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Micron-Sized Protein Patterning on Diazonaphthoquinone/ Novolak Thin Polymeric Films Dan V. Nicolau,* Takahisa Taguchi, Hiroshi Taniguchi, and Susumu Yoshikawa Osaka National Research Institute, Osaka, Japan Received July 17, 1997. In Final Form: January 27, 1998 Protein patterns at 2-µm resolution were surface-printed on the unprofiled surface of diazonaphthoquinone/ novolak thin polymeric films through positive-tone and reversed negative-tone lithography. The possible mechanisms responsible for selective protein patterning, that is, (i) linkage of the amino end of the protein to the photoinduced indenecarboxylic acid via NH2-to-COOH cross-linking mediated by carbodiimide, (ii) linkage of the amino end of the protein via in-situ addition to the photoinduced ketene, and (iii) hydrophobic interaction with unexposed surfaces, were assessed through the use of ESCA, contact angle, AFM, and fluorescence microscopy. Of these, the first two mechanisms, in conjunction with diazonaphthoquinone/ novolak photopolymers (a class of conventional microlithographic materials) and microlithographic equipment and techniques, can be used for bottom-to-top biomolecular architecture construction. The study proves that photolithographic materials (used for light-assisted selective surface protein attachment) can be easily transferred to bio-microlithography, with potential applications ranging from biodevice fabrication to diagnostic and combinatorial chemistry.
Introduction The knowledge regarding the building of molecular structures ordered vertically and incorporating biologically active molecules advanced constantly in the past decade1 with applications in the creation of electronically modulated biological functions.2 The advances in the knowledge regarding the vertical structuring of the biomolecular architectures are followed by advances regarding the lateral structuring,3 with new and reliable techniques required for patterning bioactive molecules. The patterning of bioactive molecules4-11 found applications in biosensor fabrication12 and in selective guidance of cells.13-15 Recently, bioactive molecule patterning received a new incentive from the growing field * Corresponding author. Present address: RioTinto/Research & Technology Development, Perth, 1 Turner Avenue, Technology Park, Bentley, WA 6102 (Locked Bag 347, Bentley Delivery Centre, WA 6983), Australia. Telephone: +61-89470-7828. Fax: +61894705579. E-mail:
[email protected]. (1) Nicolini, C. Biosens. Bioelectron. 1995, 10 (1/2), 105. (2) Aizawa, M.; Haruyama, T.; Kham, G. F.; Kobatake, E.; Ikaryiama, Y. Biosens. Bioelectron. 1994, 9, 601. (3) Gopel, W. Biosens. Bioelectron. 1995, 10 (1/2), 35. (4) Bhatia, S. K.; Hickman, J. J.; Ligler, F. S. J. Am. Chem. Soc. 1992, 114, 4432. (5) Hong, J. D.; Lowack, K.; Schmitt, J.; Decher, G. Prog. Colloid Polym. Sci. 1993, 102, 93. (6) Pritchard, D. J.; Morgan, H.; Cooper, J. M. Angew. Chem., Int. Ed. Engl. 1995, 34, 91. (7) Connolly, P.; Cooper, J.; Moores, G. R.; Shen, J.; Thomson, G. Nanotechnology 1991, 2, 160. (8) Bhatia, S. K.; Teixeira, J. T.; Anderson, M.; Shriver-Lake, L. C.; Calvert, J. M.; Georger, J. H.; Hickman, J. H.; Dulcey, C. S.; Schoen, P. E.; Ligler, F. S. Anal. Biochem. 1989, 178, 197. (9) Matsuda, T.; Sugawara, T. Langmuir 1995, 11, 2272. (10) Sugawara, T.; Matsuda, T. Macromolecules 1994, 27, 7809. (11) Sundberg, S. A.; Barrett, R. W.; Pirrung, M.; Lu, A. L.; Kiangsoontra, B.; Holmes, C. P. J. Am. Chem. Soc. 1995, 117, 12050. (12) Pritchard, D. J.; Morgan, H.; Cooper, J. M. Anal. Chim. Acta 1995, 310, 251. (13) Fromhertz, P.; Schaden, H. Eur. J. Neurosci. 1994, 6, 1500. (14) Dulcey, C. S.; Georger, J. H.; Krauthamer, V.; Stenger, D. A.; Fare, T. L.; Calvert, J. M. Science 1991, 252, 551. (15) Mrksich, M.; Dike, L. E.; Tien, J.; Ingber, D. E.; Whitesides, G. M. Exp. Cell. Res. 1997, 235 (2), 305. Chen, C. S.; Mrksich, M.; Huang, S.; Whitesides, G. M.; Ingber, D. E. Science 1997, 276 (5317), 1425. Mrksich, M.; Chen C. S.; Xia, Y.; Dike, L. E.; Ingber, D. E.; Whitesides, G. M. Proc. Natl. Acad. Sci. U.S.A. 1996, 93 (20), 10775.
of combinatorial chemistry,16 and techniques using lightdirected, spatially addressable, stepwise chemical synthesis of bioactive peptides11,17 are reported to be in use. High-resolution bio-microlithography would increase the multiplication of the functions of the complex biomolecular architectures. This enhanced potential will translate in a higher capability to control cell adhesion or an increase in the information per area in combinatorial chemistry. However, the reported bio-microlithographic techniques cannot easily achieve high resolution, either because of the use of a less robust imaging layer (e.g. soft protein layers6 or molecularly thin self-assembled monolayers4,8,14,15) or due to the use of less robust imaging chemistry (e.g. triazide9,10 and “caged” biotin6 photochemistry). On the other hand microlithographic materials, in particular photoresists based on diazonaphthoquinone/ novolak, are purposefully designed for the high-resolution patterning. At the same time, through their rich and reliable photochemistry, photoresists offer unexplored opportunities for high-resolution patterning of biomolecules. From this perspective, the use of diazonaphthoquinone/novolak photopolymers for functionality-induced surface printing rather than conventional photolithography, amplified by the use of advanced photolitographic equipment, may be beneficial to a seamless development of bio-microlithography. Methods and Results Materials. The photosensitive material used was a common, positive tone, commercial AZ type photoresist (S1400-17, purchased from Shipley Co.). The AZ type photoresist is a mixture of a photoactive compound based on diazonaphthoquinone (DNQ) and a novolak-based resin, in an approximate proportion of 3:7, respectively, dissolved in a solvent (e.g. ethyl cellosolve acetate), to reach 30% solid content in the polymer solution. The material is sensitive to the g-line of the Hg lamp spectrum (436 nm). Another photosensitive material was derived (16) Gallup, M. A.; Barrett, R. W.; Dower, W. J.; Fodor, S. P. A.; Gordon, E. M. J. Med. Chem. 1994, 37, 1233. (17) Fodor, S. P. A.; Read, J. L.; Pirrung, M. C.; Stryer, L.; Lu, A. T.; Solas, D. Science 1991, 251, 767.
S0743-7463(97)00802-0 CCC: $15.00 © 1998 American Chemical Society Published on Web 03/14/1998
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Figure 1. Surface hydrophobicity evolution with exposure energy for the DNQ/novolak system in standard positive-tone, image reversal negative-tone, and image reversal positive-tone lithography.
from the former, adding imidazole to the polymer solution to reach 2% of the solid content. This material, image reversal (IR) resist, was used for positive-tone as well as for negative-tone image reversal lithography. To visualize protein attachment, we used a fluorescent avidin (FITCavidin, purchased from Boehringer Mannheim Co.). To assess the light-assisted attachment mechanism, we used two peptides with high and low nitrogen content. The nitrogen-rich peptide, SFP4, is a neuropeptide synthesized in-house according to a procedure previously described18 with the sequence Gly-Ala-Cys-Gly-Arg-Gly-Asp-Ser-ProCys-Gly-Ala and an intramolecular linkage between the two Cys. The peptide with low nitrogen content is polyglutamic acid with a molecular weight higher than 8000 (purchased from Peptide Institute Inc., Japan). The cross-linker for anchoring the protein or peptide amino ends to the carboxylic sites created on the photoresist surface was (1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide)/HCl (EDC, purchased from Pierce Co.). Substrate preparation. The 15-mm glass substrates were liquid-primed with hexamethyldisilazane (purchased from Aldrich Co.) through spinning at low speed just prior to the photoresist deposition. The polymer solution was spin-coated on 15-mm glass substrates using a spin coater (Mikasa Spinner 1H-2D, Mikasa Co.) for 15 s at a rotation speed of 500 rpm for deposition and for 1 min at a rotation of 3000 rpm for thinning and drying. This procedure allows the deposition of uniform films approximately 0.6µm thick to occur. The films were further soft-baked for 1 h at 85 °C in a convection oven. Contact Angle Measurements. The polymeric films, both doped and undoped with imidazole, were irradiated using a Hg lamp, with a band-pass filter for below 365 nm, at exposure energies denominated as low (30 mJ/ cm2), medium (60 mJ/cm2), and high (120 mJ/cm2). In the case of imidazole-doped resist processed in the Image (18) Taguchi, T.; Bo, X.-X.; Taniguchi, H.; Kiyosue, K.; Kudoh, S. N.; Yumoto, N.; Tatsu, Y.; Yamamoto, H.; Kasai, M.; Yoshikawa, S. In Peptide Chemistry 1994; Ohno, M., Ed.; Protein Research Foundation: Osaka, 1995; p 145.
Reversal mode, the substrates were reversal-baked in a convection oven at 100 °C for 1 h. The polymer films were postexposure baked at 80 °C for 30 min to remove the possible molecular tensions. The contact angles of 0.5-µL water drops deposited on the unexposed, exposed, and exposed-and-reversal-baked polymer surfaces were measured with a Kyowa Kagaku Co. Ltd. anglemeter. The variation of contact angle with exposure energy is presented in Figure 1. Minor variations of the processing conditions may result in different values of the contact angle, but the overall variation versus exposure energy will remain similar. Protein Patterning Techniques. Two techniques, based on two different attachment mechanisms, were found useful for protein patterning: direct attachment and EDC-mediated attachment. The direct patterning technique consisted of (i) depositing a drop of a solution of (FITC) avidin at a concentration of 25 µg/mL on the resist surface, (ii) pressing the surface with a mask, to form a thin liquid layer of avidin solution between the resist surface and the mask, (iii) exposing the resist surface through the mask and the liquid layer of avidin solution, and (iv) washing the surface with deionized water and drying at 30 °C for 30 min. This procedure is technically similar to the one previously reported in the literature,6 but the chemical mechanism of attachment is entirely different. A sample of the 10µm line of FTIC avidin printed with this technique is presented in Figure 2a. The EDC-mediated attachment technique consisted of (i) exposing the resist through a patterned mask to form on the photoresist surface the chemical sites for anchoring, (ii) placing the exposed slides on the bottom of the wells of a Clostar cluster dish, (iii) adding 400 µL/well of a solution of FITC avidin with a concentration of 25 µg/mL, (iv) flooding the wells with 100 µL/well of a solution of EDC at a concentration of 2 mg/mL, (v) leaving the dish for incubation for 2 h at 37 °C, and (vi) washing thoroughly with deionized water followed by soft drying. A sample
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Figure 2. (a, top) Ten-micrometer lines/spaces of FITC avidin patterned on a standard photoresist surface (positive tone) using a direct attachment mechanism. (b, bottom) Ten-micrometer lines/spaces of FITC avidin patterned on an image reversal photoresist surface (reversed negative tone) using EDCmediated attachment.
of the 10-µm line of FTIC avidin printed with this technique is presented in Figure 2b. ESCA Spectrometry. The mechanisms of protein attachment were assessed by measuring the oxygen and nitrogen concentrations on the resist surface using an ESCA spectrometer (Escascope, VG Scientific). The probe peptides, that is, SFP4 and polyglutamic acid, were chemically linked on fully exposed photoresist surfaces, using the techniques described above. An illustration of the change in the ESCA spectrum for the fully exposed
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photoresist surface, before and after probe peptide chemical linkage, is presented in Figure 3a (upper and lower graph, respectively). The atomic oxygen content for surfaces with the probe peptides attached compared with the evolution of oxygen content on the peptide-free photoresist surface as a function of exposure energy is presented in Figure 3b. A significant content of nitrogen on the photoresist surface was found for the case of EDCmediated attachment of the probe peptides, compared with a low nitrogen content for direct attachment and insignificant levels for all other cases. High-Resolution Biolithography. To prove the feasibility of our concept, we initially used an in-house made contact printer and emulsion masks (sample images of the patterns in Figure 2). Following the successful validation of the concept, we attempted micron-size resolution and multiple printing lithography, using an exposure aligner (Mikasa manipulator MA-8, Mikasa Co.) and e-beam-patterned chromium masks. The exposure energy was also optimized to obtain the highest resolution. The microlithographic techniques used in conjunction with direct and EDC-mediated attachment of the model protein were (i) positive-tone patterning using either a standard or an image reversal resist and (ii) image reversal (negative-tone) patterning. The technological paths for protein patterning are presented in Figure 4. Standard positive-tone patterning uses the exposure of the DNQ/novolak resist surface through a mask with an energy of 40 mJ/cm2 and the attachment of the model protein, either simultaneous with or after exposure. Positive-tone lithography with an IR resist uses a similar process flow but with higher exposure energy (60 mJ/ cm2). After washing and drying, the patterned surface can be presented to successive patterning processes with different masks. Image reversal (negative-tone) patterning using an IR resist consists of the exposure through a mask at 80 mJ/ cm2, followed by a reversal bake at 100 °C for 30 min in a convection oven, a flood exposure through a clear mask at 100 mJ/cm2, and the attachment of the model protein, either simultaneous with or after flood exposure. Image reversal patterning using a standard resist follows a similar procedure but uses a higher patterning exposure energy (120 mJ/cm2) and ammonia atmosphere for the reversal bake, as well as a higher flood exposure energy (150 mJ/cm2). AFM Profiling. The avidin patterns on the resist surface were mapped in the tapping mode with an atomic force microscope (SPI 3700, Seiko Instruments Co.). The step height of the avidin patterns is about 13 nm. An AFM profile of a 2-µm line of avidin obtained in negativetone image reversal lithography using an IR resist is presented in Figure 5. The observed artifacts are due to the contamination-prone nature of contact printing (as opposed to e.g. projection printing). Discussion DNQ/Novolak Chemistry. It is generally accepted19 that the pronounced surface hydrophobicity of the unexposed DNQ/novolak mixture as compared with exposed one is due to an unusually strong hydrogen bonding between the hydroxylic groups of the novolak and the diazo groups of the photoactive DNQ. Upon exposure with nearUV light, the DNQ molecule reacts with a high quantum yield, expelling a N2 molecule, presumably to form a highly unstable carbene biradical. Although the formation of (19) Dammel, R. Diazonaphthoquinone-based Resists; SPIE Optical Engineering Press: Bellingham, WA, 1993.
1930 Langmuir, Vol. 14, No. 7, 1998 Figure 3. (a, left) ESCA spectra for a fully exposed standard photoresist surface (upper graph) and after probe peptide (SFP4) attachment on this surface using an EDC-mediated mechanism. Peak assignment: C@287, O@536, and N@403. (b, right) Oxygen and nitrogen concentration on the photoresist surface for different processing conditions and for different peptides attached on the surface (ESCA measurements).
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Figure 4. Technological avenues for the patterning of proteins, using standard positive-tone, image reversal negative-tone, and image reversal positive-tone lithography.
Figure 5. AFM profile of a protein (avidin) feature printed using image reversal negative-tone lithography. The width of the feature is 2 µm (its depth is profiled in Figure 8).
this biradical has not yet been confirmed, the subsequent Wolf rearrangement, leading to the formation of a less reactive ketene derivative, is certain. In normal conditions, the ketene reacts with water molecules present in the atmosphere to form an indenecarboxylic acid (ICA). The formation of carboxylic groups in the exposed regions of the polymer induces an increase in hydrophilicity, which in turn allows the diffusion of the aqueous basic solution
(developer) and selective dissolution of the exposed areas. This classical physicochemical pathway for standard positive-tone lithography is presented in detail elsewhere.19 If water is not present in sufficient amounts in the atmosphere, the ketene reacts with the hydroxylic groups of the surrounding novolak to form an ester. This esterification produces only small increases in surface hydrophobicity as compared to that of the unexposed
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Figure 6. Chemistry of the DNQ/novolak system with the mechanisms responsible for protein attachment on a photoresist surface.
photoresist surface but important increases in the molecular weight of the polymer. The blockage of the diffusion of aqueous developer in the exposed areas, due to important increases in molecular weight, can be obtained through an alternative technique (negative-tone image reversal) which relies on the decarboxylation of the ICA (leading to a hydrophobic indene derivative). The decarboxylation temperature (in the exposed areas) is reduced below the DNQ decomposition temperature (in the unexposed areas) by basic catalysis. This catalysis can be achieved either by addition of a basic additive (usually an imidazole derivative) in the original photoresist formulation or during the reversal bake in an ammoniasaturated atmosphere. All negative-tone image reversal techniques require a “blanket” or flood exposure to induce the selective dissolution of the previously unexposed areas. A detailed presentation of the chemical mechanisms related to the technological alternatives to standard positive-tone lithography can be found elsewhere.19 Although the addition of imidazole was intended for negative-tone image reversal lithography, an important improvement in the patternability for positive-tone lithography with an imidazole-doped photoresist was reported.20,21 A chemical mechanism which explains the increase of the apparent lithographic resolution has been recently proposed.22 The above description of DNQ chemistry has been restricted to the chemical pathways relevant to protein patterning. Many other chemical reactions related to the photochemistry of diazo ketene compounds have been reviewed recently,23 and the chemistry and physicochemical mechanisms involved in the various microlithographic processes using the DNQ/novolak system have been extensively treated.19,24 Also, the surface chemistry of
the photoresists was recently used to control the selective attachment and metabolism of living cells, that is, neuronal cells25 and marine bacteria.26 The most common chemical pathways used by microlithography are presented in the upper section of the chemical scheme in Figure 6. Light-Assisted Mechanisms for the Selective Attachment of Proteins. Three mechanisms are potential candidates for an effective protein patterning on DNQ/ novolak polymeric surfaces: (i) photoinduced creation of carboxylic groups on the polymer surface and subsequent linkage of the amino groups through EDC-mediated crosslinking (two-step process), (ii) photoinduced creation of ketene groups and in-situ linkage through amido groups, when the polymer surface is in contact with the bioactive molecule solution (one-step process; alternatively, the ketene groups react with water present in solution and the link between the protein and the surface is a loose one, through the ionic association of the carboxylic and amino groups), and (iii) hydrophobicity-induced attachment due to the different chemical functionalization of the exposed and unexposed surfaces, respectively. The photochemistry of the DNQ/novolak photoresist with the proposed chemical avenues for anchoring proteins is presented in Figure 6. Light-Assisted Surface Chemistry of the DNQ/Novolak System. The first step of the selective attachment of proteins is the formation of the sites for protein anchoring, along the lines of the classical DNQ photochemistry (Figure 6). The evolution of the oxygen content on the surface (Figure 3.2) and the evolution of the contact angle (Figure 1) with exposure energy are in line with the surface functionalization mechanism proposed previously.25 Briefly, the increase in exposure energy increases the
(20) Nicolau, D. V.; Dusa, M. Microelectron. Eng. 1990, 11, 557. (21) Dusa, M.; Nicolau, D. V. Optical/Laser Microlithography II, Proc. SPIE 1989, 1088, 539. (22) Nicolau, D. V.; Dusa, M.; Nicolau, D.; Yoshikawa, S. J. Photopolym. Sci. Technol. 1995, 12, 187. (23) Ye, T.; McKervey, M. A. Chem. Rev. 1994, 94, 1091.
(24) Ersov, V. V.; Nikiforov, G. A.; de Jonge, C. R. H. I. Quinone Diazides; Elsevier Science Press: Amsterdam, 1981. (25) Nicolau, D. V.; Taguchi, T.; Tanigawa, H.; Yoshikawa, S. Biosens. Bioelectron. 1996, 11, 1237. (26) Ivanova, P. E.; Nicolau, D. V.; Yumoto, N.; Taguchi, T.; Okamoto, K.; Yoshikawa, S. J. Marine Biol., in press.
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concentration of carboxylic groups up to a point where all DNQ moieties on the photoresist surface are photolized. Consequently, the oxygen content and the surface hydrophilicity increase. After this point, and in the advanced stages of exposure, other surface chemistries intervene. The main process is novolak cross-linking on the surface, with a subsequent increase in hydrophobicity. The decrease in the oxygen content can be linked to the incipient decarboxylation. However, it is not expected that DNQ photodecomposition (and its subsequent transformation to ICA) and the novolak surface cross-linking are perfectly sequential. Hence, to assess the protein attachment mechanism, we preferred to work with a higher exposure energy to ensure that all DNQ is photolized. The nitrogen content on the photoresist surface was undetectable by ESCA spectrometry, even in the unexposed state. This is rather unexpected, but it can be explained on the grounds of (i) the low content of nitrogen in the bulk (estimated to be less than about 3% of that in the solid polymer mixture), (ii) the hydrogen bonding between DNQ moieties containing nitrogen and the hydroxyl groups of the novolak and consequently the molecular reorganization of DNQ moieties far from the surface (the strong NtN‚‚‚HO hydrogen bonding is reported to be responsible for the high resolution of the photoresist19), (iii) the small depth of ESCA probing in the range of several nanometers, and (iv) the parasitic reactions responsible for DNQ depletion during the microlithographic process, for example the esterification reaction between DNQ and novolak (mechanism responsible for the photoresist “skin” effect19). EDC-Mediated Protein Patterning. The second step for the selective attachment of proteins using EDC linkage involves (i) the creation of EDC-activated sites and (ii) the reaction of the amino ends at these sites (Figure 6). EDC-mediated patterning was expected to exhibit a high attachment yield, as EDC cross-linking is the classical method for linking amino groups to carboxylic ones. Indeed, both oxygen and nitrogen content increased substantially after the EDC-mediated attachment (Figure 3b). Moreover, the increase in the oxygen content on the surface is higher for the attachment of an oxygen-rich probe peptide (i.e. polyglutamic acid, with an oxygen content of 32.6%) than for that of an oxygen-poor one (i.e. SFP4, with an oxygen content of 16.8%), from 45.3% oxygen on the surface before to 60.4% and 49.2% after the attachment of the respective probe peptides. Consequently, the increase in the nitrogen content on the surface is higher for the attachment of the nitrogen-rich probe peptide (i.e. SFP4, with a nitrogen content of 13.4%) than for that of the nitrogen-poor one (i.e. polyglutamic acid, with a nitrogen content of 9.52%), from no nitrogen on the surface before to 13.5% and 10.4% after the attachment of the respective probe peptides. The above mechanism for the EDC-mediated protein attachment is valid for both standard and image reversal resist processed in the positive tone. The small amount of imidazole (less than 1%) added to the resist is unlikely to change fundamentally the nature of protein attachment, but it may have an important positive influence on the resolution.20,21 The EDC-mediated protein attachment produces avidin layers with a thickness of about 13 nm. The smooth surface of the protein coverage observed by AFM suggests an uniform density of the protein layer. The thickness of the protein layer in the range of 10 nm and its high fluorescence suggest the compact nature of the protein layer. For comparison, other authors9,10 patterned protein features using a less specific chemical anchoring of avidin
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to photoinduced radicals on a polymer surface and obtained uneven surfaces for protein layers with an average thickness in the range of 100 nm. The most probable explanation for the relative compactness reported here consists of the synergism between the specificity of the binding mechanism, the high COOH concentration on the polymer surface, the possible preordering of the protein molecules with the NH2 groups toward the surface, and the competition for available anchoring sites. Direct Linkage through in-Situ Attachment. A second mechanism considered for protein patterning relied on the direct linkage to ketene moieties before they are able to turn into carboxylic groups (Figure 6). This alternative technique comprises the photoinduced generation of the active anchoring sites and their linkage to the protein amino groups simultaneously, in one patterning step (Figure 4). However, the competition between the addition of the protein amino groups and water molecules present in solution to ketene decreased the apparent yield of the selective attachment. This decrease in yield translates to reduced printing selectivity, which can be observed in the lower quality of the images (Figure 7b compared to Figure 7a). Consequently, the increase in oxygen and nitrogen content on the surface of the resist, following direct linkage, is consistently reduced as compared to the respective increase for the EDC-mediated linkage (Figure 3b, for pGA only). This observed reduced yield may also be connected to the loose nature of the link proteinphotoinduced sites. The presence of water molecules in the protein solution leads to the formation of carboxylic sites, which in turn can be loosely connected to protein amino groups through an ionic linkage (as proposed in Figure 6). This loose linkage may be broken when the polymer surface is washed and dried, and probably even more for ESCA measurement conditions. Hydrophobicity-Induced Protein Attachment. A third mechanism considered for protein patterning was hydrophobicity-induced selective attachment. Hydrophobicitydriven protein patterning has been employed by the “stamping” soft lithography12 (positive-tone images) with the potential to print 100-nm features.27 Two hundred nanometer protein features were obtained23 (negativetone images), induced by differences in hydrophobicity by patterning e-beam exposure of poly(tert-butyl methacylate). However, in the context of the present work, the photoinduced surface hydrophobicity manipulation (Figure 1) is not selective enough for effective protein patterning, generating low-contrast features. This is probably due to the relatively small variations in the surface hydrophobicity induced by DNQ/novolak photochemistry as compared to those for other hydrophobicityinduced protein printing and probably due to the weak linkage between proteins and the unexposed photoresist surface. For protein patterning on photoresist surfaces the hydrophobic interaction between protein and unexposed resist acts as a parasitic effect, decreasing the contrast of the patterned images (i.e. as seen more clearly in the low-resolution imagessthe background glow in Figure 2sand less clearly in the high-resolution imagess Figure 7). Further optimization of the concentration of the protein solution and controlled blanket exposure at the end of the patterning process may be used to moderate this parasitic effect. Comparison of the Processability of Various Patterning Techniques. DNQ/novolak photopolymer is the classical material for photolithography used to create profiled features. In contrast to this use, the proposed (27) Whitesides, G. M. Personal communication.
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Figure 7. (a, left) High-resolution patterns of FITC-avidin printed using EDC-mediated cross-linking. The protein is patterned on standard and image reversal photoresists (top and bottom rows, respectively) in positive and negative tone (left and right columns, respectively). (b, center) High-resolution patterning of FITC-avidin printed using direct linkage. The protein is patterned on an image reversal photoresist in positive and negative tone (top and bottom rows, respectively). (c, right) Superimposed multiple patterns of FITC-avidin printed using EDC-mediated cross-linking.
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Table 1. Advantages and Drawbacks of the Protein Attachment Mechanisms and Techniques
Figure 8. AFM profile of a 2-µm protein line. The maximum height of the protein layer is 13 nm. The height of the protein layer (in inset) at the corners of the pattern reproduces the variation in the concentration of the carboxylic groups on the surface (induced by diffraction-controlled, submicron scale variations of the local density of the aerial image).
technique uses the exposure to create new chemical functionalities on the polymer surface that are further used as anchoring sites for proteins. In a sense, our technique is exactly the opposite of classical photolithographic process, as we (i) confine the whole printing process to the polymer surface, instead of printing on the basis of bulk photochemical reactions, (ii) “grow” features from the surface up, instead of etching features from the surface down, and (iii) use the surface of DNQ/novolak as a scaffold for building protein features, instead of using the resists to print other materials (e.g. SiO2-on-Si, metal patterns, etc.). With these considerations in mind, the actual protein-patterning processability and resolution (either achieved or potential) are products of the characteristics of the “photolithography” stage (i.e. exposure, reversal bake, etc.) and the “protein attachment” stage (direct or EDC-mediated attachment). The presented protein-patterning techniques, that are spin-offs from classical photolithography, are not equal in terms of process characteristics, that is, robustness of the process, yield of the protein linkage, resolution, contrast, and availability of multiple patterning. The
advantages and drawbacks of the protein-patterning mechanisms and techniques are summarized in Table 1. From the point of view of protein attachment mechanisms, EDC-mediated linkage is superior in terms of linkage yield but also in terms of technological flexibility. The technological flexibility of the EDC-mediated linkage allows the decoupling of the exposure stage from the attachment stage. The exposure can be made on highresolution (projection optics) tools, in conditions similar to those for semiconductor manufacturing, and the substrates can be transported or stored to another site for further attachment of biologically active molecules. On the other hand, the direct linkage mechanism would allow in-situ processing, suitable for stepwise chemical synthesis used in combinatorial chemistry. The superior contrast of the protein features obtained with EDC-mediated attachment can be observed by comparing the images in parts a and b of Figure 7, for EDC-mediated and direct linkage, respectively. From the point of view of lithographic patterning tones, it is recognized that negative-tone lithography presents
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a better inherent optical resolution capability29 than positive-tone lithography. However, in the frame of this study the increased inherent resolution comes at the price of a single pattern printed via multiple technological steps. Using an EDC-mediated linkage and an image reversal resist, the superior contrast of negative-tone patterning can be observed by comparing the images in the bottom row in Figure 7a, left and right columns, for negative and positive tone, respectively. From the point of view of photolithographic materials, the DNQ/novolak/imidazole system presents the highest resolution capability and process latitude30 (in both positive and negative tone). These benefits come however with the price of reduced shelf life for the material due to parasitic, basic-induced destruction of DNQ and slightly reduced sensitivity. Superior contrast is obtained using an image reversal resist (Figure 7a, bottom row) as compared to that for the images obtained with a standard resist (Figure 7a, top row). Moreover, the positive images printed on an image reversal resist are almost defectfree, whereas the images printed on a standard resist have a higher level of accidental features, presumably due to a higher incidence of parasitic exposure and subsequent protein adsorption (the image reversal resist has a different response to exposure than the standard one30). It was expected that additional technological steps would reduce the quality of the patterning, and indeed multiple patterning (Figure 7c) decreases the contrast of the protein images. However, the effective resolution of bio-microlithography is higher than that of classical semiconductor microlithography using similar microlithographic materials and exposure equipment due to the bottom-up patterning (preservation of the aerial image through surface imaging) as opposed to the top-down patterning used in semiconductor fabrication.29 The beneficial effect of confining the exposed latent image at the top of the resist translated in increased resolution up to the printing of diffraction features (in the range of 0.6 µm). These features can be observed at the corners of the (28) Nicolau, D. V.; Taguchi, T.; Dusa, M.; Yoshikawa, S. Smart Materials, Structures and Integrated Systems, Proc. SPIE 1997, 3241, 222. (29) Dusa, M.; Nicolau, D. V.; Fulga, F. Optical/Laser Microlithography III, Proc. SPIE 1990, 1264, 439. (30) Nicolau, D. V.; Taguchi, T.; Taniguchi, H.; Yoshikawa, S. J. Photopolym. Sci. Technol. 1996, 9, 645.
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rectangles printed with image reversal positive-tone lithography (Figure 7a, bottom right corner) and more clearly on the AFM-profiled image of the same pattern (Figures 5 and 8, in inset). Furthermore, it would be expected that the height of the protein layer be proportional with the surface concentration of COOH groups at the edges of the aerial image (where the COOH concentration switches from 1 for a fully exposed surface to 0 for a totally unexposed surface). To this end, the AFM profile shows that the height of the protein layer at the corners of the patterned features reproduces the diffractioncontrolled variations of the local density of the aerial image. This reproducibility of the diffraction features at the corners of the patterned image strongly suggests that the reported method has submicron capability if appropriate optical tools are used (e.g. projection printing instead of contact/proximity printing). Conclusions Diazonaphthoquinone/novolak materials (the common formulation for photoresists) were successfully used as photoassisted functional materials for micron-sized protein patterning. Two linkage mechanisms can be used for the selective attachment of the proteins to the photoinduced anchoring sites on the DNQ/novolak polymeric surface, namely EDC-mediated cross-linking and direct linkage. Of these, the former allows superior yield and contrast, and the latter allows easier processability. Hydrophobicity-induced protein attachment was unable to induce effective patterning. Positive and negative tone, as well as multiple patterning, with micron resolution was demonstrated using these techniques. AFM profiles of the height of the patterned protein layer suggest that the reported techniques have submicron resolution capability. Acknowledgment. One of the authors (D.V.N.) wishes to acknowledge the support of Science and Technology Agency, MITI, Japan, through an STA Fellowship. Some of the masks for high-resolution patterning and AFM profiling were obtained through the courtesy of Florin Fulga (Institute of Microtechnology) and Seiko Instruments, respectively. Finally, the authors wish to thank George Whitesides for discussion during the final preparation of the manuscript. LA970802G
