. (23) Maskos, U.; Southern, E. M. Nucleic Acids Res. 1992, 20, 16791684.
Figure 1. Apparatus for the synthesis of oligonucleotides on a flat support: (A) plan and side views of mask and piping; (B) steps involved in the synthesis of an oligonucleotide array (1) the support is sealed onto the mask using the screw clamp to create the flow cell, (2) the synthesis reagents are added, (3) the clamp is slackened, (4) the support is displaced by the required distance.
trimethoxysilane in xylene with a trace of diisopropylethylamine for seven different time periods (1, 15, 30, 60, 120, 180, 360 min). Ellipsometry measurements were performed on these samples to provide further insight into the silanation process. In the second reaction step each wafer was heated to 90 °C in polyethylene glycol (average molecular weight ca. 200), containing a catalytic amount of concentrated H2SO4, for 10 h. The wafers were washed in ethanol and acetone and dried under nitrogen before ellipsometry readings were taken. All of the derivitization process was carried out in a class 1000 clean room environment. (iii) Synthesis of Oligonucleotide Arrays. We performed synthesis directly onto the support using a flow cell which defined the area of probes to be synthesized. In this way, by coupling and then flushing, purging, and moving the cell so that it overlapped an area of previous synthesis and performing further couplings, we were able to generate a simple oligonucleotide array. This method, which has been described in detail previously,24 is summarized in Figure 1. (24) Southern, E. M.; Case-Green, S. C.; Elder, J. K.; Johnson, M.; Mir, K. U.; Wang, L.; Williams, J. C. Nucleic Acids Res. 1994, 22, 13681373.
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Figure 2. Cycle used in the synthesis of oligonucleotides on a solid support using phosphoramidite chemistry. The initial reaction is the attachment of the first monomer to the hydroxyl groups of the linker surface, each subsequent cycle adding monomers to the growing oligonucleotide chain. Final deprotection in ammonia solution removes the cyanoethyl protecting groups of the phosphates and any base protecting groups.
Two flow cells (42 and 30 mm diameter), machined from Teflon with a cell depth of ca. 0.5 mm, have been used. Inlet and outlet ports were made by drilling 1.0 mm diameter holes at the top and bottom of the circular wall and fitting sawed-off 19 gauge (1.1 mm o.d.) syringe needles through from the back. A “G” clamp, fitted with a dished polyethylene cushion was used to apply pressure to the silicon wafer support and form the seal to the Teflon reaction cell. This arrangement was mounted onto the frame of an Applied Biosystems 381A oligonucleotide synthesizer so that delivery lines normally connected to the column could be connected to the reaction cell. Oligonucleotide synthesis used standard reagents for phosphoramidite chemistry (Figure 2), omitting the capping step. The scale was for 0.2 mmol synthesis, adjusted slightly to provide volumes that would just fill the reaction chamber. Three different specimens were prepared in this way. The first, and simplest, involved synthesizing a circular 42 mm patch of oligonucleotide probes 10 deoxythymidine nucleotides (oligo dT10) in length. In the second, a circular 42 mm patch containing 4 dT couplings was followed by a mask displacement of 10.5 mm before a further four couplings of dT. This procedure was repeated twice more with the displacement of the mask being 10.5 mm in the same direction each time. This protocol produces the array of different length oligonucleotide probes (oligo dT4-16) shown in Figure 3a. The third array was designed to distinguish between the common ∆F508 cystic fibrosis transmembrane regulator (CFTR) mutation and the normal gene. This mutation is a specific deletion of three base pairs, which results in the loss of a phenylalanine residue from the
Figure 3. Layout of oligonucleotide arrays formed on silicon wafer substrates: (a) stepped array formed by moving the mask along at intervals of four dT bases; (b) CF array formed by synthesizing 3′-ATCAT-5′ moving the mask and synthesizing 3′-CTT-5′, then moving the mask to the original position and synthesizing 3′-TGGTGT-5′ to form oligonucleotides complementary to mutant and normal genes.
normal polypeptide product.25 In the region of interest the normal sequence is 3′-ATCATCTTTGGTGT-5′. However, in the cystic fibrosis (CF) gene the sixth, seventh, and eighth bases of this sequence (3′-CTT-5′) are missing
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to give 3′-ATCATTGGTGT-5′. These sequences were generated on an array by coupling the first five bases from the 3′ end to the solid support in a circular 30 mm patch (namely, 3′-ATCAT-5′). The reaction cell was then moved 18 mm to the right and the three CF deletion bases (3′-CTT-5′) were synthesized before moving the mask back to its original position and coupling the last segment of the sequence (3′-TGGTGT). As shown in Figure 3b, this protocol produces a three-element array in which the left area contains the CF mutation, the middle represents the normal sequence, and the right has the three-base omission. Finally, the protecting groups used to mask potentially reactive sites on the base and phosphate moieties throughout oligonucleotide synthesis were removed. The arrays containing only base T sequences were treated in ammonia (30%) at room temperature for 15 min while the CF array was treated in ammonia (30%) at 55 °C for 5 h. The different treatments arise as base T does not require protection and the 2-cyanoethyl protecting group on the phosphate is rapidly removed in ammonia, while the benzoyl and isopropyl base protecting groups on the A, G, and C bases require the more aggressive deprotection step. After ammonolysis the wafers were washed in ethanol and acetone and dried under nitrogen before ellipsometry readings were taken. (iv) Hybridization Conditions. Molecular hybridization was used to confirm that a successful synthesis had occurred during the fabrication of the CF array. Oligonucleotides containing sequences complementary to those on the array (A, 5′-ACACCAATGATATTTTCTTTATTGGTGCCAGGCATAATC-3′; B, 5′-ACACCAAAGATGATATTTTCTTTATTGGTGCCAGGCATAATC-3′) were synthesized using an Applied Biosystems 392A DNA synthesizer employing phosphoramidite chemistry and labeled using γ32P-ATP (Amersham International) in the presence of polynucleotide kinase. The hybridization was performed by injecting 200 µL of a 1 M NaCl solution containing ca. 3 pmol of oligonucleotide in 50 mM Tris HCl (pH 8.0), 1 mM EDTA, and 0.01% SDS, between the array and a plain glass plate placed against its surface. The hybridization was carried out in a moist container at 22 °C for 30 min after which time the array was briefly washed in 1 M NaCl solution containing 50 mM Tris HCl (pH 8.0) and 1 mM EDTA at 22 °C. The array was wrapped in “clingfilm” and exposed to a phosphor storage screen (Fuji ST-III) which was later imaged on a Molecular Dynamics phosphorimager. Hybridized oligonucleotides were stripped from the surface of the array by heating in a solution of 50 mM Tris HCl (pH 8.0) and 1 mM EDTA at 80 °C for 15 min. (v) Ellipsometry. Ellipsometry measurements were taken in air using a SENTECH SE400 rotating analyzer ellipsometer with a 632.8 nm HeNe laser. The angle of incidence of the laser beam was 70° during all the readings, this angle being sufficiently near the Brewster angle of the substrate to yield accurate optical parameters and thickness values of the surface layers. Theoretical manipulation of the data obtained was carried out using both in-house and commercial software (Optichem Ltd.). The ellipsometer has a manual positioning stage accurate to 0.02 mm. Readings were taken by mapping the surface of the wafer along two orthogonal axes of the wafer and on a third diagonal line between these. The spot of illumination was ∼1 mm2 and an average of three readings (25) Riordan, J. R.; Rommens, J. M.; Kerem, B.; Alon, N.; Rozmahel, R.; Grzelczak, Z.; Zielenski, J.; Lok, S.; Plavsic, N.; Chou, J.-L.; Drumm, M. L.; Iannuzzi, M. C.; Collins, F. S.; Tsui, L.-C. Science 1989, 245, 1066-1073.
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Figure 4. Thickness variation of silanation layer with respect to reaction time and concentration of silane. All the reactions were carried out at room temperature on silicon wafer substrates in xylene containing varying concentrations of (3-glycidoxypropyl)trimethoxysilane and a trace of diisopropylethylamine.
was taken at 0.2 mm intervals to provide cross-sectional profiles of the wafer surface. The ellipsometric parameters delta (∆) and psi (ψ) describe the change in polarization of light which occurs on reflection, where ψ is the amplitude attenuation and ∆ is the phase difference between the s and p polarization waves. These parameters are related to the complex refractive index and the thickness of a thin film on a known reflective surface.6 Theoretical modeling using software, based on that described by McCrackin,26 was carried out to determine the thicknesses and the complex refractive indices present in the system. The complete oligonucleotide arrays can be thought of as a multilayer system with at least four layers. The system consists of a silicon substrate, a silicon dioxide layer, a linker layer made up of a silanation layer and a glycol layer, and finally layers of oligonucleotides. To enable an accurate model to be made, readings were taken after each stage of their manufacture and each layer was fully characterized before proceeding to the following step. This meant that each model was based on a known substrate with one additional unknown film on top of it. A complex refractive index of 3.858-0.019i was used for the bulk silicon substrates and a refractive index of 1.459 was used for the silicon dioxide film, these values fitted well with experimental results and corresponded to those previously reported.27 These values provided a model which fitted extremely well to initial ellipsometric readings carried out on the bare wafers. No assumptions for the thicknesses or the refractive indices of the linker or the oligonucleotides were used. A fast, automated process for mapping the whole wafer was provided by Instruments SA. Using their polarization modulated ellipsometer (UVISEL, Jobin Yvon) with an automated stage it was possible to provide a complete image of the surface of the wafer in less than 20 min (a single cross-sectional profile achieved using the SE400 ellipsometer took typically 40 min to obtain manually). These readings were taken in air using a wavelength of (26) McCrackin, F. L. NBS Tehn. Note 1969, No. 479. (27) Palik, E. D. Handbook of opitcal constants of solids; Academic Press: London, 1985 (ISBN 0-12-544420-6).
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Figure 5. A potential structure and mechanism for the linker formed on silanation of a silicon dioxide substrate with (3-glycidoxypropyl)trimethoxysilane.
563 nm at an angle of incidence of 70°. The spot of illumination was ∼1 mm2 and readings were taken every 2 mm in both the x and y direction. Processing of the ellipsometric data was carried out using incorporated software, with readings again taken at each stage of array manufacture to give the maximum possible accuracy in the model. The UVISEL ellipsometer also allowed spectroscopic measurements to be made from 250 to 825 nm. Analysis of these spectra allowed the optimal wavelength to be chosen for the monochromatic mapping of the surface (563 nm) and provided confirmation of the complex refractive indices used in modeling the results from the SE400 ellipsometer. Mapping of the surface through the UVISEL’s complete wavelength range, although practicable, was not carried out as such readings would have taken three times longer than the single wavelength readings which already provided sufficient information to characterize our samples completely. (vi) Interferometry. Interferometric readings were carried out on a Micromap interferometer, courtesy of Burleigh Instruments. Measurements were made at 553.4 nm using a 5× objective lens. The instrument was sensitive to 0.1 nm changes in height and is capable of
