Deep-UV

Negative and Positive Tone Protein Patterning on E-Beam/. Deep-UV Resists. Dan V. Nicolau,* Takahisa Taguchi, Hiroshi Taniguchi, and Susumu Yoshikawa...
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Langmuir 1999, 15, 3845-3851

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Negative and Positive Tone Protein Patterning on E-Beam/ Deep-UV Resists Dan V. Nicolau,* Takahisa Taguchi, Hiroshi Taniguchi, and Susumu Yoshikawa Osaka National Research Institute, Osaka, Japan Received July 21, 1998. In Final Form: February 25, 1999 The patterning of protein features on poly[(tert-butyl methacrylate)-co-(methyl methacrylate)] via e-beam lithography was achieved using two mechanisms which control the radiation-assisted, spatially addressable, selective attachment of the protein on the polymer surface. The hydrophobicity-controlled adsorption on the unexposed hydrophobic polymeric surface produces negative tone, high-contrast, high-resolution images. The chemical linkage of the protein amino groups to the carboxylic groups generated by the e-beam radiolysis produces images with a contrast tunable by the exposure energy. The performance measures of protein patterning, that is, the contrast, resolution, and level of defects, were discussed in the context of the respective mechanisms. As PtBuMA is the radiation sensitive component of a class of deep-UV resists, the study applies to the protein patterning via deep-UV lithography, with potential impact on the fabrication of biodevices and combinatorial chemistry.

Introduction We have witnessed in recent years a convergence between semiconductor manufacturing technology and biomicroanalytical devices, the former being applied with an increasing rate of success in the latter field of application. Bioactive molecule patterning1-7 found application in the fabrication of biosensors,8 and it was recently developed as a high-throughput combinatorial chemistry analytical technique.9 More specifically, techniques using light-directed, spatially addressable stepwise chemical synthesis of bioactive peptide10 and high-density oligonucleotide arrays11 have been reported. Because the microlithographic materials and techniques are purposefully designed for high-resolution patterning, their straightforward application to the fabrication of biomicrodevices, if technologically feasible, would require a minimal reengineering effort. Interestingly, the photoresists have been recently used for constructing highdensity oligonucleotide libraries11 but not for peptide libraries.

In a previous work12 we reported on the use of diazonaphthoquinone (DNQ)/novolak photoresists for printing protein features. It was found that micron-sized protein features could be printed using a chemically controlled mechanism. However, the change of photoresist surface hydrophobicity induced by exposure was not sufficient to ensure an efficient physically controlled protein patterning. Furthermore, the use of photoresists would be limited to above-micron-resolution patterning. Finally, photoresists have an inherently, albeit mild, fluorescence which will limit their use in fluorescence-based diagnostic devices. In contrast, the polymers used for e-beam/deepUV lithography are fluorescent-free and are capable of high-resolution printing. To this end, the present study explores the feasibility of the usage of a common e-beam/ deep-UV sensitive polymeric material with no background fluorescence for more versatile, high-resolution protein patterning.

* 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. Phone: +61-89470-7828. Fax: +61-894705579. E-mail: [email protected].

Materials. The radiation sensitive material used to demonstrate the potential of the two radiation-induced mechanisms for protein patterning was a copolymer of tert-butyl methacrylate (tBuMA) with methyl methacrylate (MMA). The copolymer P(tBuMA-co-MMA) can be synthesized following procedures described in detail elsewhere.13,14 Briefly, P(tBuMA-co-MMA) was obtained via the copolymerization of a 4:1 tBuMA/MMA mixture in toluene. The mixture was gently heated at 50 °C for 24 h with azoisobutyronitrile as a catalyst. At the end of the copolymerization reaction the polymer was poured in a large amount of rapidly stirred water and then further purified via repeated precipitation from an acetone/water solution and drying in a vacuum. This procedure gives a copolymer with a molecular weight of approximately 106. Both monomers and the catalyst were purchased from Aldrich Co. Pure polymers of PtBuMA and PMMA with a similar molecular weight to that of the synthesized copolymer, used to assess the response of the two components to e-beam radiation, were also purchased from Aldrich Co.

(1) 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. (2) Connolly, P.; Cooper, J.; Moores, G. R.; Shen, J.; Thomson, G. Nanotechnology 1991, 2, 160. (3) Bhatia, S. K.; Hickman, J. J.; Ligler, F. S. J. Am. Chem. Soc. 1992, 114, 4432. (4) Hong, J. D.; Lowack, K.; Schmitt, J.; Decher, G. Prog. Colloid Polym. Sci. 1993, 102, 93. (5) Pritchard, D. J.; Morgan, H.; Cooper, J. M. Angew. Chem., Int. Ed. Engl. 1995, 34, 91. (6) 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. (7) Chen, C. S.; Mrksich, M.; Huang, S.; Whitesides, G. M.; Ingber, D. E. Science 1997, 276, 1425. (8) Morgan, H.; Pritchard, D. J.; Cooper, J. M. Biosens. Bioelectron. 1995, 10, 841. St John, P. M.; Davis, R.; Cady, N.; Czajka, J.; Batt, C. A.; Craighead, H. G. Anal. Chem. 1943, 15, 1108. (9) Gallup, M. A.; Barrett, R. W.; Dower, W. J.; Fodor, S. P. A.; Gordon, E. M. J. Med. Chem. 1994, 37, 1233. (10) Fodor, S. P. A.; Read, J. L.; Pirrung, M. C.; Stryer, L.; Lu, A. T.; Solas, D. Science 1991, 251, 767. (11) McGall, G.; Labadie, J.; Brock, P.; Wallraff, G.; Nguyen, T.; Hinsberg, W. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 13555.

Experimental Section

(12) Nicolau, D. V.; Taguchi, T.; Taniguchi, H.; Yoshikawa, S. Langmuir 1998, 14, 1927. (13) Gipstein, E.; Ainslie, H.; Levine, H. A. U.S. Patent 3,779,806, 1973. (14) Cortellino, C. A.; Gipstein, E.; Hewett, W. A.; Johnson, D. E.; Moreau, W. M. U.S. Patent 3,996,393, 1976.

10.1021/la980914n CCC: $18.00 © 1999 American Chemical Society Published on Web 05/06/1999

3846 Langmuir, Vol. 15, No. 11, 1999 The model protein used for the patterning was a fluorescent avidin (FITC-avidin, purchased from Boehringer Mannheim Co.). 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, purchased from Pierce Co.), a NH2-to-COOH linker, was used to link the protein to the carboxylic-rich surfaces. Substrate Preparation. Four-inch silicon wafers were liquidprimed with hexamethyldisilazane (purchased from Aldrich Co.) and then the polymer solution was spin-coated on the wafers 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 0.6 µm thick. The films were further soft-baked for 2 h at 85 °C in a convection oven. Measurements of the Radiation-Induced Changes on the Polymeric Surface. Pure PMMA and PtBuMA polymeric films were e-beam-irradiated at several exposure energies. The contact angles of 0.5 µL water drops on the unexposed, partially exposed (i.e. 15 and 30 µC/cm2, respectively), and fully exposed (i.e. around 45 µC/cm2) polymer surfaces were measured with a Kyowa Kagaku Co. Ltd. angle meter. The change of the atomic concentration of oxygen and carbon on the polymer surface versus exposure energy was measured with an ESCA spectrometer (Escascope, VG Scientific). E-Beam-Assisted, Spatially Addressable, Selective Functionalization of the Polymer Surfaces. The patterning exposure of the deep-UV/e-beam sensitive resist P(tBuMA-coMMA) was performed using an e-beam exposure machine (ZBA 21, Jenoptik, Germany) and a test pattern. The exposure energy varied from 1 to 50 µC/cm2. The wafers were cut in 1 cm2 squares to accommodate the further processes of the selective attachment of the proteins. Selective Attachment of Proteins on Functionalized Polymer Surfaces. The radiation-induced surface-functionalized patterns on the P(tBuMA-co-MMA) resist create the layout for the selective attachment of the protein. Protein patterning has been achieved using two mechanisms: (i) a hydrophobicity selective attachment based on the differences in the hydrophobicity-induced by e-beam exposure (physically controlled mechanism) and (ii) a chemically selective attachment using the linking of the protein amino groups to the radiationinduced carboxylic groups on the exposed resist (chemically controlled mechanism). The protein patterning using the physically controlled mechanism consisted of (i) positioning the patterned 1 cm2 slides on the bottom of the wells of a Clostar cluster dish, (ii) flooding the wells with a solution of FITC-avidin at a concentration of 25 µg/mL, (iii) incubation for 2 h at 37 °C, and (iv) washing with deionized water followed by soft drying. Protein patterning using the chemically controlled mechanism followed the same steps, with the addition of about 100 µL/well of a solution of EDC at a concentration of 2 mg/mL immediately after the avidin solution was put in contact with the patternexposed resist surface. Image Processing. Where necessary, the images of the fluorescent protein features were analyzed with image-processing software (Optimas 6.1 from Optimas Corp.).

Results and Discussion Radiation-Induced Changes of the Polymeric Surface. The radiation-induced changes of the two components of the chosen resist were assessed independently for PtBuMA and PMMA. The radiation-induced evolutions of the surface hydrophobicity and oxygen content are presented in Figure 1 and Table 1, respectively. PMMA is rather insensitive to the exposure energy with respect to the surface hydrophobicity. In contrast, the presence of tBuMA moieties in the PtBuMA induces an important change in the hydrophobicity. The contact angle of the PtBuMA surface varies in the range 90°-75°, according to the exposure energy, whereas the contact angle of the PMMA surface varies within 79°-75°. The hydrophilization of the PtBuMA surface continues with the increase of the exposure energy, but with a much slower rate after reaching an exposure energy of around

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Figure 1.

Figure 2. Table 1. Evolution of the ESCA Signals versus Exposure Energy sample

% oxygen

% carbon

PMMA unexposed PMMA fully exposed PtBuMA unexposed PtBuMA fully exposed

64.01 63.90 60.83 64.61

35.99 36.10 39.17 35.39

15 µC/cm2. The atomic content of the oxygen of the surface does not vary with exposure energy for PMMA but exhibits an important change for PtBuMA. The ratio of oxygen/ carbon content on the resist surface increases by 18% for fully exposed versus unexposed resist for PtBuMA, compared with virtually no change for PMMA. Protein Patterning and Tonality of the Images. The technological steps used for protein patterning are presented in Figure 2. Negative and positive tone protein features were produced on a patterned-exposed P(tBuMAco-MMA) resist, using hydrophobicity selective attachment (physically controlled mechanisms) and chemical linkage/ EDC-mediated attachment (chemically controlled mechanism), respectively. Samples of the protein patterns on the P(tBuMA-co-MMA) surface using physically controlled and chemically controlled selective attachment are pre-

Protein Patterning on E-Beam/Deep-UV Resists

Figure 3.

sented in Figure 3. The fluorescent images were denominated as positive tone if the brighter areas are located on exposed surfaces (the opposite is true for the images denominated as negative). The physically controlled patterning (left side of Figure 3) produced negative tone features with sharp contrast and high resolution. The protein adheres on unexposed, hydrophobic surfaces (bright fluorescent areas) and is completely rejected from exposed, hydrophilic surfaces (black areas). The images present a notable number of defects (brightest globular areas). The chemically controlled patterning (right side of Figure 3) produced positive tone features with a lower contrast and, as a result, a lower resolution. The protein attached on exposed, carboxylic-rich surfaces (brighter fluorescent areas) but also (albeit to a lesser extent) on unexposed, hydrophobic surfaces (less bright areas). The images present a lower degree of defects. Radiation-Induced Physicochemistry of the P(tBuMA-co-MMA) Surface. The P(tBuMA-co-MMA) copolymer has been used as an e-beam resist15 and more recently as a 193 nm deep-UV resist.16 The radiation chemistries of its constitutive components, that is, PtBuMA and PMMA (presented in Box 1 in Figure 4) are markedly different. The tert-butyl ester moiety in PtBuMA is sensitive to low exposure energies15 generating carboxylic groups.16 On the other hand, PMMA requires high exposure energies necessary for the cutting of the bonds in the polymer backbone.17 Hence, when the surface of a thin film of the copolymer P(tBuMA-co-MMA) is exposed, two processes occur in sequence: (i) in the first stages of exposure only the tert-butyl ester moiety reacts (with butanol, with a carbon/oxygen atomic ratio of 4:1, being expelled from the surface, as presented on the left side of Box 1 in Figure 4), inducing the decrease of the hydrophobicity and the generation of carboxylic groups on the polymer surface; (ii) at higher energies of exposure, and after the depletion of tert-butyl ester moieties, the classical e-beam-induced PMMA chemistry intervenes (i.e. the (15) Miller, L. J.; Brault, R. G.; Granger, D. D.; Jensen, J. E.; Van Ast, C. I.; Lewis, M. M. J. Vac. Sci. Technol., B 1989, 7, 68. (16) Allen, R. D.; Wallraff, G. M.; Hinsberg, W. D.; Simpson, L. L.; Kunz, R. R. Methacrylate Terpolymer Approach in the Design of a Family of Chemically Amplified Positive Resists. In Polymers for Microelectronics; Thompson, L., Willson, C. G., Tagawa, S., Eds.; ACS Symposium Series 537; American Chemical Society: Washington, DC, 1994; Chapter 11. (17) Hatzakis, M. J. Electrochem. Soc. 1969, 116, 1033.

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breakdown of the backbone polymer chain and subsequent decrease of the polymer molecular weight, as presented on the right side of Box 1 in Figure 4) with little influence on the hydrophobicity and functionality of the polymer surface. The proposed mechanism is supported by (i) the experimental evolution of the surface hydrophobicity versus exposure energy (presented in Figure 1), that is, the high sensitivity of the hydrophobicity versus exposure energy for PtBuMA, as opposed to the relative lack of sensitivity for PMMA, (ii) the comparison of the experimental ESCA signals (presented in Table 1), that is, an important increase of the ratio of oxygen/carbon content versus exposure energy for PtBuMA, compared with virtually no change for PMMA; and (iii) the thoroughly documented15 high e-beam sensitivity of the P(tBuMAco-MMA) copolymers compared with the low sensitivity of the PMMA. Under the conditions of e-beam exposure, that is, advanced vacuum, it is possible that adjacent carboxylic groups form anhydride groups due to the water-depleted environment. However, these anhydride groups on the surface hydrolyze when in contact with nonneutral solutions of the protein and EDC. Apart from this possible but minor difference, the described processes are similar for e-beam and deep-UV exposure. Therefore, the following results and discussion can be entirely applied for the case of the high-resolution patterning of the proteins on P(tBuMA-co-MMA) via either e-beam or deep-UV lithography. Mechanisms Responsible for the Protein Patterning. The intensity of the fluorescence is a relative indication of the concentration of the protein on the functionalized polymer surface. In the case of a high concentration of the protein, the intensity of fluorescence may deviate from the proportionality with respect to concentration. Moreover, different formulations of the e-beam/deep-UV sensitive polymer and different processing conditions will result in different concentrations of the protein on the functionalized resist surface. Therefore, the capability of the alternative mechanisms responsible for the protein patterning can be judged in relative terms, using the intensity of the fluorescence of the protein features as a relative measure of the protein concentration on the patterned resist surface. In a previous work12 we reported on the printing of micron-sized protein features using DNQ/novolak photoresists using only a chemical linkage mechanism. The change of photoresist surface hydrophobicity induced by exposure was not sufficient to ensure an efficient physically controlled protein patterning. However, in the case of the P(tBuMA-co-MMA) polymer, the decrease of the surface hydrophobicity induced by low exposure energies blocks almost entirely the attachment of the protein on the resist surface. Subsequently, this physically controlled mechanism (outlined in Box 2 in Figure 4) is responsible for the patterning of high-contrast, negative tone protein features (left side of Figure 3). The drawbacks of this mechanism with respect to protein patterning are the generation of defects on the surface and a longer time required for printing (in general negative tone lithography is more time-consuming than the positive tone method). The chemically controlled, EDC-mediated attachment of the protein consists of the linking of the amino groups of the protein to the radiation-induced carboxylic groups on the exposed areas (the respective chemistry is presented in Box 3 in Figure 4). First, EDC activates the carboxyl groups previously generated on the surface. Subsequently, the attack of a protein amino group leads to the formation of can amide bond and the release of a urea derivative.

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Figure 4.

In parallel with the chemically controlled attachment mechanisms, the hydrophobicity-induced attachment occurs on the unexposed areas (as described above). Therefore, this twin-process mechanism (outlined in Box 4 in Figure 4) produces lower contrast, positive tone protein features (right side of Figure 3). It was reported5,8 that avidin preserved its biotin activity when using a patterning mechanism that is also physically controlled (i.e. avidin fully covered a hydrophobic substrate, and subsequently photobiotin locked-in on the avidin layer). In the case of chemically controlled selective attachment, it was proved12 that the density of avidin is extremely high. This high density of avidin would enable enough sites to be available for biotin specific interaction. Finally, another physically controlled mechanism (i.e. LB film deposition) was used18 to build molecularly ordered, highly packed avidin and biotin surfaces. This comprehensive study proved that indeed the forces between avidin and biotin monolayer surfaces are modulated by the pH and by the colloidal properties of the model surfaces (i.e. membranes). In the context of this study, the pH and the hydrophobicity of the surface can be tuned (the latter via exposure energy) to optimize the effective avidin-biotin molecular recognition. Contrast of the Protein Patterning. The contrast is an essential (together with the resolution and the level of defects) performance measure of any patterning process. The actual patterning contrast (i.e. the difference between protein concentration on protein-rich and protein-poor areas) can be inferred from the contrast of the fluorescent images of the protein features (i.e. the difference between the intensity of the fluorescence on protein-rich and (18) Leckband, D. E. Adv. Biophys. 1997, 34, 173-190.

Figure 5.

protein-poor areas). Conceptually, the relation between the attachment mechanisms and the resultant contrast is presented in Figure 4, in Box 2 and Box 4 for physically controlled and twin-process mechanism, respectively, and in Box 5 for resultant contrast. A more accurate understanding emerges from the measurement of the spatial distribution of the relative level of fluorescence for the physically controlled mechanism and the twin-process mechanism (presented in Figure 5, top and bottom row, respectively). The physically controlled mechanism exhibits a threshold-like evolution versus protein attachment, as inferred from the only two distinguishable levels of the fluorescence

Protein Patterning on E-Beam/Deep-UV Resists

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Figure 6.

intensity for the unexposed, and medium and highly exposed areas, respectively. Apart from the globular defects, the background fluorescence (i.e. on exposed, hydrophilic areas) is almost nil. This threshold-like feature of the physically controlled mechanism accounts for the sharp contrast of the negative tone images. In contrast with the physically controlled mechanism, the twin-process mechanism is more sensitive to exposure energies. It would be expected that the concentration of the protein on the surface would increase with the concentration of the carboxylic groups, which in turn will translate to an increase in the exposure energy. This assumption is validated by the measurement of the intensity of the fluorescence of the avidin versus exposure energy (bottom row in Figure 5). The higher efficiency of the chemically induced protein attachment reverses the tonality of the image at the expense of a decrease in contrast. The sensitivity of the chemically controlled mechanism regarding the exposure energy is demonstrated by the three distinguishable levels of the fluorescence intensity (on the right of the bottom row in Figure 5) for the unexposed (i.e. physically controlled attachment) and medium and highly exposed areas (i.e. chemically controlled attachment), respectively. Hence, more complex features with variable protein concentration may be produced through the variation of the exposure energy (as presented on the left of the bottom row in Figure 5). The contrast of the features printed with the twinmechanism is however decreased by the competition between the chemical linkage and the hydrophobicitycontrolled attachment (as explained in Box 5 in Figure 4). Although the chemical linkage produces positive features with a similar contrast to that for the images obtained via the hydrophobicity-controlled mechanism (comparing the ratio between the levels of the fluorescence intensity in Figure 5), the “background” fluorescence in the unexposed areas effectively decreases the overall optical contrast. A similar competition between the hydrophobicitycontrolled and chemical linkage selective protein attachments has been found to be at play in the patterning of proteins using the DNQ/novolak resist.12 The DNQ/ novolak system which produces carboxylic groups upon near-UV photolysis can be used in a similar manner, albeit with different exposure techniques and subsequent processing, to print positive tone features. However, in opposition to the DNQ/novolak system, where only the chemically controlled mechanism can be used to pattern

proteins, the P(tBuMA-co-MMA) system proved that both mechanisms are successful in printing positive and negative protein features. Resolution of the Protein Patterning. The resolution is another essential performance measure of any patterning process. In our previous work12 the resolution of the protein patterning was limited by the achievable resolution of the photoresists (usually in the above-micron range). Moreover, photoresists have a background, albeit mild, fluorescence. This background fluorescence of the base layer will limit the apparent resolution in fluorescence-based diagnostic devices. In contrast, the P(tBuMAco-MMA) used for e-beam/deep-UV lithography is fluorescent-free and capable of high-resolution printing. A robust patterning process requires that the deviation of the actual size of the printed feature be as small as possible compared with the size of the feature on the mask. In the case of e-beam lithography, the size of the feature on the mask is actually the size of the scanned pattern area on the resist surface, but we will use the former terminology for convenience. Furthermore, a robust patterning process requires that the size of the printed lines and spaces be as equal as possible. Both requirements are increasingly difficult to meet for finer features. Therefore, they constitute qualifying characteristics of the resolution capability. Finally, from an optical point of view, the resolution is in direct proportionality with the contrast of the patterned images. As a result, the twin-process mechanism is unlikely to produce high-resolution features. Furthermore, the decrease of image contrast makes a thorough analysis of the attainable resolution difficult. Hence, in the context of this study, only the physically controlled mechanism allowed a more detailed analysis of the resolution. Figure 6 presents the resolution attained when printing lines and spaces via physically controlled selective attachment (the black areas are exposed, hydrophilic, protein-free regions). Figure 7 presents the evolution of the printed feature size for lines and spaces (above and below the diagonal, respectively) versus the size of the feature on the mask. The logarithmic scale was used for axes because the test patterns (lines and spaces) decrease in a logarithmic ratio (i.e. x2). The diagonal dotted line represents an ideal, perfect patterning. The best resolution was achieved for an e-beam exposure energy of around 5 µC/cm2. Although the ultimate resolu-

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Figure 7.

tion was not the aim of this study, submicron features (spaces approximately 700 nm) have been printed. The size of the printed spaces is consistently and constantly lower than the size of the feature on the mask (the lines for the perfect printing and the actual printing of spaces are parallel in Figure 7). In contrast with the evolution of the resolution for spaces, it is more difficult to print finer features for lines (in Figure 7 the curve of the printed lines diverges considerably from the line of perfect printing for features below 3 µm). Simultaneous strong rejection and attachment of the protein on hydrophilic and hydrophobic surfaces in a confined environment may be used to explain this behavior. In the case of spaces (schematically presented in the bottom right corner of Figure 7) the exposed, hydrophilic spaces effectively reject the protein layer and therefore are only marginally overlapped by the protein layer. Because the rejection of the protein is very effective, this overlap is constant irrespective of the size of the space on the mask. On the other hand, in the case of lines (schematically presented in the top left corner of Figure 7) a too narrow hydrophobic feature will promote the protein adhesion, but the adjacent hydrophilic areas will also promote a strong rejection of the protein. These competitive processes would induce the aggregation of “cylindrical” features that present a proportionally larger overlap of the exposed, hydrophilic areas with the advance toward finer features (as presented schematically in the top left corner of Figure 7). To counterbalance this effect, deep submicron printing of the protein lines would require the biasing of the mask toward a greater than 1:1 ratio of the actual features and those on the mask, respectively. Further optimization of the formulation of the resist may also improve the resolution.

Level of Defects. A robust patterning process also requires a low level of defects, together with a good contrast and high resolution. The physically controlled mechanism produces a notable level of defects. A likely explanation relies on the presence of the particles electrostatically attached on the resist surface. These particles may act as “nucleation” centers for the attachment of the protein or as effective hydrophobic, fine “patterning features”. In this case the proposed mechanism explaining the nonlinearity of the relationship between the size of the printed lines and the size of the features on the mask (presented in the upper left corner of Figure 7) can also explain the higher level of defects for the physically controlled mechanism. The twin-process mechanism produces a lower level of defects, presumably because of the complete coverage of the resist surface (i.e. the particles are embedded in the protein layer). In all cases, the printing of proteins in a controlled environment similar to that of the semiconductor industry would drastically reduce the level of defects. Conclusion The high-resolution patterning of protein features on a poly[(tert-butyl methacrylate)-co-(methyl methacrylate)] resist via e-beam lithography was investigated. Two mechanisms can be used to control the radiation-assisted, spatially addressable selective attachment on the polymer surface. The mechanism based on the chemical linkage of the protein amino groups to the carboxylic groups generated by the e-beam radiolysis can be used to print positive tone protein images, while the mechanism based on the

Protein Patterning on E-Beam/Deep-UV Resists

adsorption of the protein on the unexposed hydrophobic polymeric surface can be used to print negative tone protein images. The described mechanisms can also be applied to the high-resolution patterning of the proteins via deep-UV lithography, because the poly[(tert-butyl methacrylate)-co-(methyl methacrylate)] material is used as a deep-UV resist.

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Acknowledgment. The present work was partially performed during D.V.N.’s Science & Technology Agency of Japan Fellowship. The authors thank the technical personnel from Jenoptik and Drs. Fulga and Iorgulescu from the Institute of Microtechnology for e-beam exposures. LA980914N