Production of Periodic Arrays of Protein Nanostructures Using

Optical micrographs were acquired using an Olympus model BH-2 phase contrast .... The thickness of the protein nanostructures as measured from the cur...
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Production of Periodic Arrays of Protein Nanostructures Using Particle Lithography Jayne C. Garno,†,‡ Nabil A. Amro,† Kapila Wadu-Mesthrige,‡,§ and Gang-Yu Liu*,† Department of Chemistry, University of California, Davis, California 95616, and Department of Chemistry, Wayne State University, Detroit, Michigan 48202 Received June 3, 2002. In Final Form: July 30, 2002 We introduce a new method of nanoparticle lithography to produce periodic arrays of protein nanostructures. The resulting protein arrays are characterized using atomic force microscopy. The periodicity and detailed morphology of these arrays can be varied by selecting latex particles with a desired size and the protein-to-latex ratio. Particle lithography offers the advantages of nanometer precision and high throughput. The proteins within the nanostructures retain the ability to bind corresponding specific antibodies. This approach provides an alternative means for production of a new generation of nanostructurebased biochips and sensors.

I. Introduction Ultrasmall protein patterns are used in biosensing, control of cell adhesion and growth, and biochip fabrication.1-4 Miniaturization of feature sizes offers the rewards of reduced quantities of analytes and reagents, increased density of sensor and chip elements, and faster reaction/ response time.5-10 At the micrometer level, protein patterning has been accomplished using techniques such as photolithography9,11-13 and microcontact printing.14-22 In these approaches, a surface is fabricated to include discrete * To whom correspondence should be addressed. Tel: 530-7549678. Fax: 530-752-8995. E-mail: [email protected]. † University of California. ‡ Wayne State University. § Present address: Department of Chemistry, Delphi Research Laboratories, Troy, MI 48098. (1) Blawas, A. S.; Reichert, W. M. Biomaterials 1998, 19, 595-609. (2) Scouten, W. H.; Luong, J. H.; Brown, R. S. Trends Biotechnol. 1995, 13, 178-185. (3) Zhang, S. G.; Yan, L.; Altman, M.; Lassle, M.; Nugent, H.; Frankel, F.; Lauffenburger, D. A.; Whitesides, G. M.; Rich, A. Biomaterials 1999, 20, 1213-1220. (4) Ravenscroft, M. S.; Bateman, K. E.; Shaffer, K. M.; Schessler, H. M.; Jung, D. R.; Schneider, T. W.; Montgomery, C. B.; Custer, T. L.; Schaffner, A. E.; Liu, Q. Y.; Li, Y. X.; Barker, J. L.; Hickman, J. J. J. Am. Chem. Soc. 1998, 120, 12169-12177. (5) Marvin, J. S.; Corcoran, E. E.; Hattangadi, N. A.; Zhang, J. V.; Gere, S. A.; Hellinga, H. W. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 4366-4371. (6) Bergveld, P. Sens. Actuators, A 1996, 56, 65-73. (7) Polzius, R.; Schneider, T.; Bier, F. F.; Bilitewski, U.; Koschinski, W. Biosens. Bioelectron. 1996, 11, 503-514. (8) Eggers, M.; Hogan, M.; Reich, R. K.; Lamture, J.; Ehrlich, D.; Hollis, M.; Kosicki, B.; Powdrill, T.; Beattie, K.; Smith, S.; Varma, R.; Gangadharan, R.; Mallik, A.; Burke, B.; Wallace, D. BioTechniques 1994, 17, 516. (9) O’Brien, J. C.; Jones, V. W.; Porter, M. D. Anal. Chem. 2000, 72, 703-710. (10) Kunz, R. E. Sens. Actuators, B 1997, 38-39, 13-28. (11) Jones, V. W.; Kenseth, J. R.; Porter, M. D. Anal. Chem. 1998, 70, 1233-1241. (12) Nicolau, D. V.; Taguchi, T.; Taniguchi, H.; Yoshikawa, S. Langmuir 1998, 14, 1927-1936. (13) Dontha, N.; Nowall, W. B.; Kuhr, W. G. Anal. Chem. 1997, 69, 2619-2625. (14) Bernard, A.; Delamarche, E.; Schmid, H.; Michel, B.; Bosshard, H. R.; Biebuyck, H. Langmuir 1998, 14, 2225-2229. (15) James, C. D.; Davis, R. C.; Kam, L.; Craighead, H. G.; Isaacson, M.; Turner, J. N.; Shain, W. Langmuir 1998, 14, 741-744. (16) Kane, R. S.; Takayama, S.; Ostuni, E.; Ingber, D. E.; Whitesides, G. M. Biomaterials 1999, 20, 2363-2376.

regions of reactive terminal groups, such as, self-assembled monolayers (SAMs), organic thin films, or polymers, for subsequent adsorption. Chemistries have been developed to tailor surface reactivities for immobilizing proteins onto surfaces, such as the photoactivation of SAM surfaces,23 and biotin-derivatized polymers.24,25 Recently, microfluidic networks have been used for protein patterning: poly(dimethylsiloxane) (PDMS) stamps, which are typically used for microcontact printing, have been used to direct small volumes of protein solutions into networks of channels to create protein patterns on various surfaces.26-28 These techniques provide a high throughput means for assembling proteins at a size scale of hundreds of nanometers or larger.16,17 Electron beam lithography has achieved submicrometer-sized patterns with SAMs, which were subsequently backfilled with cysteamine for linking fluorescent antibodies via biotinylation.29 To further miniaturize the feature size, atomic force microscopy (AFM) based lithography was developed, which can pattern SAMs at the dimension of nanometers.30-33 (17) Bernard, A.; Renault, J. P.; Michel, B.; Bosshard, H. R.; Delamarche, E. Adv. Mater. 2000, 12, 1067-1070. (18) Lahiri, J.; Ostuni, E.; Whitesides, G. M. Langmuir 1999, 15, 2055-2060. (19) Ta, T. C.; McDermott, M. T. Anal. Chem. 2000, 72, 2627-2634. (20) Willner, I.; Shlittner, A.; Doron, A.; Joselvich, E. Langmuir 1999, 15, 2766-2772. (21) Garrison, M. D.; McDevitt, T. C.; Luginbuhl, R.; Giachelli, C. M.; Stayton, P.; Ratner, B. D. Ultramicroscopy 2000, 82, 193-202. (22) Bieri, C.; Ernst, O. P.; Heyse, S.; Hofmann, K. P.; Vogel, H. Nature 1999, 17, 1105-1108. (23) Delamarche, E.; Sundarababu, G.; Biebuyck, H.; Michel, B.; Gerber, C.; Sigrist, H.; Wolf, H.; Ringsdorf, H.; Xanthopoulos, N.; Mathieu, H. J. Langmuir 1996, 12, 1997-2006. (24) Yang, Z.; Frey, W.; Oliver, T.; Chilkoti, A. Langmuir 2000, 16, 1751-1758. (25) Hengsakul, M.; Cass, A. E. Bioconjugate Chem. 1996, 7, 249254. (26) Delamarche, E.; Bernard, A.; Schmid, H.; Bietsch, A.; Michel, B.; Biebuyck, H. J. Am. Chem. Soc. 1998, 120, 500-508. (27) 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, 7252-7257. (28) Kim, Y.-D.; Park, C. B.; Clark, D. S. Biotechnol. Bioeng. 2001, 73, 331-337. (29) Harnett, C. K.; Satyalakshmi, M.; Craighead, H. G. Langmuir 2001, 17, 178-182. (30) Lee, K.-B.; Park, S.-J.; Mirkin, C. A.; Smith, J. C.; Mrksich, M. Science 2002, 295, 1702-1705. (31) Wadu-Mesthrige, K.; Amro, N. A.; Garno, J. C.; CruchonDupeyrat, S.; Liu, G.-Y. Appl. Surf. Sci. 2001, 175-176, 391-398.

10.1021/la020518b CCC: $22.00 © 2002 American Chemical Society Published on Web 09/14/2002

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Langmuir, Vol. 18, No. 21, 2002 8187 Table 1. Latex and BSA Mixtures Used in Particle Lithography latex diameter (nm)

[BSA] (µg/mL)

[latex] (particles/ mL)

BSA/latex ratios

503 ( 4 304 ( 5 204 ( 6

20 20 24

1.4 × 1011 6.6 × 1011 2.1 × 1012

26000:1 to 67000:1 9000:1 to 31000:1 7000:1 to 9000:1

To produce arrays of nanometer-sized structures of metals, oxides, and polymers, particle or nanosphere lithography has been developed.36-40 Monodisperse particles are closely packed into 2D or 3D periodic structures. These structures are then used as a template/frame, where the void space can be filled with the material of interest. Particles are then removed by either calcination or solvent dissolution. Arrays of nanostructures produced include 2D arrays of metal nanoparticles;41-44 3D arrays of nanoparticles,45 metal oxides,46 and silica;47 porous membranes of polyurethane;48 and 3D opal or reversed opal photonic band gap materials using silicon and titanium dioxide.49 In this work, we investigate if periodic arrays of protein nanostructures can be produced using nanoparticle lithography. Various proteins and latex particles are employed for this study. The procedure is optimized to produce low-defect, periodic arrays of protein nanostructures. II. Experimental Section 1. Preparation of Substrates. Two types of substrates were used for this study: mica(0001) (clear ruby muscovite mica, S&J Trading Co., NY) and ultraflat gold thin films. Mica is a common substrate for preparing biological samples, because of the hydrophilic nature of its surface.50,51 In addition, cleavage provides clean and atomically flat surfaces with single crystalline domains up to millimeters. For protein deposition, mica was cut into 1 cm2 pieces and then cleaved just before protein deposition. Ultraflat thin gold films were prepared following a previously reported procedure.52 Gold (Alfa Aesar, 99.99%, Ward Hill, MA) was deposited in a high-vacuum evaporator (Denton Vacuum Inc., Moorestown, NJ, model DV502-A) at ∼10-7 Torr onto mica substrates. The mica was freshly cleaved immediately before being mounted on the substrate holder inside the vacuum chamber. The substrates were preheated to 325 °C using quartz lamps. The evaporation rate was typically 3 Å/s, with a final film

Figure 1. Schematic diagram of the basic procedure to produce protein nanopatterns using particle lithography.

Dip-Pen nanolithography was used to construct protein arrays with 100-350 nm features.30 Nanostructures of proteins, as small as 10 nm × 150 nm, which contain three lysozyme molecules were produced on solid substrates.34,35 AFM-based lithography offers the highest spatial precision but has the limitation of low throughput. (32) Kenseth, J. R.; Harnisch, J. A.; Jones, V. W.; Porter, M. D. Langmuir 2001, 17, 4105-4112. (33) Liu, G.-Y.; Amro, N. A. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 5165-5170. (34) Wadu-Mesthrige, K.; Amro, N. A.; Garno, J. C.; Xu, S.; Liu, G.Y. Biophys. J. 2001, 80, 1891-1899. (35) Wadu-Mesthrige, K.; Xu, S.; Amro, N. A.; Liu, G.-Y. Langmuir 1999, 15, 8580-8583.

(36) Xia, Y.; Gates, B.; Yin, Y.; Lu, Y. Adv. Mater. 2000, 12, 693-713. (37) Frey, W.; Woods, C. K.; Chilkoti, A. Adv. Mater. 2000, 12, 15151519. (38) Wu, M.-H.; Whitesides, G. M. Appl. Phys. Lett. 2001, 78, 22732275. (39) Hulteen, J. C.; Van Duyne, R. P. J. Vac. Sci. Technol., A 1995, 13, 1553-1558. (40) Jiang, P.; Hwang, K. S.; Mittleman, D. M.; Bertone, J. F.; Colvin, V. L. J. Am. Chem. Soc. 1999, 121, 11630-11637. (41) Haynes, C. L.; Van Duyne, R. P. J. Phys. Chem. B 2001, 105, 5599-5611. (42) Winzer, M.; Kleiber, M.; Dix, N.; Weisendanger, R. Appl. Phys. A 1996, 63, 617-619. (43) Jensen, T. R.; Malinsky, M. D.; Haynes, C. L.; Van Duyne, R. P. J. Phys. Chem. B 2000, 104, 10549-10556. (44) Rossi, R. C.; Tan, M. X.; Lewis, N. S. Appl. Phys. Lett. 2000, 77, 2698-2700. (45) Tessier, P. M.; Velev, O. D.; Kalambur, A. T.; Lenhoff, A. M.; Rabolt, J. F.; Kaler, E. W. Adv. Mater. 2001, 13, 396-400. (46) Holland, B. T.; Blanford, C. F.; Do, T.; Stein, A. Chem. Mater. 1999, 11, 795-805. (47) Velev, O. D.; Jede, T. A.; Lobo, R. F.; Lenhoff, A. M. Chem. Mater. 1998, 10, 3597-3602. (48) Gates, B.; Yin, Y.; Xia, Y. Chem. Mater. 1999, 11, 2827-2836. (49) Subramania, G.; Constant, K.; Biswas, R.; Sigalas, M. M.; Ho, K.-M. Adv. Mater. 2001, 13, 443-446. (50) Amrein, M.; Mu¨ller, D. J. Nanobiology 1999, 4, 229-256. (51) Wagner, P. FEBS Lett. 1998, 430, 112-115. (52) Wagner, P.; Hegner, M.; Guntherodt, H. J.; Semenza, G. Langmuir 1995, 11, 3867-3875.

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Figure 2. AFM topograph of a latex film on mica, prepared by mixing BSA and 503 nm latex and then depositing the mixture on mica. thickness of 150-200 nm. The temperature was maintained for an additional 15 min after deposition for annealing. Thin glass slides were glued to the gold films using Epotek 377 (Epoxy Technologies, Inc., Billerica, MA) and then cured at 150 °C for 2 h. For particle lithography, the glass pieces were peeled to reveal a fresh Au(111) surface, just prior to protein deposition. This procedure produces surfaces with flat Au(111) areas as large as 300 × 300 nm2 in lateral dimension, with a mean roughness as small as 2-5 Å according to AFM measurements. 2. Protein and Latex Particle Solutions. Bovine serum albumin (BSA, fraction V, 98% purity) and rabbit immunoglobulin G (IgG, 95% purity) were commercially available (Sigma Biochemicals, St. Louis, MO) and used as received. Phosphate buffered saline (PBS) was prepared as follows: 1.6332 g of NaCl, 0.0447 g of KCl, 0.0381 g of KH2PO4, and 0.1221 g of Na2HPO4 in 200 mL of deionized water (18 MΩ), for final concentrations of 140 mM NaCl, 3 mM KCl, 1.4 mM KH2PO4, and 4.3 mM Na2HPO4. The pH measured 7.2 for this PBS buffer. The concentrations of proteins ranged from 10 to 200 µg/mL, depending on the desired protein/latex ratio. Latex particles were either obtained commercially (Duke Scientific, Palo Alto, CA) or synthesized using surfactant-free emulsion polymerization of styrene.46 The latex particles were washed in deionized water by centrifugation. The latex pellets were resuspended with deionized water and vortex mixing, Finally, protein solution was added to the latex solution according to the selected protein/latex ratio. Experimental requirements for particle lithography include the use of monosized latex spheres, which are free of additives such as charge stabilizers or surfactants. Surfactants were removed by centrifuging and rinsing the samples with deionized water. Large latex particles (diameter, 500-800 nm) were centrifuged for 10 min at 10 000 rpm, and smaller particles (diameter, 200-300 nm) formed a pellet within 20 min at 14 000 rpm. Using small volumes (0.1-2 mL) of diluted (1-10%) latex solutions, the particles were readily resuspended in deionized water or protein solutions using vortex mixing. Further requirements include determining the appropriate ratios and concentrations for latex and proteins. Table 1 provides the solution conditions used for successful lithography of BSA arrays. 3. Optical Microscopy. Optical micrographs were acquired using an Olympus model BH-2 phase contrast microscope. Digital images were acquired with a Sony DXC-107 color video CCD camera, with a 768 × 494 pixel chip. The computer interface includes an Osprey 100 video capture card and its driver (version 1.2.00), in conjunction with the VidCap 32 software within Windows 98. Samples were immersed in Cargill type DF immersion oil (Electron Microscopy Sciences, Washington, PA) for examination under a 100× objective. The video-capture images were further magnified for the CCD camera using a 2.5× objective.

Figure 3. [A] Optical micrograph (65 × 45 µm2) of a 2D BSA array on mica(0001). The BSA/latex ratio is 398 000:1. This micrograph was acquired under immersion oil, using a phase contrast objective. The micrograph displays broad regions of periodic dots, spanning 5-40 µm2 areas. The removal of latex was not complete in this case, as identified by the white opaque contrast in the images. [B] AFM topograph of an array of BSA nanostructures. This image was acquired in water using CRI, at a modulation frequency of 37.8 kHz and an amplitude of 23 Å. 4. Atomic Force Microscopy. An atomic force microscope with a home-constructed scanner was used for imaging, which employs the optical beam-deflection configuration.53,54 The electronic controllers and software used to operate the scanner are from RHK Technology, Inc. (Troy, MI). Images were acquired either in solution or in air. Commercially available Si3N4 cantilevers were used for imaging, with force constants of 0.03 and 0.1 N/m (microsharpened cantilever, Thermo Microscopes, Sunnyvale, CA) or 0.38 N/m (regular microlever, Digital Instruments, Santa Barbara, CA). When latex or proteins were imaged in liquid, the total force applied ranged from 0.2 to 0.6 nN. When latex was imaged in air, the measured total force ranged from 0.3 to 1.5 nN. The magnitude of the imaging force was determined from the corresponding force-distance curves and includes both the capillary/meniscus contribution and the force of cantilever bending. The AFM scanner was calibrated using commercial calibration grids, with a periodicity of 3.0 µm and heights of 22.0, (53) Kolbe, W. F.; Ogletree, D. F.; Salmeron, M. B. Ultramicroscopy 1992, 42, 1113-1117. (54) Xu, S.; Cruchon-Dupeyrat, S. J.; Garno, J. C.; Liu, G.-Y.; Jennings, G. K.; Yong, T.-H.; Laibinis, P. E. J. Chem. Phys. 1998, 108, 50025012.

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Figure 4. Periodic arrays of BSA nanostructures produced via particle lithography. [A] AFM topograph of a 3 × 3 µm2 area of a BSA pattern after the 304 nm latex was removed, acquired using CRI at 37.6 kHz and an amplitude of 19 Å. The BSA/latex ratio is 9000:1. [B] High-resolution view of a 1 × 1 µm2 area of (A). [C] Cursor profile for the line in (B). [D] Topograph of a 3 × 3 µm2 area for a BSA pattern from 204 nm latex, acquired in ethanol using CRI at 29.3 kHz and an amplitude of 38 Å. The BSA/latex ratio is 9000:1. [E] Topograph for a 1 × 1 µm2 scan area. [F] Cursor profile for the line in (E). 100, and 485 nm (NT-MDT, Moscow, Russia). The AFM calibration was further verified using latex particles with known sizes ranging from 102 ( 3 to 503 ( 4 nm (NIST traceable as measured by TEM, Duke Scientific, Palo Alto, CA). Potential problems when imaging soft and hydrophilic proteins include the stick-slip motion during the scan and/or tip contamination. We employed three strategies to minimize these effects and to improve AFM resolution: (1) imaging in a liquid environment, which substantially decreases capillary adhesion between the tip and surface; (2) minimizing the imaging force in order to prevent the displacement of proteins; (3) using contactresonance imaging (CRI) to minimize tip-surface adhesion and stick-slip behavior.31 In CRI, the sample is modulated at the resonance frequency of the tip-sample contact, while the average position of the tip remains in contact with the surface. The modulation is driven by supplying a designated voltage and frequency to the scanning piezo.

III. Results and Discussion 1. Basic Procedure of Particle Lithography. The procedure of particle lithography to produce protein nanostructures is illustrated in Figure 1. First, the protein and latex are mixed together in an aqueous solution (Figure 1A). For best results, the solution containing protein and latex is allowed to remain at room temperature for time intervals ranging from 10 min to 4 h. In the second step (Figure 1B), a small volume of the colloidal suspension is taken and then deposited at the center of the substrate surface, using a volumetric hand pipettor. The dropletcovered surfaces are allowed to dry in a clean and isolated environment. The volume to be deposited was chosen so that the liquid spreads across more than 90% of the surface area (∼1 × 1 cm2). The protein and latex mixture forms ordered assemblies supported by mica or Au(111). During drying (Figure 1C), the convective motion of water as it evaporates pulls the latex together into close-packed

assemblies, as has previously been described.36,38,40,41,43,55-57 After the deposits have dried, the latex is rinsed away with deionized water, resulting in periodic arrays of protein nanostructures on the substrate, shown schematically in Figure 1D. Typically, 0.5 mL of water was sufficient to completely rinse away the latex particles on 1 × 1 cm2 substrates. The assembly of latex particles and the protein nanostructures can be characterized using AFM throughout the fabrication process. For this protocol to succeed, the protein needs to exhibit strong adhesion, whereas latex needs to adhere weakly to the substrate. Further, the adhesion between the protein and latex needs to be relatively weak, in order for the latex to be released by rinsing. Hydrophobic polystyrene latex spheres are good candidates due to their relatively weak adhesion to hydrophilic mica in comparison to the proteins, which thus can be selectively removed. Another requirement is for the substrate to be flat, to minimize defect formation during the assembly of latex spheres. A few key experimental precautions follow. First, the latex particles must be prewashed (see Experimental Section) to ensure that the particles are free of a surfactant coating. Second, to maintain protein activity, the solutions should be freshly prepared. Third, the protein-to-latex particle ratio and concentrations affect the outcome of the periodicity and the morphology of the nanopatterns. 2. Periodic Arrays of BSA Nanostructures on Mica. The packing of latex particles in the presence of proteins is shown in Figure 2. The AFM topograph in Figure 2 reveals a two-dimensional close-packed structure with (55) Denkov, N. D.; Velev, O. D.; Kralchevsky, P. A.; Ivanov, I. B.; Yoshimura, H.; Nagayama, K. Langmuir 1992, 8, 3183-3190. (56) Butt, H.-J.; Gerharz, B. Langmuir 1995, 11, 1735-4741. (57) Rakers, S.; Chi, L. F.; Fuchs, H. Langmuir 1997, 13, 71217124.

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Figure 6. Applicability of nanoparticle lithography to IgG systems. [A] A 1 × 1 µm2 topograph of nanostructures of normal rabbit IgG, prepared using 503 ( 4 nm latex particles. The image was obtained in water using CRI at 39.5 kHz and an amplitude of 12 Å. The IgG/latex ratio is 15 000:1. [B] Cursor profile as indicated in (A). [C] After introduction of anti-rabbit IgG, the pattern morphology is still evident. [D] Cursor profile as indicated in (C). Figure 5. Periodic arrays of BSA nanostructures produced on Au(111) using particle lithography. [A] AFM topograph of a BSA nanopattern after 304 nm particles were removed, acquired in PBS. The BSA/latex ratio is 30 000:1. [B] A cursor profile corresponding to the line in (A).

long-range order, for latex particles of diameter 503 ( 4 nm. In the 12 × 12 µm2 area shown, only five missing particles and three phase-shifted rows are present. The observation of low defect density is typical for monodisperse latex. With a latex ratio of 65 000:1, the freshly prepared film has a measured periodicity of 603 ( 70 nm, which is slightly larger than the native diameter of the latex due to the surrounding BSA molecules. The periodicity was found to decrease with longer drying periods of the films. The long-range order and periodicity of the 2D array are maintained after removal of the latex template. Figure 3A is an optical microscopy image of BSA arrays immediately after removal of the latex template, 802 ( 6 nm in diameter. The morphology spans most of the areas covered by the films on the mica substrate, with disordered regions only at the outermost edges of the periphery. Larger areas of 2D arrays can also be achieved following a similar protocol. Figure 3A also demonstrates the high throughput nature of particle lithography. For latex particles smaller than 800 nm, AFM was employed because it has higher resolution than optical microscopes. Figure 3B shows a 12 × 12 µm2 area of an array of BSA nanostructures formed after removing a latex template made of arrays of 503 ( 4 nm particles. Under a BSA/ latex ratio of 61 000:1, particle lithography yields a large area of a 2D BSA array with the periodicity of 556 ( 65 nm. With the use of smaller latex spheres, 2D arrays of BSA with smaller periodicity can be produced. Using 304 ( 5 nm diameter latex particles as templates, the resulting

BSA nanostructures are shown in Figure 4A,B. The periodicity in this case is 335 ( 37 nm. The thickness of the protein nanostructures as measured from the cursor in Figure 4C is 3.9 ( 0.3 nm, corresponding well with the diameter of BSA (4.0 nm).58 Nanoparticle lithography also works well with latex spheres as small as 204 nm, Figure 4D-F. The cursor in Figure 4F shows a height measurement of 3.8 ( 0.5 nm. The measured periodicity is 189 ( 24 nm. The arrays of BSA nanostructures are stable under ambient conditions. In buffer solution, the stability was tested by imaging a sample immersed in PBS buffer over time. The pattern remained unchanged for the duration of the experiment, i.e., at least 16 h. 3. Periodic Arrays of BSA on Gold Surfaces. To test the generality of this method, thin films of Au(111) were used as a solid support. Gold is commonly used as a substrate for AFM sample preparation.51 Gold is a good material for electrodes in electrochemical measurements and applications, such as for electrochemical-based biosensors, because of its conductivity and chemical stability. Various protein immobilization strategies have been developed for gold surfaces to preserve protein conformation and activity.59 An array of BSA nanopatterns prepared on a Au(111) substrate is shown in Figure 5. The same BSA/latex ratios and conditions as optimized for mica were also successful with gold substrates. In the 5 × 5 µm2 area shown in Figure 5A, very few defects are present. The pattern height measures 3.0 ( 0.5 nm (Figure 5B), which is smaller than the diameter of BSA. This is likely because the AFM tip cannot reach the very bottom of the substrate at this scale. The protein array has a periodicity of 289 ( 30 nm. (58) Rosenoer, V. M.; Oratz, M.; Rothschilde, M. A. Albumin Structure and Function; Pergamon Press: New York, 1977. (59) Willner, I.; Katz, E. Angew. Chem., Int. Ed. 2000, 39, 11801213.

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Figure 7. Effect of protein/latex ratios on the structure of 2D protein arrays. [A] AFM topograph of a 2 × 2 µm2 area of a BSA assembly produced using a BSA/latex ratio of 61 000:1. [B] Cursor profile corresponding to the line in (A). [C] Schematic diagram illustrating a top and side view of the latex packing arrangement. [D] AFM topograph (2 × 2 µm2) of BSA nanostructures prepared using a ratio of 26 000:1. [E] Cursor profile corresponding to the line in (D). [F] Schematic diagram (top and side views) depicting the latex particle deformation within the 2D assembly: lateral compression and flattening.

4. Applicability of Particle Lithography to Antibodies. In addition to simple proteins such as BSA, less symmetric molecules such as IgG were tested with particle lithography. Antibodies are used in sensing and bioassays. IgG molecules exhibit a Y-shaped geometry. There are two antibody binding domains (Fab) at the ends of the Y-like fork, which are spaced 14.5 nm apart, according to X-ray diffraction studies.60,61 The tail of the Y-shaped molecule is the Fc domain, which contains one or more carbohydrate chains. The distance between the two Fab domains and the end of the Fc domain is 8.5 nm. The thickness of the molecule is 4.0 nm. Hinge regions, composed of disulfide bridges, link amino acid chains at the center of the molecule and confer additional flexibility through bending. Our method of nanoparticle patterning is directly applicable to IgG. Arrays of nanostructures of IgG are shown in Figure 6A. The nanostructures of IgG exhibit an interesting donut shape, in which the proteins are packed near the base of the spheres to form craters. The centerto-center distance between two nanocraters measures 540 nm. Similar circular morphologies have been observed previously for inorganic nanostructures formed using particle lithography.45 Diffusion in a vacuum42 or capillary forces during the drying process62 were proposed as the cause of the morphology formed from inorganic molecules depositing at the base of the latex particles. We think that the liquid motion during drying is likely the cause for the crater-shaped IgG morphology in IgG lithography. (60) Silverton, E. W.; Navia, M. A.; Davies, D. R. PNAS 1977, 74, 5140-5144. (61) Sarma, V. R.; Silverton, E. W.; Davies, D. R.; Terry, W. D. J. Biol. Chem. 1971, 246, 3753-3759. (62) Boneberg, J.; Burmeister, F.; Schafle, C.; Leiderer, P.; Reim, D.; Fery, A.; Herminghaus, S. Langmuir 1997, 13, 7080-7084.

Using the uncovered areas in the center of the nanorings as a reference, the measured height of these patterns ranged from 2.5 to 4.2 nm. The representative cursor in Figure 6B shows a height of 3.8 ( 0.6 nm. To test the activity of the immobilized proteins, a solution containing anti-rabbit IgG was introduced to the AFM sample cell. Within 2 h of incubating the IgG nanopatterns in 2.4 µg/ mL anti-rabbit IgG in PBS buffer, a height increase was observed, as shown by the areas of brighter contrast in Figure 6C and by the corresponding cursor measurement in Figure 6D. Though the image frame has shifted from the original position before the introduction of secondary antibodies, the measurement unambiguously shows an increase in height ranging from 5.0 to 10.0 nm. A representative cursor in Figure 6D shows a height of 8.2 ( 1.5 nm. The height increase is indicative of specific antibody-antigen binding after the injection of specific antibodies. The original morphology is also preserved, suggesting the spatial selectivity of this nanofabrication method, as well as the activity of the primary antibodies. Work is in progress to fully optimize the experimental conditions for more precise quantitative assessment of the changes in nanopattern dimensions and in surface coverage for antigen-antibody binding. 5. Effect of Protein-to-Particle Ratios. During latex particle lithography, proteins are deposited in the void spaces among surrounding latex particles in the 2D assembly. Thus, the periodicity of the resulting nanopatterns primarily depends on the separation of latex spheres, in most cases corresponding to the particle diameter. However, we have observed that the periodicity may be adjusted around the latex particle diameter by varying the protein/latex ratio and drying time. To avoid denaturation of the proteins, we recommend using freshly

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prepared samples. Therefore, the protein/latex ratio is the key to controlling the periodicity. In the examples shown in Figure 7, 503 ( 4 nm latex particles are used, in which the ratios of BSA/latex are 61 000:1 in Figure 7A and 29 000:1 in Figure 7D. The rationale behind the selected ratios is as follows: if all BSA molecules adsorb onto the surface of the latex particle, a 61 000:1 ratio corresponds to monolayer coverage of the sphere, while a 29 000:1 ratio is half of a monolayer. BSA molecules and latex co-deposit onto the substrate surface during drying. In Figure 7A, using a high BSA/latex ratio, a homogeneous layer of BSA is observed punctuated by pits, which were formerly occupied by the latex template. The cursor profile in Figure 7B shows a height of 3.5 ( 0.5 nm for the BSA area, in agreement with the dimensions of BSA particles. The periodicity measured 495 ( 22 nm, which corresponds well to the expected latex diameter. Thus, the array of BSA nanostructures is formed from a latex template with little deformation. The schematic diagram in Figure 7C depicts the corresponding closely packed arrangement of spheres and the void spaces occupied by BSA molecules. At the lower BSA/latex ratio of 29 000:1, a honeycomb morphology is observed as shown in Figure 7D. The periodicity, as more quantitatively shown in the cursor profile (Figure 7E), is measured to be 407 ( 35 nm. This periodicity is 20% smaller than the latex diameter. The observed periodicity and the hexagonal shape are rationalized by the deformation of latex spheres upon deposition on mica. The deformation of latex particles has been observed previously.63-67 Spherical latex particles are (63) Routh, A. F.; Russel, W. B. Ind. Eng. Chem. Res. 2001, 40, 43024308. (64) Dobler, F.; Pith, T.; Lambla, M.; Holl, Y. J. Colloid Interface Sci. 1992, 152, 1-11. (65) Sperry, P. R.; Snyder, B. S.; O’Dowd, M. L.; Lesko, P. M. Langmuir 1994, 10, 2619-2628. (66) Tzitzinou, A.; Keddie, J. L.; Geurts, J. M.; Peters, A. C. I. A.; Satguru, R. Macromolecules 2000, 33, 2695-2708. (67) Lin, F.; Meier, D. J. Langmuir 1995, 11, 2726-2733.

Garno et al.

closely packed and then are compressed along the lateral direction, resulting in a periodicity smaller than the expected 503 ( 4 nm diameter. The area in contact with mica may also undergo deformation, such as, flattening. The morphology of the pattern is thus hexagonal, rather than circular. The height of the nanostructures measures 4.1 ( 0.5 nm (Figure 7E), corresponding to a BSA monolayer, and the nanopattern boundaries are narrower than that in Figure 7A. Figure 7F depicts the possible deformation of latex particles within the initial 2D assembly. IV. Conclusion Particle lithography using monodisperse latex nanoparticles can produce highly organized, periodic arrays of protein nanostructures. The advantages of this approach include its simplicity, mild environments (near-physiological conditions and ambient temperatures), and high throughput. The removal of latex templates is rapid and complete. The heights of protein nanostructures typically correspond to the dimensions of single protein molecules. The detailed morphology and periodicity of protein nanostructures are determined by the latex diameter and the protein/particle ratio. Arrays of protein nanopatterns are produced on both Au(111) and mica substrates, using sphere sizes ranging from 200 to 800 nm. These patterned protein arrays maintain their activity. Work is in progress to investigate if this method can be developed into a more generic patterning method for other proteins or substrates, to systematically test the reactivity of the patterned proteins, and to further reduce the size. Acknowledgment. The authors thank the University of California, Davis for financial support. Partial support for J.C.G. and N.A.A. was provided by the National Science Foundation (IGERT-970952 and IGERT-9972741). We also thank Maozi Liu, Guohua Yang, William Price, James Benigna, and Adrienne Brown for helpful discussions. LA020518B