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Photochemical Modification and Patterning of SU-8 Using Anthraquinone Photolinkers Gabriela Blagoi,* Stephan Keller, Fredrik Persson, Anja Boisen, and Mogens Havsteen Jakobsen Department of Micro and Nanotechnology, Technical UniVersity of Denmark, DTU Nanotech, DK-2800 Kongens Lyngby, Denmark ReceiVed March 26, 2008. ReVised Manuscript ReceiVed June 25, 2008 Bioactive protein patterns and microarrays achieved by selective localization of biomolecules find various applications in biosensors, bio-microelectromechanical systems (bio-MEMS), and in basic protein studies. In this paper we describe simple photochemical methods to fabricate two-dimensional patterns on a Novolac A derivative polymer (SU-8) and, subsequently, their functionalization with biomolecules. Anthraquinone (AQ) derivatives are used to chemically modify and pattern SU-8 surfaces. Features as small as 20 µm are obtained when using uncollimated light. The X-Y spatial resolution of micropatterned AQ molecules is improved to 1.5 µm when a collimated light source is used. This micropatterning process will be important for the functionalization of MEMS-based biosensors. The method saves several processing steps and can be integrated in cleanroom fabrication thus avoiding contamination of the sensor surfaces.
Introduction Polymeric materials are increasingly used in micro- and nanotechnology because their low-cost and high-throughput fabrication methods.1 Applications ranges from microarrays,2 micro- and nanosized biosensors,3 to miniaturized cell-based assays with integrated microfluidic networks.4 Chemical patterning of surfaces is necessary when systematic modification of interfaces is required. Patterning is not only compulsory when well-defined arrays of biosensing molecules are immobilized in a controlled and spatially addressable pattern, but also when reduction of assay volumes, miniaturization, and parallelization of the assay formats are sought.5 Patterned polymers have been used for example as scaffolds for tissue engineering,6 optical devices,7 and as etch resist masks.8 Moreover, proteins and cell-patterned surfaces have been proved to be important in several fields, such as the development of biosensors9 and cellular biology studies.10 There is a great number of available patterning methods such as automated microspotters,11 microcontact printing using poly(dimethylsiloxane) stamps,12 patterning along microfluidic channels,13 bead-based lithography,14 dip-pen lithography,15 and light-guided methods including in situ synthesis of biomolecules on the substrate.16,17 Each of these methods has advantages and limitations concerning * Corresponding author. E-mail:
[email protected]. (1) Liu, C. AdV. Mater. 2007, 19, 3783–3790. (2) Ito, Y. Biotechnol. Prog. 2006, 22, 924–932. (3) Hoa, X. D.; Kirk, A. G.; Tabrizian, M. Biosens. Bioelectron. 2007, 23, 151–160. (4) Liu, M. C.; Ho, D.; Tai, Y. C. Sens. Actuators, B: Chem. 2008, 129, 826–833. (5) Foley, J.; Schmid, H.; Stutz, R.; Delamarche, E. Langmuir 2005, 21, 11296– 11303. (6) Southgate, J.; Rohman, G.; Pettit, J. J.; Isaure, F.; Cameron, N. R. Biomaterials 2007, 28, 2264–2274. (7) Mele, E.; Di Benedetto, F.; Persano, L.; Cingolani, R.; Pisignano, D. Nano Lett. 2005, 5, 1915–1919. (8) Cho, S. H.; Kim, S. H.; Lee, J. G.; Lee, N.-E. Microelectron. Eng. 2005, 77, 116–124. (9) Hodgson, L.; Chan, E.W. L.; Hahn, K. M.; Yousaf, M. N. J. Am. Chem. Soc. 2007, 129, 9264–9265. (10) Yap, F. L.; Zhang, Y. Biosens. Bioelectron. 2007, 22, 775–778. (11) Howbrook, D. N.; Van der Valk, A. M.; O’Shaughnessy, M. C.; Sarker, D. K.; Baker, S. C.; Lloyd, A. W. Drug DiscoVery Today 2003, 8, 644–651.
the resolution of the patterned structures, the majority of them being incompatible with restrictions imposed by the cleanroom technology flow. Photolithography is capable of high-resolution patterned structures and is fully compatible with micro- and nanofabrication process flows and automation, in contrast to, e.g., microfluidics network-mediated patterning, bead-based lithography, dip-pen, and in situ photo-mediated synthesis of molecules. The SU-8 photoresist is an important material for fluidic networks and microelectromechanical system (MEMS)-based sensors because of its ability to produce high-aspect-ratio structures, its chemical inertness, and low Young’s modulus.18 Recently, UV-mediated grafting of poly(acrylic acid) and watersoluble monomers onto the surface of the negative photoresist SU-8 was demonstrated.19 An X-Y spatial resolution of 2 µm polymer micropatterns was achieved. However, this method relies on the residual photoacid generator of the SU-8 polymer limiting the possibility of using it for fully processed SU-8 layers. In addition, sensing layers with a thickness of several microns are not desirable for applications where the analyte-receptor interaction should take place close to the sensor surface. To address these problems we have developed a novel photolithographic method for high-resolution chemical patterning of the SU-8 that can be integrated in the cleanroom fabrication process. The method is based on anthraquinone (AQ) photolinkers. (12) Bernard, A.; Delamarche, E.; Schmid, H.; Michel, B.; Bosshard, H. R.; Biebuyck, H. Langmuir 1998, 14, 2225–2229. (b) Lahiri, J.; Ostuni, E.; Whitesides, G. M. Langmuir. 1999, 15, 2055–2060. (13) Delamarche, E.; Bernard, A. A.; Schmid, R.; Bietsch, A.; Michel, B.; Biebuyck, H. J. Am. Chem. Soc. 1998, 120, 500–508. (14) Lin, K.-H.; Crocker, J. C.; Prasad, V.; Schofield, A.; Weitz, D. A.; Lubensky, T. C.; Yodh, A. G. Phys. ReV. Lett. 2000, 85, 1770–1773. (15) Salaita, K.; Wang, Y.; Mirkin, C. A. Nat. Nanotechnol. 2007, 2, 145–155. (16) Bock, C.; Coleman, M.; Collins, B.; Davis, J.; Foulds, G.; Gold, L.; Greef, C.; Heil, J.; Heilig, J. S.; Hicke, B.; Hurst, M. N.; Husar, G. M.; Miller, D.; Ostroff, R; Petach, H.; Schneider, D.; Vant-Hull, B.; Waugh, S.; Weiss, A.; Wilcox, S. K.; Zichi, D. Proteomics 2004, 4, 609–618. (17) Pease, A. C.; Solas, D.; Sullivan, E. J.; Cronin, M. T.; Holmes, C. P.; Fodor, S. P. A. Proc. Natl. Acad. Sci. 1994, 91, 5022–5026. (18) Johansson, A.; Blagoi, G.; Boisen, A. Appl. Phys. Lett. 2006, 89, 173505/ 1–173505/3. (19) Wang, Y.; Bachman, M.; Sims, C. E.; Li, G. P.; Allbritton, N. L. Langmuir 2006, 22(6), 2719–2725.
10.1021/la800948w CCC: $40.75 2008 American Chemical Society Published on Web 08/19/2008
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Figure 1. (a) Chemical structures of the photolinkers and Alexa 647cadaverine used for functionalization and patterning of SU-8 films. (b) Simplified photoreaction between the AQ-based photolinker modified with electrophilic group (E) and SU-8 polymer surface (R-H).
AQ photolinkers are highly reactive when irradiated in the UV-A range where photochemical damage of biomolecules and polymers are minimized.20 A fluorescently labeled diamine (Alexa 647-cadaverine) and amino-modified biotin (biotin-NH2) are covalently bound to the AQ patterned SU-8 in order to prove the possibility of patterning with biomolecules on the SU-8 surface.
Materials SU-8 2002 photoresist and its developer were bought from Microresist Technology, Germany. Alexa Fluor 647 cadaverine (Alexa 647 cadaverine) was obtained from Molecular Probes. Cy3Streptavidin and bovine serum albumin were obtained from Sigma, Germany. High-purity Milli-Q water was used to prepare the all aqueous solutions. AQ linkers with and electrophilic group (AQ-E) and amino group (AQ-NH2) were purchased from Exiqon A/S, Vedbaek, Denmark. All photolinker solutions were prepared in Milli-Q water.
Results and Discussion Figure 1a shows the chemical structures of the two AQ photolinkers and the simplified chemical structure of the fluorescently labeled Alexa 647 cadaverine used in this work. Alexa 647-cadaverine and Biotin-NH2 were utilized as model biomolecules in this work. The AQ-NH2-modified SU-8 can be used for further covalent coupling of phosphate or carboxylic acid-containing biomolecules such as DNA, polysaccharides, and proteins. The AQ-E photolinker contains an electrophilic group that reacts with amino and thiol groups at different pH. It also contains a flexible spacer of several ethylene glycol units that facilities preservation of the active sites of the biomolecules bound to the SU-8. Moreover, because of the ethylene glycol spacer’s amphiphilic nature, the AQ-E modified surfaces have (20) Suzuki, A.; Hasegawa, M.; Ishii, M.; Matsumura, S.; Toshima, K. Bioorg. Med. Chem. Lett. 2005, 15, 4624–4627.
Figure 2. Dependence of the contact angle of AQ-NH2 modified SU-8 surface on (a) concentration of the photolinker, 30 min exposure time with 2 mW/cm UV light; and (b) exposure time (100 µg/mL AQ-NH2).
antifouling properties, thereby minimizing nonspecific adsorption. No blocking step is required in sandwich immunoassays on substrates modified with AQ-E when detergent is used with the detection antibody.21 A simplified schematic of the reaction between the anthraquinone (AQ-E) derivative used in our experiments and the SU-8 surface (R-H) is presented in Figure 1b. In the presence of light, the carbonyl oxygen of the lowest excited nπ*triplet state (I) readily reacts with C-H σ-bonds of the polymer substrate (R-H), resulting in hydrogen abstraction to form a semiquinone radical.22 The semiquinone radical (II) can recombine with the alkylradical generated in the substrate (III) or abstract another hydrogen (IV). Overall, the AQ linkers attach with the O atom to the substrate, forming a new C-O bond (Figure 1b). Therefore, the electrophile and amino group of the AQ photolinkers used in this study are available for further bioconjugation. The reaction of AQ photolinkers with the SU-8 surface was monitored by contact angle measurements. The contact angle is expected to drop significantly when SU-8 is treated with AQNH2 due to the hydrophilic nature of the amino groups that are photografted on the SU-8 surface. Figure 2a shows the measured contact angle as a function of AQ-NH2 photolinker concentration in aqueous solution. The highest grafting efficiency of this photolinker in solution is obtained for a concentration of 100 µg/ml AQ-NH2. For concentrations above 500 µg/mL the contact (21) Koch, T.; Jacobsen, N.; Fensholdt, J.; Boas, U.; Fenger, M.; Jakobsen, M. H. Bioconjugate Chem. 2000, 11, 474–483. (22) Jauho, E. S.; Boas, U.; Wiuff, C.; Wredstrom, K.; Pedersen, B.; Andresen, L. O.; Heegaard, P. M. H.; Jakobsen, M. H. J. Immunol. Methods 2000, 242, 133–143.
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Figure 3. Dependence of the contact angle of AQ-E modified SU-8 surface on the concentration of the photolinker when the photolinker (a) was dissolved in aqueous solution, and (b) was preadsorbed on the SU-8 surface; 2 mW/cm2 30 min exposure.
angle is increasing. Possibly, multilayers of AQ-NH2 are formed by reaction of the AQ-NH2 photolinker with the side chain of AQ-NH2 modified SU-8 surface. Moreover, possible isomeric ring closure reaction may take place; the photoexcited AQ molecule might react with the methylene hydrogen of the glycine residue from the AQ-NH2 side chain.23 To determine the optimal UV exposure time for the AQ-NH2 coated (100 µg/ml) SU-8 films various UV exposure times were used. The resulting contact angles of the SU-8 surfaces after 0-35 min exposure time are presented in Figure 2b. The contact angle decreases as the exposure time is increased. However, increasing the exposure time from 30 to 60 min does not change the contact angle, and we therefore conclude that the reaction has reached its maximum effect after an exposure for 30 min. Similar experiments were performed for the AQ-E molecule to determine the optimal concentration for the maximal grafting efficiency of this photolinker. The SU-8 surface is saturated with AQ-E at a concentration of approximately 1 µg/mL in solution (Figure 3a). This photolinker does not show contact angle recovery in the range of the concentrations studied (up to 8 µg/mL). The saturation concentration of AQ-E is approximately 100 times lower than for AQ-NH2. This result might be explained by the possibility that AQ-E, unlike AQ-NH2, can not form multilayers. The amphilic AQ-E molecules could be involved in a selfassembly phenomenon at the polymer surface-aqueous solution (23) Maruyama, K.; Hashimoto, M.; Tamiaki, H. J. Org. Chem. 1992, 57, 6143–6150.
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interface. It is possible that the PEG linkers grafted on the SU-8 surface do not come in contact with the AQ moiety of the AQ-E molecules, therefore the cross-linking reaction does not take place. The same contact angles (73 ( 1.5) as obtained at the saturation condition in solution are achieved when the SU-8 film is incubated overnight with 50 µg/mL AQ-E and then exposed to UV light (Figure 3b). Slightly lower contact angles (71.6 ( 1.5) are obtained for 100 µg/mL AQ-E. This indicates that high AQ-E concentrations and long incubation times of the AQ-E photolinker with the polymer substrate can increase the density of photolinkers on the SU-8 surface. Further experiments will be undertaken in order to clarify the formation of mono- or multilayers when AQ-E is preadsorbed on the SU-8 films. Two different methods were developed to pattern and functionalize the SU-8 surface with model fluorescent biomolecules. The two methods are summarized in Figure 4. Method I is performed in an aqueous solution and does not require cleanroom facilities. Method II is performed in a dry phase and uses UV-aligner facilities. Details about the experimental conditions can be found in the Supporting Information section. In Method I, a pyrex wafer with a gold pattern is used as a photomask. The substrate and the mask are introduced in a 1 µg/mL AQ-E solution. The mask contains several rectangles with thin beams attached at the ends with different sizes. The photoreaction is performed in a home-built photoreactor with an uncolimated light source. After UV exposure (2 mW/cm2), the SU-8 sample is placed in a 10 µg/mL solution of Alexa 647cadaverine at pH ) 9.4 for 2 h. The free amino group of the Alexa 647-cadaverine reacts with the grafted electrophile (E) moieties on the SU-8 surface. After the reaction the substrate is rinsed to remove nonspecifically adsorbed Alexa 647-cadaverine. The resulting pattern is imaged using a fluorescent scanner. Figure 5a shows a beam pattern of Alexa 647-cadaverine on the surface of SU-8, the square structure is 50 µm × 50 µm. Figure 5b shows a pattern obtained when AQ-NH2 is used as the photolinker and the biotin-NH2 was covalently bound to this pattern. The surface was stained with Cy3-streptavidin. The beams have a width of 50 µm, lengths of 100 and 500 µm, with a pitch of 20 µm. As seen in Figure 5a,b, the pattern on the mask is transferred to the SU-8 surface and is easily reproduced. The smallest line widths obtained using uncollimated light are 20 µm. In Method II, the goal is to make the patterning process cleanroom compatible. It is difficult to introduce liquid into a standard UV aligner process, and we therefore developed a dry process. First, the SU-8 surface is incubated overnight with 50 µg/mL AQ-E, and subsequently dried. Then, the substrate is photopatterned using a standard UV lithography mask inside the cleanroom. After exposure, the substrate is extensively cleaned in order to remove any none specifically adsorbed AQ-E. Finally, the covalent binding of the Alexa 467 cadaverine is performed in the same conditions as for the patterns obtained in Method I. A fluorescence microscopy image of resolution lines is shown in Figure 5c. The smallest line width is 1.5 µm. In order to investigate the stability of the generated cadaverine pattern, the substrate has been extensively rinsed and sonicated in ethanol. The fluorescence signal from the rinsed surface has been reduced by only 2%, which indicates that the Alexa 647cadaverine has been selectively and stably immobilized. Moreover, the selective immobilization of cadaverine onto the AQ pattern was stable for more than 1 month (fluorescence measurements results not shown).
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Figure 4. Schematic of the micropatterning procedures of biomolecules on SU-8 using a simple photoreactor (I) and a mask aligner (II).
Figure 5. Fluorescence image of patterns of (a) Alexa 647-cadaverine on AQ-E modified SU-8 (fluorescent scanner, Cy5 channel, 500 ms exposure), (b) biotin-NH2 Cy3-streptavidin on AQ-NH2-modified SU-8 (fluorescent scanner, Cy3 channel, 500 ms exposure, and (c) Alexa 647-cadaverine resolution marks using a standard aligned in the cleanroom (fluorescence microscope, 60× water immersion objective, FITC channel).
photochemical modification of the SU-8 surface that is immersed in an AQ photolinker solution. The other method is based on the photoreaction of the AQ photolinkers preadsorbed and dried on SU-8. The resolution limits are compatible with state-of-the-art standard photolithography. The advantages of our proposed methods are manifold. The used AQ photolinkers are nontoxic and water-soluble molecules, therefore this approach can be carried out safely in any laboratory space, including cleanrooms. Moreover, using preadsorbed AQ-E photolinkers the photopatterning process, followed by reaction with biomolecules, is fully compatible with a micro- and nanofabrication process flow that can be completely automated. Moreover, using AQ photolinkers, sensing layers with a thickness of a few nanometers can be obtained. This is highly interesting for applications where it is required that the sensing layer-analyte interaction should take place close to the solid surface. The reported methods can be optimized for any type of polymer containing C-H or C-F on their surface. Acknowledgment. This work was supported by the EC project funded by Novopoly (Contract # STRP 013619).
Conclusion We have demonstrated two reliable and straightforward methods for functionalization and micropatterning of biomolecules on a SU-8 photoresist surface that involve the use of AQ photolinkers. One method takes advantage of the possibility of
Supporting Information Available: Detailed information about the experimental procedures used in this article is available free of charge via the Internet at http://pubs.acs.org. LA800948W
