pubs.acs.org/Langmuir © 2009 American Chemical Society
Site-Specific Immobilization of DNA in Glass Microchannels via Photolithography† TuHa Vong,‡,§ Jurjen ter Maat,‡ Teris A. van Beek,‡ Barend van Lagen,‡ Marcel Giesbers,‡ Jan C. M. van Hest,§ and Han Zuilhof*,‡ ‡ Laboratory of Organic Chemistry, Wageningen University, Dreijenplein 8, 6703 HB Wageningen, The Netherlands, and §Laboratory of Bio-Organic Chemistry, Radboud University, Toernooiveld 135, 6525 AJ Nijmegen, The Netherlands
Received April 30, 2009. Revised Manuscript Received June 18, 2009 For the first time, a microchannel was photochemically patterned with a functional linker. This simple method was developed for the site-specific attachment of DNA via this linker onto silicon oxide surfaces (e.g., fused silica and borosilicate glass), both onto a flat surface and onto the inside of a fused silica microchannel. Sharp boundaries in the micrometer range between modified and unmodified zones were demonstrated by the attachment of fluorescently labeled DNA oligomers. Studies of repeated hybridization-dehybridization cycles revealed selective and reversible binding of cDNA strands at the explicit locations. On average, ∼71011 fluorescently labeled DNA molecules were hybridized per square centimeter. The modified surfaces were characterized with X-ray photoelectron spectroscopy, infrared microscopy, static contact angle measurements, confocal laser scanning microscopy, and fluorescence detection (to quantify the attachment of the fluorescently labeled DNA).
Introduction DNA immobilization is widely used in microchip technology for medical diagnostic tools, genetic analysis, and hybridization studies. The ability of DNA to hybridize and dehybridize reversibly is utilized for DNA amplification or for studying binding interactions.1,2 Although this technology is still under development, it has proven to be robust, and it is therefore interesting to use this reversible binding property to anchor for example, protein-DNA conjugates.3 Although microchip technology such as DNA microarray devices allows multiple analyses at the same time, the microchip can be regenerated and reused only in a batchwise manner, because multiple spots are situated on a open flat surface. By implementing the immobilization of the DNA into a microchannel, flow-through processes can also be studied. This allows the reloading and thus reuse of the microfluidic device and also minimizes the required number of protein-DNA conjugates, which are often hard to produce. Glass substrates are particular popular for DNA microchips because of their low cost, high stability toward different temperatures, inertness to many chemicals, high optical transparency, and low fluorescence absorbance.4 The last two factors are especially important because fluorescence detection is generally used to analyze the hybridization efficiency, and these two characteristics allow a high signal-to-noise ratio. Modification of the glass is necessary to attach the DNA to the surface. This is currently almost exclusively done with organosilanes. These silanes may contain reactive functionalities, which can be used † Part of the “Langmuir 25th Year: Self-assembled monolayers: synthesis, characterization, and applications” special issue. *To whom correspondence should be addressed. E-mail: Han.Zuilhof@ wur.nl. Telephone: (þ31) 317 482361.
(1) Seela, F.; Budow, S. Mol. Biosyst. 2008, 4, 232–245. (2) Ramsey, G. Nat. Biotechnol. 1998, 16, 40–44. (3) Schweller, R. M.; Constantinou, P. E.; Frankel, N. W.; Narayan, P.; Diehl, M. R. Bioconjugate Chem. 2008, 19, 2304–2307. (4) Zammateo, N.; Jeanmart, L.; Hamels, S.; Courtois, S.; Louette, P.; Hevesi, L.; Remacle, J. Anal. Biochem. 2000, 280, 143–150.
13952 DOI: 10.1021/la901558n
as coupling agents for bio-organic moieties (e.g., DNA). Amine, epoxide, aldehyde, and polylysine are the most common functionalities that allow the subsequent mild attachment of DNA to the glass surface.5-8 Furthermore, the attachment of silanes is easy, and has been reported to take place in only a few minutes.9 However, silane chemistry is not compatible with constructive photolithography, and photopatterning has been achieved only by local photochemical degradation of silanes by 172 nm light under vacuum conditions.10 Nevertheless, a number of soft lithographic techniques such as microcontact printing is available to directly create chemically patterned surfaces.11,12 These techniques often require mechanical contact with the substrate; thus, they cannot be used to pattern enclosed surfaces such as the inside of microchannels. Patterning of microchannels is therefore most frequently achieved via soft lithography-inspired methods, which combine patterning, protection of the patterned areas, and (relatively) mild bonding procedures13,14or use local heating-induced15 or electrode-induced deposition.16 Very recently, an alternative method was developed that does allow the covalent modification of a glass surface by means of photolithography.17 This method is based on a photochemical (5) Grainger, D. W.; Greef, C. H.; Gong, P.; Lochhead, M. J. In Micorarrays Volume 1: Synthesis Methods, 2nd ed.; Rampal, J. B., Ed.; Methods in Molecular Biology; Humana Press Inc.: Totawa, NJ, 2007; Vol. 381, Chapter 2. (6) Pirrung, M. C. Angew. Chem., Int. Ed. 2002, 41, 1276–1289. (7) Rusmini, F.; Zhong, Z.; Feijen, J. Biomacromolecules 2007, 8, 1775–1789. (8) Miyazaki, M.; Kaneno, J.; Kohama, R.; Uehara, M.; Kanno, K.; Fujii, M.; Shimizu, H.; Maeda, H. Chem. Eng. J. 2004, 101, 277–284. (9) Jang, L. S.; Liu, H. J. Biomed. Microdev. 2009, 11, 331–338. (10) Sugimura, H; Nakagiri, N. Appl. Phys. 1997, A 66, S427–S430. (11) Onclin, S.; Ravoo, B. J.; Reinhoudt, D. N. Angew. Chem., Int. Ed. 2005, 44, 6282–6304. (12) Gates, B. D.; Xu, Q.; Stewart, M.; Ryan, D.; Willson, C. G.; Whitesides, G. M. Chem. Rev. 2005, 105, 1171–1196. (13) Martin, C. R.; Aksav, I. A. J. Mater. Res. 2005, 20, 1995–2003. (14) Goto, M.; Tsukahara, T.; Sato, K.; Kitamori, T. Anal. Bioanal. Chem. 2008, 390, 817–823. (15) Yamamoto, M.; Yamada, M.; Nonaka, N.; Fukushima, S.; Yasuda, M.; Seki, M. J. Am. Chem. Soc. 2008, 130, 14044–14045. (16) Kaji, H.; Hashimoto, M.; Nishizawa, M. Anal. Chem. 2006, 78, 5469–5473. (17) ter Maat, J.; Regeling, R.; Yang, M.; Mullings, M. N.; Bent, S. F.; Zuilhof, H. Langmuir 2009, accepted for publication.
Published on Web 07/21/2009
Langmuir 2009, 25(24), 13952–13958
Vong et al.
Article
reaction of 1-alkenes with the silica surface. In the following text, this mild method is used to provide the proof of principle of a locally patterned microchannel via the local photochemical attachment of a tailor-made linker, 2,2,2-trifluoroethyl undec10-enoate. Subsequently, this linker molecule is functionalized with short DNA oligomers, and its stability under repeated hybridization and dehybridization conditions is shown. Finally, quantification of the amount of immobilized DNA is performed by measuring the fluorescence emission in hybridization and dehybridization experiments.
Materials and Methods Chemicals and Materials. All chemicals and solvents were purchased from Sigma-Aldrich (The Netherlands) and used without additional purification unless stated otherwise. The chemicals involved for coupling and hybridization experiments were dilutions of 20 SSC buffer at pH 7 (= 3 M NaCl in 3 M sodium citrate; molecular biological grade, VWR; note that 2 SSC is a 10-fold dilution of this 20 stock buffer, etc.), Tween-20, and sodium dodecyl sulfate (SDS, Fluka). Dichloromethane (DCM, Fisher) and petroleum ether 40/60 were distilled prior to use. Other solvents used were methanol (HPLC grade, BioSolve), hydrochloric acid (p.a. 37%, Riedel de Hae.n), absolute ethanol (Merck), acetone (semiconductor grade, Riedel de Hae.n,) and ultrapure water (18.3 MΩ cm). Synthetic fused silica substrates (10 20 mm2) used for surface modification were purchased from Praezisions Glas & Optik GmbH (Germany), large-diameter synthetic fused silica capillaries (i.d.=1 mm, 100 mm length) were obtained from Vitrocom, and fused silica capillaries (i.d. = 100 μm) with a Teflon AF fluoro-polymer external coating, TSU100375, were purchased from Polymicro. 2,2,2-Trifluoroethyl undec-10-enoate (TFEE) was synthesized as described elsewhere.18,19 DNA Probes and Targets. The end-modified DNA oligonucleotides used as probes and targets were purchased from IBA GmbH (Germany) and consisted of 21 bases with the following sequence: H2N-(CH2)6-50 -CCA CGG ACT ACT TCA AAA CTA-30 -Cy3. Two targets were used: both were modified at the 50 end with an amine group, and one was additionally labeled at the 30 end with Cy3. They are further denoted as A-NH2 and ANH2-Cy3. In addition, two probes were used:one as a positive and the other as a negative control, both labeled at the 30 -end position with Cy5 and Atto488, respectively, and referred to as AC-Cy5 and NC-Atto488. The sequence for the complementary strand, AC-Cy5, is 50 - TAG TTT TGA AGT AGT CCG TGG-30 Cy5 and that for the non-complementary strand, NC-Atto488, is 50 -AGT ATT GAC CTA AGT ATT GAC-30 -Atto 488. Pretreatment Reaction Vessels. Prior to use, the glassware used for surface modification was cleaned and etched overnight in basic detergent, followed by thorough rinsing with ultrapure water and drying for g2 h at 120 °C. Cleaning and Hydroxyl Formation. A fused silica slide was cleaned by sonication in acetone for 5 min. After it was dried with argon, the slide was immersed in a freshly prepared 1:1 (v/v) mixture of HCl and methanol for 45 min. The cleaned slide was ready for modification after it was rinsed with ultrapure water and dried with argon. The same cleaning procedure was applied for the large-diameter fused silica capillaries, but these were additionally dried at 120 °C for 30 min. Cleaning of small-diameter fused silica capillaries was performed by flushing for 20 min at 20 μL/min with the following solvents: acetone, ultrapure water, 1 M NaOH (1 h), ultrapure water, 1 M HCl, ultrapure water, and acetone, respectively. Afterwards, the capillaries were dried with argon. (18) De Smet, L. C. P. M.; Pukin, A. V.; Sun, Q. Y.; Eves, B. J.; Lopinski, G. P.; Visser, G. M.; Zuilhof, H.; Sudh€olter, E. J. R. Appl. Surf. Sci. 2005, 252, 24–30. (19) Rosso, M.; Giesbers, M.; Arafat, A.; Schroen, K.; Zuilhof, H. Langmuir 2009, 25, 2172–2180.
Langmuir 2009, 25(24), 13952–13958
Surface Modification. The modification of a fused silica slide was performed in a specially designed quartz flask as described previously.17 Neat TFEE (1.5-2 mL) was deoxygenated by three consecutive freeze-pump-thaw cycles, after which the liquid was frozen again under argon. The cleaned fused silica sample was added and vacuum was applied again until the TFEE was completely molten and the slide could be immersed. Two lowpressure mercury lamps (254 nm, 6.0 mW/cm2, Jelight) were placed in front of the fused silica slide at a distance of approximately 0.5 cm, and the sample was irradiated for 10 h. The setup was wrapped in aluminum foil and kept under a slight argon overpressure during the entire process. After illumination, the sample was removed from the reaction flask and cleaned by rinsing with petroleum ether 40/60, dichloromethane, and ethanol. Subsequently, it was sonicated in ethanol and dichloromethane (5 min per solvent) and then finally dried with argon. The modification of cleaned and dried large-diameter capillaries was performed in a glovebox (MBraun MB20G,
