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Direct Synthesis of Oligonucleotides on Nanostructured Silica Multilayers Ilaria Rea,*,† Giorgia Oliviero,‡ Jussara Amato,‡ Nicola Borbone,‡ Gennaro Piccialli,‡ Ivo Rendina,† and Luca De Stefano† Institute for Microelectronics and Microsystems, National Council of Research, Via P. Castellino 111, I-80131 Naples, Italy, and Department of Chemistry of the Natural Substances, UniVersity of Naples “Federico II”, Via D. Montesano 49, I-80131 Naples, Italy ReceiVed: September 1, 2009; ReVised Manuscript ReceiVed: January 4, 2010
In this work, we have quantified the functionalization of some porous silicon multilayers that optically act as Bragg reflectors: 1.3 × 10-3 mol/g, corresponding to 3.25 nmol/cm2, of a ten base oligonucleotide has been estimated by a chemical procedure, based on the dimethoxytrityl monitoring. The oligonucleotide has been synthesized directly on two different porous silicon structures, one oxidized by a thermal treatment, the other by a chemical process based on exposure to I2/pyridine. We have also monitored the oligonucleotide synthesis by a label-free technique: a 11 nm red-shift of the optical spectrum can be observed by spectroscopic reflectometry. 1. Introduction Significant efforts are being devoted to the development of more efficient DNA chip technology for applications in areas of social interest, such as gene expression analysis, medical diagnostics, and personal therapies.1 Commercial DNA chips are constituted by single-stranded DNA probes covalently attached to solid supports by a proper immobilization process2 or directly synthesized on them.3 In these devices a key role is played by the surface passivation strategies, which should ensure chemical stability to the support and uniform distribution of the DNA probes. The commercial devices use probes or targets labeled with chromophores or radioisotopes4 so that the molecular recognition events, i.e., the hybridization of probes with targets, are monitored by fluorescence microscopes. Even if the fluorescence based technique is a standard in genomic applications, this method is strongly dependent on the labeling procedures, which sometimes can give undesirable effects on the molecular interactions. Moreover, reading and quantifying hundreds of thousands of fluorescence spots in a single run is not straightforward nor always precise. To overcome these limitations, a new class of label-free DNA chip has been recently proposed, which uses direct electrical5 and optical6,7 detection methods. The porous silicon (PSi), fabricated by electrochemical etching of doped crystalline silicon, is an ideal support for biochip fabrication. PSi is a nanostructured material characterized by a high specific surface area of the order of hundreds of square meters per cubic centimeter,8 which makes it a sensible sensing platform. The porous silicon can be simply described as a network of air holes in a silicon matrix: the dielectric properties, and in particular the refractive index, of a PSi layer depend on the content of voids in the silicon and can be calculated by using an effective medium approximation such as the Bruggeman model.9 Several high quality optical structures (Fabry-Perot interferometer,10 Bragg mirror,11 optical microcavity,12 aperiodic multilayered sequences,13 optical wave* Corresponding author. Tel.: +390816132594. Fax: +390816132598. E-mail address:
[email protected]. † National Council of Research. ‡ University of Naples “Federico II”.
guide14) can be obtained by means of the PSi technology. The application of the PSi structures to biomolecular screening is a topic of large interest and actuality, as demonstrated by several papers,15-19 showing that PSi-based sensors are generally characterized by a linear response curve20 and can detect biomolecules in low concentrations.21,22 Even if the PSi has been used as support for biomolecules,2,23,24 there are only a few works reporting direct synthesis of DNA on PSi monolayers.25 In this study we have synthesized a 10base oligonucleotide (ON) directly on two different porous silicon multilayered platforms. With respect to monolayers only a few micrometers thick, PSi stacks are constituted by several layers with different porosities: the interfaces between adjacent layers are mechanical barriers for liquid and gas penetration, so that solution based chemical processes must be verified on these much thicker structures. Moreover, the PSi surface functionalization has been quantified by a label-free detection method based on dimethoxytrityl (DMT) measurements and the results have been compared with those obtained by Fourier transform infrared spectroscopy (FTIR) and by spectroscopic reflectometry. 2. Material and Methods 2.1. Fabrication of the Porous Silicon Structures. We have used as solid support for the ON synthesis a twin pair of PSi Bragg reflectors obtained by alternating 20 high (H) refractive index layers (low porosity) and low (L) refractive index layers (high porosity) whose thicknesses satisfy the Bragg relationship nHdH + nLdL ) mλB/2, where m is an integer and λB is the Bragg resonant wavelength. The Bragg reflectors were produced by electrochemical etching of highly doped p+-silicon, 〈100〉 oriented, 0.003 Ω cm resistivity, 400 µm thick, using a HF (50% in weight):ethanol ) 1:1 solution in the dark and at room temperature. Before the anodization, the silicon substrate was placed in HF solution to remove the native oxide. Since the PSi fabrication process is self-stopping, it is possible to obtain adjacent layers with different porosities by changing the current density during the electrochemical etching.26 A current density of 200 mA/cm2 for 1.2 s was applied to obtain the low refractive index layers (nL ) 1.542; dL ) 125 nm) with a porosity of 72%,
10.1021/jp908440u 2010 American Chemical Society Published on Web 01/22/2010
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Figure 1. Scheme of the solid phase synthesis of the 10 base oligonucleotide 4 on the PSi-OH surface 1 using 5′-(dimethoxytrityl)thymidine-phosphoramidite 2. i: standard automatic synthetic cycle.
while a current density of 100 mA/cm2 was applied for 1.4 s for the high index layers (nH ) 1.784; dH ) 108 nm) with a porosity of 64%. Both the Bragg reflectors have been etched on the bottom of a microchamber (1 µm deep) created by an electropolishing current pulse (800 mA/cm2 for 5 s) to avoid the presence of a thin parasitic film27 (about 40 nm thick measured by spectroscopic ellipsometry) on the surface of the samples. The structures have been oxidized against uncontrolled environmental aging and corrosion in alkaline biological solutions. One of the Bragg reflectors (TOBR) was thermally oxidized in pure O2 by a two-step process: a preoxidation at 400 °C for 30 min followed by an oxidation at 900 °C for 15 min. The other Bragg (COBR) has been dipped in an oxidizing solution (0.1 M I2 in tetrahydrofuran/pyridine/H2O) for 2 h, then washed by acetonitrile, and dried in a stream of N2. After the oxidation processes, the PSi devices were immersed in freshly prepared Piranha solution (H2SO4/H2O2 4:1, v/v) for 40 min at room temperature, rinsed with deionized water, and dried in a stream of nitrogen gas. This treatment creates Si-OH groups on the PSi surface. Caution: piranha solution is aggressive and explosive. Never mix piranha waste with solvents. Check the safety precautions before using it. 2.2. Oligonucleotide Synthesis. Chemicals and solvents were purchased from Fluka-Sigma-Aldrich. Reagents and phosphoramidites for DNA synthesis were purchased from Glen Research. Solid phase ON syntheses were performed on a PerSeptive Biosystems Expedite 8909 DNA automated synthesizer. The reaction scheme of the ON synthesis on the PSi platform is reported in Figure 1. The PSi structure, characterized by surface Si-OH bonds, was introduced in a suitable column reactor before the synthesis (1, Figure 1). The DNA oligomer was assembled on the chip following standard phosphoramidite chemistry by ten growing cycles thus obtaining polymer bound T10 (4, Figure 1). Coupling. The first reaction of ON growing cycles involves the coupling of the 5′-(dimethoxytrityl)-thymidine-phosphoramidite (2, Figure 1) dissolved in dry acetonitrile (45 mg/mL). The reaction occurs in 1 h in the presence of tetrazole (0.45 M in acetonitrile) as coupling activator. Oxidation. The newly formed phosphite triester internucleotide bond is then converted to the corresponding phosphodiester with the standard oxidizing solution of iodine in pyridine/ acetonitrile (5 min). Capping. In this step the unreacted 5′-OH groups were capped by the standard solution of acetic anhydride in THF/pyridine solution (5 min). Detritylation. The removal of the 5′-dimethoxytrityl protecting group from the support-bound 5′-terminal nucleotide was performed by using the deblocking solution of trichloroacetic acid in dichloromethane (3% w/w). The amount of DMT cation released by acid treatment was used as a direct measure of the efficiency of the ongoing synthesis. The release of the protecting group generates a bright red-orange color solution in which the quantity of the DMT cation could be measured online by UV-vis spectroscopy at 503 nm (ε ) 71 700 M-1 cm-1). After
the each step the support is thoroughly washed with acetonitrile before the beginning of the successive reaction. The described synthetic cycle is repeated until the full-length ON has been synthesized. 2.3. Samples Characterization. The sample’s morphology was investigated by scanning electron microscopy (SEM) using a field emission instrument (Zeiss-Supra 35). We have monitored all the surface modifications of the Bragg reflectors by FTIR (Thermo Scientific - Nicholet Continum XL) and reflectivity spectroscopy. The reflectivity measurements have been performed by a very simple experimental setup: a white light was sent on the PSi samples through a Y-fiber. The same fiber was used to guide the output signal to an optical spectrum analyzer (Ando AQ6315A). The spectra were acquired at normal incidence over the range 600-1200 nm with a resolution of 0.2 nm. 3. Results and Discussion In Figure 2 the SEM top view images of the high porosity layers of the PSi Bragg reflectors chemically oxidized (Figure 2a) and thermally oxidized (Figure 2b) are reported together with the pore diameter histograms estimated directly from these micrographs. In the case of the COBR sample, the pore size ranges from 10 to 45 nm and the mean value is about 22 nm. This value is comparable with those reported in the literature for the “as etched” p+ porous silicon,28 so that we can conclude that chemical oxidation does not affect the pore size. In the case of the thermal oxidation, a strong reduction in the pore size can be observed: the distribution is included between 5 and 30 nm and the mean value is 14 nm. This phenomenon is due to an isotropic silica expansion quantified by a factor of 2.27, which implies the growth of the silica also into the PSi pores.29 The reflectivity spectra of the Bragg reflectors COBR (Figure 3a) and TOBR (Figure 3b) before and after the oxidation processes are shown in Figure 3. The oxidations induce a blue shift in the reflectivity spectra and a decrease in the full width at half-maximum (fwhm) of the high reflectivity stop bands. These effects are due to the substitution of the silicon by the silica, which is characterized by a lower value of the refractive index (1.4 compared to 3.4 of the silicon). In particular, we observe a higher decrease (about 14%) of the fwhm for the TOBR with respect to the case of COBR (10%): in the thermally oxidized sample some voids have been substituted by silica so that the refractive index contrast between layer pairs is much more decreased than in the case of chemical oxidation. The oxide growth is in both cases uniform in the whole multilayer: an inhomogeneous distribution of oxide inside the PSi would give a distorted optical spectrum.30,31 Since the spectra undergo rigid shifts toward lower wavelengths maintaining unaltered their shape, we can conclude that the oxide film is homogeneously distributed in the porous matrix. Therefore, we can use the top view images of the samples as representative of the whole devices.
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Figure 2. SEM top view images and pore diameter histograms of porous silicon devices chemically oxidized (a) and thermally oxidized (b).
Figure 3. Reflectivity spectra of the porous silicon Bragg reflectors COBR (a) and TOBR (b) before (solid line) and after (dashed line) the oxidation processes. Blue shifts are observed due to the substitution of the silicon with silica.
The presence of a larger amount of silica in the thermally oxidized sample has also been demonstrated by FTIR measurements. In Figure 4 we have reported the FTIR spectra of the Bragg reflectors before and after the oxidation processes. Even if siloxane (Si-O-Si) peaks can be observed at 1100 and 480 cm-1 in both cases, a higher value of the peak area of the silica grown in the TOBR sample has been calculated, 1750 counts cm-1 compared to 670 counts cm-1 for COBR. The Si-Hx bonds (at 680-630 cm-1), characteristic of the freshly etched porous silicon, disappear in the case of the thermally oxidized sample, while they can be still found after the chemical oxidation process.
Figure 4. FTIR spectra in the range 1400-400 cm-1 of the Bragg reflectors COBR (a) and TOBR (b) before (solid line) and after (dashed line) the oxidation processes. The absorption peaks at 1100 and 980 cm-1 are due to Si-O-Si antisymmetric stretching. The absorption at 800 cm-1 is attributed to the Si-O-Si symmetric stretching vibration. The peak at 480 cm-1 corresponds to the out-of-plane rocking of Si-O-Si.32
After the treatment in Piranha solution, peaks related to the presence of silanol groups (Si-OH) on the PSi device’s surface appear in the FTIR spectra in the range between 955 and 830 cm-1, as shown in Figure 5. Peaks areas of 166 and 126 counts cm-1 have been calculated for the TOBR and COBR samples, respectively. The presence of Si-Hx bonds is still observed in the case of the chemically oxidized device. We have quantified the amount of ON synthesized on the PSi surface by DMT measurements. The results of the analysis after each synthesis
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Figure 5. FTIR spectra in the range 1100-600 cm-1 of the Bragg reflectors COBR (solid line) and TOBR (dashed line) after the immersion in Piranha solution. Vibrational modes relating to Si-OH bonds appear.
Figure 6. DMT measurements performed on the sample COBR (9) and TORB (b) after each synthesis cycle. Higher values have been obtained for the thermally oxidized sample.
cycle are reported in Figure 6. Higher absorbance values, due to a higher amount of DMT cation released in solution after the detritylation process, can be observed in the case of the thermally oxidized PSi structure. Furthermore, the amount of DMT released after each coupling cycle indicated reaction yields over 98%. These values were almost steady during the ON growing process, confirming the stability of the chip surface and the high accessibility of ON 5′-OH end groups for the TOBR sample. By averaging over these values, we have estimated a functionalization of 1.1 × 10-3 mol/g in the case of the COBR device, and of 1.3 × 10-3 mol/g for the TOBR. The thermally oxidized sample shows not only a higher degree of functionalization due to a higher concentration of -OH groups, as stated in the aforementioned comments to Figure 5, but also a better quality of the oxide layer in terms of density and surface roughness.33 If we want to express these results in terms of mol/cm2, we have to consider the specific surface area of the PSi devices. In the case of chemical oxidation, we can assume that the porosity and the pore size distributions do not change with respect to the “as etched” sample, since the oxide layer is a few nanometers,34 so that we can use the value measured for the fresh sample by the BET method (data not shown here), which is about 100 m2/g. For the thermally oxidized sample, we have to take into account the pore size reduction (36%) observed by
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Figure 7. Reflectivity spectra of the Bragg reflectors COBR (a) and TOBR (b) before (solid line) and after (dashed line) the oligonucleotide synthesis.
Figure 8. FTIR spectra in the range 2000-600 cm-1 of the PSi samples COBR (solid line) and TOBR (dashed line) after the oligonucleotide synthesis. Vibrational modes relating to ON synthesized on the chips are observed.
SEM analysis, which corresponds to a decrease in specific surface area of about 60% (from 100 to 40 m2/g) calculated using a simplified model based on the cylindrical shape of the pores. This value of the surface area reduction is in agreement with those reported by Salonen et al. on thermally oxidized PSi samples.35,36 The functionalization degree of COBR and TOBR can thus be expressed as 1.1 and 3.25 nmol/cm2, respectively. These results are 3 orders of magnitude higher than those obtained on flat surfaces37,38 and in very good agreement with those reported by Voelker et al. on similar samples of porous silicon.39 After the synthetic cycle, the presence of ON chains bonded on the chips has been also verified by reflectometry and FT-IR spectroscopy measurements. The biological molecules, attached to the PSi pore walls, induce an increase in the average refractive indexes of the layers, causing a red shift in the reflectivity spectra of the Bragg reflectors. The magnitude of the shift increases with the increase of the pore surface coverage with the organic matter. The reflectivity spectra of the PSi multilayered structures before and after the ON synthesis are reported in Figure 7. Red shifts of 7 and 11 nm are observed for COBR and the TOBR, respectively. The higher value observed in the case of the structure thermally oxidized is due to the superposition of two effects: the higher functionalization of this surface, as demon-
Oligonucleotides on Nanostructured Silica strated by the measurements reported in Figure 6, and the lower pore size of the TOBR, which makes the device more sensible.40 In Figure 8, we report the FT-IR spectra of the devices after the oligonucleotide synthesis: the characteristic peak of the double bond vibrations of the bases can be observed in both cases in the range between 1780 and 1530 cm-1. A peak area of 450 counts cm-1 has been estimated in the case of the COBR sample, and of 1300 counts cm-1 for the TOBR. 4. Conclusions In this work, we have characterized by several techniques the direct synthesis of a 10 base oligonucleotide on two different porous silicon supports: a thermally oxidized Bragg reflector and a Bragg mirror oxidized by a chemical process based on exposure to I2/pyridine. Even if SEM analysis shows a pore size reduction in the case of the thermally oxidized sample, the DMT measurements, which quantify the amount of oligonucleotide covalently bound on the porous silicon surfaces, have demonstrated a higher functionalization with respect to the chemical oxidized sample. This result has also been confirmed by normal incidence spectroscopic reflectometry and FTIR spectroscopy. Acknowledgment. We gratefully acknowledge Dr. P. Dardano of IMM-CNR in Naples, Italy, for SEM images. References and Notes (1) Mastrangelo, C. H.; Burns, M. A.; Burke, D. T. Proc. IEEE 1998, 86, 1769–1787. (2) Di Francia, G.; La Ferrara, V.; Manzo, S.; Chiavarini, S. Biosens. Bioelectron. 2005, 21, 661–665. (3) Lawrie, J. L.; Xu, Z.; Rong, G.; Laibinis, P. E.; Weiss, S. M. Phys. Status Solidi A 2009, 206, 1339–1342. (4) Ramsay, G. Nat. Biotechnol. 1998, 16, 40–44. (5) Wei, F.; Sun, B.; Guo, Y.; Zhao, X. S. Biosens. Bioelectron. 2003, 18, 1157–1163. (6) Piliarik, M.; Vaisocherova, H.; Homola, J. Sens. Actuators B-Chem. 2007, 121, 187–193. (7) Furbert, P.; Lu, C.; Winograd, N.; DeLouise, L. Langmuir 2008, 24, 2908–2915. (8) Herino, R.; Bomchil, G.; Barla, K.; Bertrand, C.; Ginoux, J. L. J. Electrochem. Soc. 1987, 134, 1994–2000. (9) Aspnes, D. E.; Theeten, J. B. Phys. ReV. B 1979, 20, 3292–3302. (10) Dancil, K. P. S.; Greiner, D. P.; Sailor, M. J. J. Am. Chem. Soc. 1999, 121, 7925–7930. (11) Snow, P. A.; Squire, E. K.; Russell, P. S. J.; Canham, L. T. J. Appl. Phys. 1999, 86, 1781. (12) Mulloni, V.; Pavesi, L. Appl. Phys. Lett. 2000, 76, 2523.
J. Phys. Chem. C, Vol. 114, No. 6, 2010 2621 (13) Moretti, L.; Rea, I.; Rotiroti, L.; Rendina, I.; Abbate, G.; Marino, A.; De Stefano, L. Opt. Exp. 2006, 14, 6264–6272. (14) Arrand, H. F.; Benson, T. M.; Loni, A.; Arens-Fischer, R.; Kruger, M.; Thonissen, M.; Luth, H.; Kershaw, S. IEEE Photonics Technol. Lett. 1998, 10, 1467–1469. (15) Kilian, K. A.; Bocking, T.; Gooding, J. J. Chem. Commun. 2009, 630–640. (16) Park, J. H.; Gu, L.; von Maltzahn, G.; Ruoslahti, E.; Bhatia, S. N.; Sailor, M. J. Nat. Mater. 2009, 8, 331–339. (17) De Stefano, L.; Rea, I.; Giardina, P.; Armenante, A.; Rendina, I. AdV. Mater. 2008, 20, 1529-+. (18) Low, S. P.; Voelcker, N. H.; Canham, L. T.; Williams, K. A. Biomaterials 2009, 30, 2873–2880. (19) Low, S. P.; Williams, K. A.; Canham, L. T.; Voelcker, N. H. Biomaterials 2006, 27, 4538–4546. (20) Rendina, I.; Rea, I.; Rotiroti, L.; De Stefano, L. Physica E 2007, 380, 188–192. (21) Janshoff, A.; Dancil, K. P. S.; Steinem, C.; Greiner, D. P.; Lin, V. S. Y.; Gurtner, C.; Motesharei, K.; Sailor, M. J.; Ghadiri, M. R. J. Am. Chem. Soc. 1998, 120, 12108–12116. (22) Ouyang, H.; Christophersen, M.; Viard, R.; Miller, B. L.; Fauchet, P. M. AdV. Funct. Mater. 2005, 15, 1851–1859. (23) Vamvakaki, V.; Chaniotakis, N. A. Electroanalysis 2008, 20, 1845– 1850. (24) De Stefano, L.; Rotiroti, L.; Rea, I.; Moretti, L.; Di Francia, G.; Massera, E.; Lamberti, A.; Arcari, P.; Sanges, C.; Rendina, I. J. Opt. A: Pure Appl. Opt. 2006, 7, S540. (25) Bessueille, F.; Dugas, V.; Vikulov, V.; Cloarec, J. P.; Souteyrand, E.; Martin, J. R. Biosens. Bioelectron. 2005, 21, 908–916. (26) Lehman, V. Electrochemistry of Silicon; Wiley-VCH: New York, 2002; pp 17-20. (27) Chamard, V.; Dolino, G.; Muller, F. J. Appl. Phys. 1998, 84, 6659. (28) Khokhlov, A. G.; Valiullin, R. R.; Stepovich, M. A.; Ka¨rgen, J. Colloid J. 2008, 70, 507–514. (29) Pirasteh, P.; Charrier, J.; Soltani, A.; Haesaert, S.; Haji, L.; Godon, C.; Errien, N. Appl. Surf. Sci. 2006, 253, 1999–2002. (30) De Stefano, L.; Rendina, I.; Moretti, L.; Rossi, A. M. Sens. Act. B 2004, 100, 168–172. (31) De Stefano, L.; Rotiroti, L.; Rea, I.; De Tommasi, E.; Rendina, I.; Canciello, M.; Maglio, G.; Palumbo, R. J. Appl. Phys. 2009, 106, 023109. (32) Ogata, Y.; Niki, H.; Sakka, T.; Iwasaki, M. J. Electrochem. Soc. 1995, 142, 1595–1601. (33) Lapin, N. A.; Chabal, Y. J. J. Phys. Chem. B 2009, 113, 8776– 8783. (34) Salonen, J.; Lehto, V.-P. Chem. Eng. J. 2008, 137, 162–172. (35) Salonen, J.; Lehto, V.-P.; Bjo¨rkqvist, M.; Laine, E.; Niinisto¨, L. Phys. Status Solidi A 2000, 182, 123–126. (36) Bjo¨rkqvist, M.; Salonen, J.; Laine, E.; Niinisto¨, L. Phys. Status Solidi A 2003, 197, 374–377. (37) Pal, S.; Kim, M. J.; Choo, J.; Kang, S. H.; Lee, K.-H.; Song, J. M. Anal. Chim. Acta 2008, 622, 195–200. (38) Pal, S.; Kim, M. J.; Song, J. M. Lab Chip 2008, 8, 1332–1341. (39) Voelker, N. H.; Alfonso, I.; Reza Ghadiri, M. ChemBioChem 2008, 9, 1776–1786. (40) Ouyang, H.; Striemer, C. C.; Fauchet, P. M. Appl. Phys. Lett. 2006, 88, 163108.
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