Investigation of Approaches for the Fabrication of Protein Patterns by

binding of anti-rabbit IgG antibody is performed using scanning force microscopy. ... terminated monolayers by mechanically “scraping” patterns at...
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Langmuir 2001, 17, 4105-4112

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Investigation of Approaches for the Fabrication of Protein Patterns by Scanning Probe Lithography Jeremy R. Kenseth,† Jennifer A. Harnisch, Vivian W. Jones,‡ and Marc D. Porter* Microanalytical Instrumentation Center, Ames Laboratory-USDOE, and Department of Chemistry, Iowa State University, Ames, Iowa 50011 Received January 12, 2001. In Final Form: April 26, 2001 This paper investigates three different approaches to patterning proteins within ultrathin resist layers formed from self-assembled monolayers using scanning probe lithography (SPL) at the submicrometer length scale. The first approach uses a “nanografting” method to pattern a reactive carboxylic acid terminated thiol into a resist composed of a methyl-terminated monolayer. Rabbit IgG antigen is bound to the patterned region, and an immunoassay utilizing direct readout of the topographic change resulting from specific binding of anti-rabbit IgG antibody is performed using scanning force microscopy. To address issues related to nonspecific protein adsorption, the other two approaches investigated the patterned removal of glycolterminated monolayers by mechanically “scraping” patterns at high tip-sample forces by SPL. Protein attachment to the scraped regions was achieved either through the chemisorption of a disulfide coupling agent or by the direct adsorption of Fab′-SH antibody fragments. Results obtained from all approaches are presented and compared, and the strengths and weaknesses of each toward fabricating high-density, multiple protein arrays are discussed.

Introduction The patterned immobilization of proteins on surfaces has widespread applications ranging from fundamental studies in cell biology1,2 to the development of various “biochip” platforms.3 In the latter case, the goal is to create miniaturized arrays of proteins for use in multianalyte immunoassays,4,5 protein screening,6,7 and a host of related applications.8,9 A variety of methods have been explored for patterning proteins, including ink-jet and pipet deposition,5,7,10 photolithography,11-15 microcontact printing,16-18 and microfluidic channel networks.19-22 While * To whom correspondence should be addressed. † Current address: CombiSep, Inc. 1915 Scholl Rd., Applied Science Building II, Ames, IA 50011. ‡ Current address: Corporate Analytical Technology Center, 3M Corporate Research Laboratories, Building 201-1E-15, St. Paul, MN 55144-1000. (1) Blawas, A. S.; Reichert, W. M. Biomaterials 1998, 19, 595. (2) Kane, R. S.; Takayama, S.; Ostuni, E.; Ingber, D. E.; Whitesides, G. M. Biomaterials 1999, 20, 2363. (3) Mrksich, M.; Whitesides, G. M. Trends Biotechnol. 1995, 13, 228. (4) Ekins, R. P.; Chu, F. In Principles and Practice of Immunoassay; Price, C. P., Newman, D. J., Eds.; Stockton Press: New York, 1997; p 625. (5) Joos, T. O.; Schrenk, M.; Hopfl, P.; Kroger, K.; Chowdhury, U.; Stoll, D.; Schorner, D.; Durr, M.; Herick, K.; Rupp, S.; Sohn, K.; Hammerle, H. Electrophoresis 2000, 21, 2641. (6) Lueking, A.; Horn, M.; Eickhoff, H.; Bussow, K.; Lehrach, H.; Walter, G. Anal. Biochem. 1999, 270, 103. (7) MacBeath, G.; Schreiber, S. L. Science 2000, 289, 1760. (8) MacBeath, G.; Koehler, A. N.; Schreiber, S. L. J. Am. Chem. Soc. 1999, 121, 7967. (9) Hergenrother, P. J.; Depew, K. M.; Schreiber, S. L. J. Am. Chem. Soc. 2000, 122, 7849. (10) Mendoza, L. G.; McQuary, P.; Mongan, A.; Gangadharan, R.; Brignac, S.; Eggers, M. BioTechniques 1999, 27, 778. (11) Pritchard, D. J.; Morgan, H.; Cooper, J. M. Anal. Chem. 1995, 67, 3605. (12) Mooney, J. F.; Hunt, A. J.; McIntosh, J. R.; Liberko, C. A.; Walba, D. M.; Rogers, C. T. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 12287. (13) Jones, V. W.; Kenseth, J. R.; Mosher, C. L.; Henderson, E.; Porter, M. D. Anal. Chem. 1998, 70, 1233. (14) Brockman, J. M.; Frutos, A. G.; Corn, R. M. J. Am. Chem. Soc. 1999, 121, 8044. (15) Brooks, S. A.; Dontha, N.; Davis, C. B.; Stuart, J. K.; O’Neill, G.; Kuhr, W. G. Anal. Chem. 2000, 72, 3253. (16) St. John, P. M.; Davis, R.; Cady, N.; Czajka, J.; Batt, C. A.; Craighead, H. G. Anal. Chem. 1998, 70, 1108.

each approach has clearly demonstrated utility, the micrometer-scale resolution generally achieved using these techniques limits address density. As biochip platforms evolve toward ever-increasing levels of miniaturization in order to meet demands for high throughput screening and decreased sample consumption, new technologies for the fabrication of even smaller address sizes (down to the nanometer or even molecular scale) need to be developed. Recently, scanning probe lithographic (SPL) methods have been explored for patterning protein surfaces. Examples include the mechanical scraping of open patches within protein-resistant polymer films for subsequent protein adsorption,23 the application of scanning electrochemical microscopy to derivatize electrode surfaces,24,25 and the use of “nanografting” to incorporate reactive sites into self-assembled monolayers.26 By taking advantage of the ability of SPL to manipulate and interrogate surfaces at the nanometer length scale,27,28 such approaches offer the possibility of creating as well as reading13,29,30 ex(17) Bernard, A.; Delamarche, E.; Schmid, H.; Michel, B.; Bosshard, H. R.; Biebuyck, H. Langmuir 1998, 14, 2225. (18) Martin, B. D.; Gaber, B. P.; Patterson, C. H.; Turner, D. C. Langmuir 1998, 14, 3971. (19) Delamarche, E.; Bernard, A.; Schmid, H.; Michael, B.; Biebuyck, H. Science 1997, 276, 779. (20) Delamarche, E.; Bernard, A.; Schmid, H.; Bietsch, A.; Michel, B.; Biebuyck, H. J. Am. Chem. Soc. 1998, 120, 500. (21) Patel, N.; Sanders, G. H. W.; Shakesheff, K. M.; Cannizzaro, S. M.; Davies, M. C.; Langer, R.; Roberts, C. J.; Tendler, S. J. B.; Williams, P. M. Langmuir 1999, 15, 7252. (22) Chiu, D. T.; Li Jeon, N.; Huang, S.; Kane, R. S.; Wargo, C. J.; Choi, I. S.; Ingber, D. E.; Whitesides, G. M. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 2408. (23) Boland, T.; Johnston, E. E.; Huber, A.; Ratner, B. D. In Scanning Probe Microscopy of Polymers; Ratner, B. D., Tsukruk, V. V., Eds.; American Chemical Society: Washington, DC, 1998; p 342. (24) Wittstock, G.; Hesse, R.; Schuhmann, W. Electroanalysis 1997, 9, 746. (25) Nowall, W. B.; Wipf, D. O.; Kuhr, W. G. Anal. Chem. 1998, 70, 2601. (26) Wadu-Mesthrige, K.; Xu, S.; Amro, N. A.; Liu, G.-y. Langmuir 1999, 15, 8580. (27) Nyffenegger, R. M.; Penner, R. M. Chem. Rev. 1997, 97, 1195. (28) Liu, G.-y.; Xu, S.; Qian, Y. Acc. Chem. Res. 2000, 33, 457. (29) Quist, A. P.; Bergman, A. A.; Reimann, C. T.; Oscarsson, S. O.; Sundqvist, B. U. R. Scanning Microsc. 1995, 9, 395.

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tremely high density protein arrays, provided that suitable advances in the rapid preparation of addresses can be realized. Indeed, recent developments in the area of highspeed atomic force microscopy imaging in solution,31 along with the fabrication of tip arrays,32 have begun to meet these needs. This paper explores several different approaches for the creation of high-resolution protein patterns via SPL down to a submicrometer scale. The goal is the development of methods for fabricating ultrahigh density, multiply addressed protein surfaces for use in our recently reported height-based biosensor platform, which allows for the direct readout of antigen-antibody13,33 or DNA-protein34,35 interactions using scanning force microscopy (SFM). The first approach involves the grafting of a carboxylic acid terminated thiol into a methyl-terminated monolayer and subsequent binding of rabbit IgG antigen using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) activation chemistry. The specific binding of antibody to this patterned antigenic surface is demonstrated using SFM. To reduce the effects of nonspecific protein adsorption, two alternative methods were explored that relied upon the patterned removal of glycolterminated monolayers by mechanical “scraping” using a sharp SFM tip. Following patterning, protein binding was then accomplished by (1) chemisorption of a disulfide coupling agent or (2) the direct adsorption of Fab′-SH antibody fragments. The three different methods are compared in terms of protein binding efficiency, and the strengths and weaknesses of each approach for fabricating multiple protein arrays are briefly discussed. Experimental Section Caution: Piranha solutions are highly oxidizing and should be handled with extreme care. A. Chemicals. Modified literature procedures were utilized in the preparation of dithiobis(succinimidyl undecanoate) (DSU)36,37 and (undec-11-mercapto-1-yl) triethylene glycol methyl ether (EG3-OMe).38,39 11-Mercaptoundecanoic acid (MUA) was synthesized in-house in earlier studies. 2-(N-Morpholino)ethane sulfonic acid (MES) and 1-octanethiol (97% purity) were obtained from Aldrich, ethanol was obtained from Aaper Alcohol, and 2-mercaptoethylamine (2-MEA) was obtained from Sigma. Whole molecule polyclonal rabbit IgG and goat anti-rabbit IgG, the F(ab′)2 fragment of goat anti-rabbit IgG, and EDC were purchased from Pierce. All other reagents were obtained from Fisher and were used as received. All buffers were prepared from purified 18 MΩ water (Millipore). B. Gold Substrate Fabrication. Gold substrates were prepared by two different methods. For the nanografting experiments, a 300 nm thick gold film (99.9% purity) was resistively evaporated onto freshly cleaved mica (Ashville-Schoonmaker) at a rate of 0.1 nm/s in an Edwards 306A evaporator. The pressure was maintained below 5 × 10-6 Torr for all evaporations. Following evaporation, the gold substrates were annealed in a muffle furnace at 300 °C for 4 h. These surfaces were then placed (30) Dong, Y.; Shannon, C. Anal. Chem. 2000, 72, 2371. (31) Sulchek, T.; Hsieh, R.; Adams, J. D.; Minne, S. C.; Quate, C. F.; Adderton, D. M. Rev. Sci. Instrum. 2000, 71, 2097. (32) Minne, S. C.; Yaralioglu, G.; Manalis, S. R.; Zesch, J.; Atalar, A.; Quate, C. F. Appl. Phys. Lett. 1998, 72, 2318. (33) O’Brien, J. C.; Jones, V. W.; Porter, M. D.; Mosher, C. L.; Henderson, E. Anal. Chem. 2000, 72, 703. (34) O’Brien, J. C.; Stickney, J. T.; Porter, M. D. J. Am. Chem. Soc. 2000, 122, 5004. (35) O’Brien, J. C.; Stickney, J. T.; Porter, M. D. Langmuir 2000, 16, 9559. (36) Nakano, K.; Taira, H.; Maeda, M.; Takagi, M. Anal. Sci. 1993, 9, 133. (37) Wagner, P.; Hegner, M.; Kernen, P.; Zaugg, F.; Semenza, G. Biophys. J. 1996, 70, 2052. (38) Rondestvedt, C. S.; Thayer, G. L. J. Org. Chem. 1977, 42, 2680. (39) Prime, K. L.; Whitesides, G. M. J. Am. Chem. Soc. 1993, 115, 10714.

Kenseth et al. into a solution of dilute (1 mM) 1-octanethiol in ethanol for 1624 h. Upon removal from solution, the octanethiolate-modified substrates were rinsed with ethanol, dried under a stream of nitrogen, and placed in the SFM. For all other experiments, template-stripped gold (TSG) substrates40,41 were prepared in a multistep process utilizing 4 in. Si(100) wafers (Montco Silicon, Inc.) as the template surface.42 The Si wafers were precleaned for 20 min periods in an ultrasonic bath under deionized water, methanol, methylene chloride, and methanol. After a rinse with methanol, the Si substrates were loaded in the evaporator, and a 300 nm thick layer of gold was deposited at 0.1 nm/s. Next, glass surfaces (10 mm × 10 mm) were cut from microscope slides (Fisher) and cleaned in a solution of Piranha (3:1 H2SO4 and H2O2). A drop (10 µL) of Epo-tek 377 epoxy was extruded onto the glass, which was subsequently placed in contact with the gold-coated side of the Si wafer. The Si/Au/ epoxy/glass sandwiches were then annealed in a muffle furnace at 150 °C for 1.5 h. Just prior to use, the TSG surface was detached from the Si templating substrate using double-sided tape and immediately immersed into a 1 mM solution of EG3-OMe in ethanol for 16-24 h. The resulting samples were then rinsed in ethanol and dried under a stream of nitrogen prior to placement in the SFM. C. SFM. A Digital Instruments Multimode IIIa SFM equipped with a 125 µm scanner and liquid cell operated in the contact mode under solution was utilized for all experiments. An O-ring was used to seal the liquid cell in all cases except when using the Fab′-SH fragments, which were directly pipetted onto the surface. Silicon nitride sharpened microlevers (Thermomicroscopes, Sunnyvale, CA) were employed for both imaging and lithography. Cantilevers corresponding to vendor-specified normal force constants of 0.1 N/m were used in the grafting experiments, while 0.5 N/m normal force constant cantilevers were required to obtain the higher loads utilized in the scraping experiments. Analysis by the thermal noise method43 of several cantilevers from the wafer utilized for all experiments determined that the average force constant was ∼1.5 times that of the vendor specification. Therefore, all forces were multiplied by this factor. For all imaging and lithography experiments, the SFM was set to collect 512 line scans per image. D. Immobilization of Rabbit IgG Using EDC. Following the SPL-based grafting of MUA into the octanethiolate monolayer, the SFM cell was rinsed with several volumes of ethanol to remove excess MUA and then filled with a solution of 1 mg/mL EDC in 0.1 M MES buffer (pH 5.5) for ∼15 min. Next, the cell was flushed with MES buffer and subsequently filled with 1 mg/mL rabbit IgG in the same buffer. Following a ∼15 min incubation under rabbit IgG and a rinse with MES buffer, a solution of 0.2 mg/mL goat anti-rabbit IgG in “binding buffer” (100 mM Tris-HCl pH 7.6, 100 mM NaCl, 15 mM MgCl, 1% Tween 80) was introduced into the cell and incubated for ∼30 min. E. Immobilization of Rabbit IgG Using DSU. Following patterning of the EG3-OMe-modified surface via SPL, the SFM cell was flushed with several volumes of water, filled with a 100 µM solution of DSU in sec-butanol, and incubated for 1 h. The cell was then flushed with several volumes of sec-butanol followed by water, exposed to a solution of 0.1 mg/mL rabbit IgG in 50 mM borate buffer (pH 8.5), and incubated for ∼20 min prior to imaging. F. Preparation and Immobilization of Fab′-SH Fragments. Fab′-SH fragments of goat anti-rabbit IgG were obtained by the controlled chemical reduction of the corresponding F(ab′)2, a truncated form of whole molecule IgG, using 2-MEA. An incubation of 90 min at 37 °C using 40 mM 2-MEA was found to be optimal for reduction to the Fab′-SH form with minimal cleavage of disulfide bonds located in the specific binding regions of the antibody. These conditions were determined by preparing solutions containing various concentrations of 2-MEA and monitoring aliquots using nondenaturing sodium dodecyl sulfate polyacrylamide gel electrophoresis as previously described.33 Following reduction, the reaction mixture (75 µL) was diluted (40) Hegner, B.; Wagner, P.; Semenza, G. Surf. Sci. 1993, 291, 39. (41) Wagner, P.; Hegner, M.; Guntherodt, H.-J.; Semenza, G. Langmuir 1995, 11, 3867. (42) Stamou, D.; Gourdon, D.; Liley, M.; Burnham, N. A.; Kulik, A.; Vogel, H.; Duschl, C. Langmuir 1997, 13, 2425. (43) Hutter, J. L.; Bechhoefer, J. Rev. Sci. Instrum. 1993, 64, 1868.

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Figure 1. SFM contact mode topography (A) and friction (B) images (12 µm × 12 µm) of an octanethiolate monolayer at Au/mica obtained under 200 µM MUA in ethanol after imaging the central 5 µm × 5 µm region for 10 scans at a load of 60 nN. The increased length of the scanned region relative to its width is due to a slight amount of drift in the liquid cell. The image was acquired at a load of 5 nN with a scan rate of 12 Hz. Scheme 1. A Reactive Carboxylic Acid Terminated Thiol Is Grafted into a Methyl-Terminated Monolayer, Followed by EDC Activation and Covalent Binding of Rabbit IgG

with an additional 75 µL of phosphate buffer (PB; 0.1 M, pH 6), transferred to a low binding cellulose centrifuge filter (Sigma, nominal molecular weight limit of 5000), and spun at 6000 rpm for 20 min to remove 2-MEA and achieve a final solution volume of ∼50 µL. The Fab′-SH solution was then directly pipetted onto the EG3-OMe-modified surface after patterning by SPL.

Results and Discussion “Grafting” of a Protein Reactive Monolayer Using SPL. Scheme 1 depicts the first of the concepts investigated using SPL to fabricate viable antigen addresses. This approach involves grafting a reactive monolayer of MUA within a resist monolayer of octanethiolate. The results are presented in the images shown in Figure 1. This figure shows 12 µm × 12 µm SFM height (Figure 1A) and friction (Figure 1B) images of an octanethiolate resist layer that were obtained at a load of 5 nN after the central 5 µm × 5 µm area was scanned 10 times at an imaging force of ∼60 nN (tip scan speed 120 µm/s) in the presence of 200 µM MUA in ethanol. While no difference is detected in the central region of the topographic image, there is an appreciable, though not homogeneous, increase in the central region of the friction image relative to the

surrounding regions. Experiments using several different cantilevers and octanethiolate resist layers revealed that, in general, 10 scans at loads in the range of 15-60 nN were required to induce an irreversible increase in the friction of the scanned area. Occasionally, partial or full “head-to-head” bilayers44,45 of MUA were formed during grafting, as evidenced by areas of higher topography and low friction. The lack of detectable differences in the topography of the patterned regions versus the surrounding octanethiolate resist and the occasional presence of bilayered regions are attributed to the incorporation of MUA into the octanethiolate monolayer by the nanografting technique described by Liu and co-workers.46 In this approach, the SFM tip “plows” through a resist monolayer while another surface-active molecule is present in the contacting solution. The result is the localized replacement of the resist layer by the solution species. Thus, application of a load at the tip-sample microcontact above a “displace(44) Sun, L.; Kepley, L. J.; Crooks, R. M. Langmuir 1992, 8, 2101. (45) Green, J.-B. D.; McDermott, M. T.; Porter, M. D.; Siperko, L. M. J. Phys. Chem. 1995, 99, 10965. (46) Xu, S.; Liu, G.-y. Langmuir 1997, 13, 127.

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Figure 2. SFM contact mode topography images (12 µm × 12 µm) of the same area as in Figure 1, after exposures to 1 mg/mL EDC and 1 mg/mL rabbit IgG in 0.1 M MES buffer (A) and further exposure to 0.2 mg/mL goat anti-rabbit IgG in binding buffer (B) (see Experimental Section for details). The cross sections below each image represent the average of the line scans within the boxed areas of each image. Both images were obtained at a load of 2 nN with a scan rate of 6 Hz. The small differences in the dimensions of the modified regions in A and B are due to a slight amount of drift in the liquid cell.

ment” threshold leads to the controlled fabrication of patterns down to the nanometer scale of a given adsorbate that is surrounded by a resist layer. Liu and co-workers have reported that threshold values of up to 20 nN were needed when grafting both hydrophilic and hydrophobic thiols of various chain lengths up to 37 carbons into a variety of resist layers.28,47 The grafting step could be completed in a single scan, utilizing sharpened silicon nitride cantilevers with a tip radius of less than 20 nm. We ascribe the higher threshold values and larger number of scans required for the incorporation of MUA into our octanethiolate resist to a combination of factors. Although the same source of tips was employed herein as in the work by Liu and co-workers, differences in the tip shape and, more importantly, the thickness of the cantilevers are likely. Using thermal noise analysis,43 we found that cantilevers used from the same wafer had spring constants ∼1.5 times higher than those reported by the manufacturer. Since only manufacturer spring constants were reported in previous nanografting studies,46,47 a direct comparison of observed threshold forces is not possible. In addition, previous experiments were generally conducted on nanometer-sized, atomically flat regions of a gold surface with tip speeds on the order of 0.1-2 µm/s. The relatively large scan sizes (micrometers) and tip speeds (>20 µm/s) employed herein likely result in a less effective disruption and removal of the resist in a single scan and are reflected in part by the observed inhomogeneities within the friction image of Figure 1B. Interestingly, our attempts to graft the disulfide coupling agent DSU into methyl-terminated alkanethiolate resists of varying chain lengths (C8, C10, and C18) were unsuccessful. This result may reflect the greater steric bulk of the disulfide in comparison to a thiol, which inhibits

intercalation into the relatively small void regions created by the tip during grafting. To validate the use of SPL toward the creation of an antigenic address suitable for a height-based SFM immunoassay, the grafted carboxylate-modified region was reacted with EDC to bind covalently rabbit IgG antigen (see Experimental Section). Figure 2A is a SFM height image collected under MES buffer after exposure to EDC and rabbit IgG. There is a clear increase in topography that is consistent with the dimensions of the grafted MUA region (5 µm × 5 µm). The accompanying plot shows that the bound IgG layer is on average 4-6 nm higher than the octanethiolate in the surrounding regions; some localized regions exhibit heights up to 8-9 nm which are probably due to differing orientations and/or a small level of antigen aggregation. On average, the observed topographic difference is consistent with the immobilization of a single monolayer of rabbit IgG in the grafted region. Some nonspecific adsorption of IgG was initially observed in the surrounding octanethiolate regions but was effectively displaced by the SFM tip at forces of ∼15 nN, with the covalently bound regions remaining intact. The ability to remove nonspecifically bound protein by “wiping” the surface is one potential advantage of employing SFM in the analysis of antigen-antibody interactions and in other biological applications where complex sample media may be encountered.6,7,13 Investigations to explore this possibility are under way and are focused, in part, on determining whether the SFM tip removes only nonspecifically bound protein, specifically bound protein, or both.48 The specific recognition of the SPL-fabricated antigenic address to anti-rabbit IgG is demonstrated in Figure 2B. This image shows the same region of the surface presented

(47) Xu, S.; Miller, S.; Laibinis, P. E.; Liu, G.-y. Langmuir 1999, 15, 7244.

(48) Kenseth, J. R.; Pris, A. D.; Jones, V. W.; Porter, M. D. Manuscript in preparation.

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Scheme 2. Portions of a Glycol-Terminated Monolayer Are Mechanically Scraped, and DSU Is Chemisorbed to the Patterned Regions Followed by Covalent Binding of Rabbit IgG

Scheme 3. Portions of a Glycol-Terminated Monolayer Are Mechanically Scraped, and Goat Anti-Rabbit IgG Fab′-SH Fragments Are Directly Adsorbed to the Patterned Regions

in Figure 2A, but after a 30 min exposure to goat antirabbit IgG. In this step, Tween 80 surfactant was added to the solution to reduce nonspecific binding to the surrounding octanethiolate regions. An increase in the average height to ∼11-12 nm over the octanethiolate regions is consistent with the specific binding of the antibody to the surface-immobilized antigen and falls in line with our earlier findings on the use of height changes as a readout modality for miniaturized bioassays.13,33-35 Taken together, these data argue that SPL can be used to fabricate address elements for subsequent use in heightbased SFM immunoassays. We note that the electrostatic or covalent binding of lysozyme or IgG to grafted carboxylic acid or aldehyde-terminated thiols in a decanethiolate matrix has been recently reported,26 but subsequent specific recognition reactions were not examined. Scraping Glycol Monolayers Using SPL. While the grafting approach was successful in patterning an active protein, several issues need to be addressed in order to further exploit the high-resolution capabilities of SPL toward fabricating miniaturized immunosensing and related devices. One issue results from the nonspecific binding to regions outside of the reactive area fabricated by SPL. In the above results and previously described grafting experiments,26 the use of methyl-terminated monolayers as resists resulted in appreciable amounts of nonspecific protein adsorption. This situation potentially compromises topographic detection of the specifically bound protein layer. While the addition of a surfactant to the buffer solution can in many cases reduce this complication,13,49 it is preferable to fabricate devices using layers that are inherently more resistive to nonspecific adsorption. To this end, we have explored the use of glycol-

terminated monolayers50 as resists for fabricating protein patterns by SPL under aqueous environments. We chose EG3-OMe to serve as our resist layer, as monolayers derived from this precursor have been previously shown to resist effectively protein adsorption.51,52 To pattern reactive sites within an EG3-OMe monolayer under aqueous conditions, we explored the two approaches depicted in Schemes 2 and 3. As opposed to the direct grafting of a reactive monolayer, the glycol layer was mechanically scraped by the application of a sufficiently high load between the tip and sample. Two different methods were investigated to fill in the scraped region with protein: (1) modification with the DSU disulfide coupling agent followed by protein binding (Scheme 2) and (2) the direct adsorption of the Fab′-SH fragment of an IgG antibody (Scheme 3). The following compares these two approaches in terms of protein binding efficiencies and then discusses the strengths and weaknesses of all three strategies. Figures 3 and 4 demonstrate the patterning of three different regions (two 1 µm × 1 µm squares separated by a 0.5 µm × 1 µm rectangle) of an EG3-OMe monolayer by mechanically scraping with the tip, adsorbing DSU, and reacting with rabbit IgG antigen. To pattern the EG3OMe layer, the tip-sample force was increased until an (49) Vandenberg, E.; Elwing, H.; Askendal, A.; Lundstrom, I. J. Colloid Interface Sci. 1991, 143, 327. (50) Pale-Grosdemange, C.; Simon, E. S.; Prime, K. L.; Whitesides, G. M. J. Am. Chem. Soc. 1991, 113, 12. (51) Harder, P.; Grunze, M.; Dahint, R.; Whitesides, G. M.; Laibinis, P. E. J. Phys. Chem. B 1998, 102, 426. (52) Feldman, K.; Haehner, G.; Spencer, N. D.; Harder, P.; Grunze, M. J. Am. Chem. Soc. 1999, 121, 10134.

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Figure 3. SFM contact mode topography (A) and friction (B) images (7 µm × 7 µm) of an EG3-OMe monolayer on TSG following the scraping of three different regions at a load of 200 nN for 10 scans each with a tip speed of 20 µm/s. The images were acquired at a load of 8 nN under deionized water with a scan rate of 5 Hz.

Figure 4. SFM contact mode topography image (5 µm × 5 µm) of the same region in Figure 3 following 1 h of exposure to 100 µM DSU in sec-butanol and 20 min of exposure to rabbit IgG in borate buffer (see Experimental Section for details). The image was acquired at a load of 2 nN with a scan rate of 10 Hz. The cross section corresponds to the line marked on the image.

appreciable irreversible increase in friction was observed (Figure 3B). This increase is attributed to the substantial disordering and removal of the resist, based on comparisons to the high-friction regions that are formed within the EG3-OMe layer when using UV thiolate photolithography (data not shown). Only a slight decrease (∼0.5 nm) is evident in the corresponding topography image in Figure 3A, which is less than the height difference expected for a fully removed EG3-OMe monolayer by ellipsometry (∼2 nm).50 This finding may result from the presence of a residual partial monolayer of EG3-OMe resist. Using several different tips and samples of EG3-OMe, we found that 10 scans at loads of 130-200 nN and tip speeds of ∼20 µm/s were usually sufficient to induce an irreversible increase in friction under deionized water or buffer solution.

The ability of the modified regions to bind rabbit IgG antigen following exposure to DSU is demonstrated in Figure 4. The topographic image and single-line cross section in the figure show a general increase in thickness of 3-5 nm in the SPL-fabricated region with localized areas possessing heights up to 7 nm, consistent with the presence of a single layer of IgG. The ability to image the bound layer at forces as high as 15 nN is consistent with the covalent attachment of IgG to DSU.13 Importantly, there is no evidence for the nonspecific binding of IgG in the surrounding regions for the duration of the experiment (∼1 h). In addition, this approach allows for the incorporation of a disulfide into fabricated regions, which was not achievable using the grafting method (see above). This ability may be due to the larger void regions created by removal of the EG3-OMe resist as compared to the smaller voids created during grafting. Another possibility involves the increased solubility of EG3-OMe in the sec-butanol solvent utilized to introduce DSU to the surface, thereby furthering the removal of loosely bound resist. We add that the ability of SPL to fabricate easily addresses with different shapes, such as those shown in Figure 4, allows for the creation of “bar codes” which can serve to identify a specific address when incorporated into a SFM heightbased multiple protein array format. To create high-resolution multiple protein arrays by SPL, additional key strategies need to be employed. One key requirement is the ability to pattern separate addresses without compromising the composition and reactivity of previously patterned regions. This requirement would eliminate approaches that rely upon coupling agents such as EDC or DSU, since these compounds would react with antibodies located at previously formed addresses. One potential approach is to rely upon the direct adsorption of Fab′-SH antibody fragments (Scheme 3), which have been shown to chemisorb directly to gold surfaces via their thiol moiety.33,53,54 Such an approach would eliminate intermediate coupling steps and may also provide improved performance by more properly orienting the protein active binding site. Indeed, recent work from our laboratory has demonstrated nearly a 4-fold increase in epitope density of a rabbit Fab′-SH antigen-modified gold surface versus an antigenic layer formed from whole molecule IgG.33 (53) Droz, E.; Taborelli, M.; Descouts, P.; Wells, T. N. C.; Werlen, R. C. J. Vac. Sci. Technol., A 1996, 14, 1422. (54) Karyakin, A. A.; Presnova, G. V.; Rubtsova, M. Y.; Egorov, A. M. Anal. Chem. 2000, 72, 3805.

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Figure 5. SFM contact mode topography (A) and friction (B) images (5 µm × 5 µm) of an EG3-OMe monolayer on TSG following the scraping of two different regions at a load of 180 nN for 10 scans each with a tip speed of 20 µm/s. The image was acquired at a load of 30 nN under 0.1 M PB with a scan rate of 10 Hz.

Figure 6. SFM contact mode topography image (5 µm × 5 µm) of the same region in Figure 5 following 45 min of exposure to 0.5 mg/mL goat anti-rabbit IgG Fab′-SH in 0.1 M PB. The image was acquired at a load of 2 nN with a scan rate of 6 Hz. The cross section corresponds to the line marked on the image.

The results of the directed adsorption of goat anti-rabbit IgG Fab′-SH to a pattern scraped in a EG3-OMe monolayer by SPL are presented in the images in Figures 5 and 6. Figure 5 shows two different square regions (1 µm × 1 µm and 500 nm × 500 nm) that were scraped into an EG3OMe layer using 10 scans at a load of ∼180 nN and a tip speed of 20 µm/s under 0.1 M PB. While the patterned areas appear fairly homogeneous in the friction image (Figure 5B), the corresponding topographic image (Figure 5A) suggests the presence of residual material which may result from the incomplete removal of EG3-OMe. Exposure of the pattern to a solution containing ∼0.5 mg/mL goat anti-rabbit IgG Fab′-SH in PB yielded the image shown in Figure 6. As in the earlier EG3-OMe scraping experiment, there was no detectable evidence for nonspecific adsorption in surrounding regions. While

the adsorption of Fab′-SH is limited only to the patterned areas, an incomplete monolayer of protein was formed as shown by the topographic image and cross-sectional line scan of Figure 6. These images also reveal the presence of some protein aggregation, with observed heights ranging up to 17 nm. We also note that the adsorbed Fab′-SH was not nearly as robust as the protein layer formed by the DSU approach, as substantial removal occurred upon scanning at forces up to 15 nN. Similar results were obtained for several samples utilizing this method. From these findings, it appears that the current Fab′SH fragment patterning conditions are not sufficient to remove completely the resist layer. The presence of residual resist combined with the relatively large size of the Fab′-SH adsorbate (molecular weight ∼45 00033) likely leads to incomplete protein layer formation. This conclusion is supported by our earlier results demonstrating that UV thiolate photolithographic removal of fluorinated resist layers followed by rinsing and exposure to Fab′-SH can lead to formation of complete homogeneous protein layers.33 One potential approach to circumvent incomplete resist removal involves the addition of surfactant during the scraping step, thereby increasing the solubility of EG3OMe and facilitating more complete removal. An examination of such a strategy along with the exploration of other conditions to optimize Fab′-SH immobilization to SPL-fabricated addresses for use in constructing multiple protein arrays is currently in progress. Conclusions This paper has examined several different approaches using SPL to fabricate protein addresses down to submicrometer dimensions. The use of EG3-OMe glycolterminated monolayers as lithographic resists effectively eliminates nonspecific protein adsorption as compared to methyl-terminated resists. While exposure to sec-butanol/ DSU solutions following scraping of EG3-OMe layers by SPL served to immobilize effectively a full antigenic layer, only partial layers were formed by the direct adsorption of antigenic Fab′-SH fragments. This result was likely due to the incomplete removal of relatively insoluble EG3OMe resist by the SFM tip during the scraping process, combined with the relatively large size of the Fab′-SH adsorbate. Of the three SPL approaches investigated, the direct adsorption of Fab′-SH fragments to scraped regions of

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EG3-OMe offers the most promise toward fabricating multiple protein patterns. Optimization of EG3-OMe resist removal should facilitate this goal. The high-resolution capabilities of SPL allow for the flexible creation of different shaped patterns, allowing for the individual indexing of addresses using a bar-code approach. Additional advantages of such an approach include simplicity (i.e., no need for clean room fabrication environments), the ability to pattern proteins under nondenaturing solution environments, and the use of SFM to interrogate directly subsequent protein binding events. While the throughput of such an approach is currently rather limited, the continued development of faster scanning SFMs,31 along with tip array technologies,32 should substantially decrease imaging and SPL fabrication times. Experiments along these lines are under way, as are investigations aimed at determining the general applicability of our readout strategy to more complex samples.

Kenseth et al.

Acknowledgment. J.R.K. gratefully acknowledges the support of a Phillips Petroleum Corporation graduate research fellowship. We express our appreciation to J. C. O’Brien for helpful discussions regarding the preparation of the Fab′-SH antibody fragments and to J.-B. Green (Southern Illinois University) and H. Takano for assistance in the calibration of the SFM cantilevers. This work was supported in part by the National Science Foundation (Grant No. BIR-9601789), the 3M Corporation, the Microanalytical Instrumentation Center, and the Office of Basic Energy Science, Chemical Sciences Division of the U.S. Department of Energy. The Ames Laboratory is operated for the U.S. Department of Energy by Iowa State University under Contract W-7405-eng-82. LA0100744