178
Langmuir 2001, 17, 178-182
Bioactive Templates Fabricated by Low-Energy Electron Beam Lithography of Self-Assembled Monolayers C. K. Harnett,* K. M. Satyalakshmi, and H. G. Craighead School of Applied and Engineering Physics, Cornell University, Ithaca, New York 14853 Received July 6, 2000. In Final Form: September 28, 2000 Adhesive templates for biomolecule patterning were fabricated on silicon and gold by low-voltage (1 kV) electron beam lithography of an inert self-assembled monolayer, followed by backfilling the exposed regions with an amine-terminated monolayer. Amine-terminated monolayers selectively attached either the desired materials or linker molecules that subsequently bound other materials including antibodies. Lines (300 nm wide) of 20-nm polystyrene beads were formed on gold by exposing a mercaptohexadecanoic acid (MHDA) monolayer, then backfilling with cysteamine, and selectively attaching aldehyde-coated beads to the amines. Attachment density was found to vary sharply around a critical dose, making the technique useful for patterns such as gradients which require varying density. An optimal dose of 200 µC/cm2 was found for attaching fluorescent polystyrene spheres to MHDA-cysteamine templates. A cycling process was developed for aligning patterns of two or more kinds of polystyrene spheres. Biotin was tethered to the amine templates, making the technique applicable to high-resolution patterning of biomaterials with the widely used avidin-biotin binding system.
Introduction Self-assembled monolayer (SAM) surfaces have been intensively studied as surface coatings for nanofabrication techniques because they can be tailored to promote or resist the adhesion of specific substances at the molecular level. Applications of patterned reactive monolayers in device fabrication include metal deposition and plating for conductive paths,1 deposition of semiconductor particles for photonic devices,2 and patterning carbon nanotubes for electronic devices.3 SAMs have already seen commercial success in biotechnology.4 Biological applications for cell-based experiments include monolayer patterning for control of neuron adhesion,5 patterning of single cell adhesive regions for studying programmed cell death,6 and controlled placement of multiple cell types to study cell interactions.7 Biological applications on the molecular scale include antibody assays created by tethering antibodies to monolayers at known positions8 and controlled wetting of patterned monolayer surfaces to create microfluidic reaction volumes.9 High-resolution patterning of biological materials will lead to further miniaturization of multianalyte biological assays and better control over the chemical environment of surface-bound cells. As features approach the size (1) Brandow, S. L.; Chen, M. S.; Wang, T.; Dulcey, C. S.; Calvert, J. M.; Bohland, J. F.; Calabrese, G. S.; Dressick, W. J. J. Electrochem. Soc. 1997, 144, 3425-3434. (2) Vossmeyer, T.; Jia, S.; Delonno, E.; Diehl, M. R.; Kim, S. H.; Peng, X.; Alivisatos, A. P.; Heath, J. R. J. Appl. Phys. 1998, 84, 3664-3670. (3) Liu, J.; Casavant, M. J.; Cox, M.; Walters, D. A.; Boul, P.; Lu, W.; Rimberg, A. J.; Smith, K. A.; Colbert, D. T.; Smalley, R. E. Chem. Phys. Lett. 1999, 303, 125-129. (4) Fodor, S.; Read, J. L.; Pirrung, M. C.; Stryer, L.; Tsai L. A.; Solas, D. Science 1991, 251, 767-773. (5) James, C. D.; Davis, R. C.; Meyer, M.; Perez, A.; Turner, S.; Withers, L.; Kam, L.; Banker, G. L.; Craighead, H. G.; Isaacson, M.; Turner, J. N.; Shain, W. IEEE Trans. Biomed. Eng. 2000, 47, 17. (6) Chen, C. S.; Mrksich, M.; Huang, S.; Whitesides, G. M.; Ingber, D. E. Science 1997, 276, 1425-1428. (7) Bhatia, S. N.; Yarmush, M. L.; Toner, M. J. Biomed. Mater. Res. 1997, 34, 189-199. (8) Pritchard, D. J.; Morgan, H.; Cooper, J. M. Anal. Chem. 1995, 67, 3605-3607. (9) Gau, H.; Herminghaus, S.; Lenz, P.; Lipowsky, R. Science 1999, 283, 46-49.
regime of single protein molecules (less than 10 nm), the possibility exists of using monolayer templates to position individual macromolecules such as molecular motors.10 Precise pattern registration will enable the creation of aligned multichemical patterns and well-defined active areas on nanoelectromechanical sensors.11 Motivated by these possibilities, we have developed new methods of patterning biomaterials on monolayers using electron beam lithography. This research stems from our efforts to develop chemical systems compatible with lowenergy electron beam lithography. Energies below 2 kV will be compatible with microfabricated electron beam column arrays designed to increase throughput by exposing multiple small patterns in parallel.12 Sub-2 kV accelerating voltages turn out to be especially compatible with self-assembled monolayers because low voltage electrons deposit energy efficiently in the top surface of a material, exposing the thin monolayer without expending excess energy in the substrate. Electron beam lithography is capable of producing high-resolution patterns, and since the technology is commonly used in semiconductor manufacturing, robust alignment processes have been developed. Features of 6 nm width have been reported for electron beam lithography on SAMs.13 Like photolithography, electron beam lithography does not require direct substrate contact, which gives it an advantage over microcontact printing or scanned probe lithography techniques when patterning monolayers on surfaces with deep channels or other difficult-to-reach features. A littleexplored advantage of electron beam lithography over photolithography of monolayers is the possibility of controlling the density of monolayer functional groups by varying the exposure dose within a single pattern. (10) Montemagno, C. D.; Bachand, G. Nanotechnology 1999, 10, 225231. (11) Baselt, D. R.; Lee, G. U.; Hansen, K. M.; Chrisey, L. A.; Colton, R. J. Proc. IEEE 1997, 85, 672-680. (12) Chang, T. H. P.; Thomson, M. G. R.; Kratschmer, E.; Kim, H. S.; Yu, M. L.; Lee, K. Y.; Rishton, S. A.; Hussey, B. W.; Zolgharnain, S. J. Vac. Sci. Technol., B 1996, 14, 3774-3781. (13) Lercel, M. J.; Whelan, C. S.; Craighead, H. G. J. Vac. Sci. Technol., B 1996, 14, 4085-4090.
10.1021/la0009543 CCC: $20.00 © 2001 American Chemical Society Published on Web 12/07/2000
Bioactive Templates
Langmuir, Vol. 17, No. 1, 2001 179
In this research, templates were created by exposing an inert monolayer and then “backfilling” the exposed, cleaned areas with a reactive monolayer which selectively attached biotin-terminated linker molecules or polystyrene spheres. These spheres are highly fluorescent, may be functionalized with chemicals including proteins, and are produced in diameters from 20 nm to over 20 µm, making them good test materials for cell adhesion applications. The tone reversal inherent in the backfilling process takes advantage of electron beam lithography’s speed at writing high-resolution single-pass lines and dots, rather than requiring large filled areas to be written around small features. Applications of such structures include wires and microreaction sites. Previous work in fabricating adhesive templates through electron-beam lithography includes direct electron beam exposure of amine-terminated monolayers14 and backfilling with a reactive monolayer to pattern carbon nanotubes.3 Non-electron-beam backfilling work includes scanned probe lithography of monolayers,15,16 backfilling photopatterned hydrophobic monolayers with amines for the creation of DNA arrays,17 and the routine use of backfilling to functionalize unprinted areas in microcontact printing. We have focused on using the strengths of electron beam lithography to develop techniques for organizing biomaterials into patterns such as gratings, gradients, and multiple aligned layers. These types of patterns can take advantage of the high-resolution, variable dose, and alignment accuracy of the electron beam and are essential for many experiments in surface-based chemical and biological analysis. Experimental Section Overview. All samples described below were created on silicon or gold by writing a low-energy electron beam pattern in a nonreactive monolayer with a 16- to 18-carbon backbone. Figure 1a depicts the template-forming process. Electron beam exposure breaks the carbon chain, creating organic residue which is removed by UV ozone cleaning. In the backfilling step, the sample was immersed in a dilute solution of amine-terminated molecules which formed a monolayer in the cleaned regions. Finally, the sample was immersed in a solution of coated particles or biomolecules which preferentially adhere to the amine surface. Nonreactive monolayers which did not adhere to the materials we have chosen to pattern are shown in parts b-d of Figure 1, and amine-terminated monolayers are shown in parts e and f of Figure 1. Monolayer Surface Preparation. Gold surfaces were prepared by thermal or electron-beam evaporation of a 100200-nm gold layer onto a silicon wafer. Before monolayer deposition, silicon and gold substrates were cleaned by acetone and 2-propanol rinses, dried under nitrogen, and treated in a UV ozone cleaner (UV Ozone Cleaning Systems, Inc.) for 20 min. Monolayers of mercaptohexadecanoic acid (MHDA, Figure 1c, Aldrich Chemical) or octadecanethiol (ODT, Figure 1b, Aldrich Chemical) were formed on gold by immersing the sample overnight in a 1 mM ethanolic solution of the monolayer compound. Afterward, the sample was rinsed with ethanol and dried with nitrogen gas. All monolayer compounds in this work were used as received without further purification. For deposition of octadecyltrichlorosilane (OTS, Figure 1b, Aldrich Chemical) on silicon, the cleaned silicon wafer was first boiled in distilled and deionized (DI) water for 5 min to hydrogen-terminate the (14) Harnett, C. K.; Satyalakshmi, K. M.; Craighead, H. G. Appl. Phys. Lett. 2000, 76, 2466-2468. (15) Sugimura, H.; Nakagiri, N. J. Vac. Sci. Technol., B 1997, 15, 1394-1397. (16) Wadu-Mesthrige, K.; Xu, S.; Amro, N. A.; Liu, G.-Y. Langmuir 1999, 15, 8580-8583. (17) Brockman, J. M.; Frutos, A. G.; Corn, R. M. J. Am. Chem. Soc. 1999, 121, 8044-8051.
Figure 1. (a) Monolayer backfilling process for gold and Si substrates, (b, c, d) inert monolayers OTS (on Si), ODT (on Au), and MHDA (on Au), (e, f) amine-terminated monolayers APTS (on Si) and cysteamine (on Au), and (g, h) LC-NHS-biotin and sulfo-LC-NHS-biotin molecules for linking biotin to amine surfaces. surface and then dried under nitrogen gas. The wafer was left overnight in a 1 mM OTS solution in 80% hexadecane and 20% chloroform under a nitrogen atmosphere. The sample was rinsed with chloroform and 2-propanol and dried with nitrogen. Both Si and gold samples were annealed 5 min on a 120 °C hotplate after drying. Prepared monolayer surfaces on both Si and gold remained usable for at least 1 month. Electron Beam Exposure and Development. Electron beam exposures were conducted in a scanning electron microscope (LEO Electron Microscopy, Inc.) equipped with a pattern generator and alignment system (JC Nabity Lithography Systems). For multiexposure patterns, gold alignment marks were produced outside the pattern area using photolithography. Patterns were typically exposed at 1 kV accelerating voltage and doses of 5-500 µC/cm2. After exposure, samples were developed for 45 s in the UV ozone cleaner. Water contact-angle studies have shown that an ozone exposure dose sufficient to remove electron-damaged monolayers does not significantly reduce the hydrophobicity of intact methyl-terminated monolayers.18 Exposed patterns could be kept safely for several days before development but were typically developed within 30 min of electron beam exposure. Backfilling with Amine-Terminated Monolayers. Immediately after development, samples were immersed in dilute solutions of amine-terminated monolayers. Gold samples were immersed in a freshly prepared 10 mM water-based solution of (18) Lercel, M. J. Ph.D. Thesis, Cornell University, Ithaca, NY, 1995.
180
Langmuir, Vol. 17, No. 1, 2001
cysteamine (Figure 1f, Fluka BioChemika) for 2 h and then sonicated for 3 min in water in a Branson ultrasonic cleaner to remove excess cysteamine. Ethanol-based solutions containing 1 mM cysteamine were also successfully used with the same process. However, the water-based solution was required for multiexposure experiments with polystyrene spheres because ethanol leaches fluorescent dye from them. Si surfaces were backfilled with (3-aminopropyl)triethoxysilane (APTS, Figure 1e, Pierce) to amine-terminate the exposed regions according to a procedure previously established for functionalizing large areas.19 Developed samples were boiled in DI water for 5 min to hydrogen-terminate the surface and then without drying were immersed for 15 min in a methanol-based solution containing 2% APTS, 4% acetic acid, and 4% water (all percentages by volume). Samples were then rinsed with methanol and dried with nitrogen gas. To avoid contamination of the freshly created reactive surfaces, amine-backfilled samples were immersed in the next solution as soon as possible. Attaching Polystyrene Beads to Amines. Amine-backfilled samples were left for 2 h in solutions consisting of 5 µL fluorescent beads (aldehyde-coated beads, 2% solids, carboxylic acid coated beads, 2-5% solids, Molecular Probes, Inc) and 1 mL of 10 mM pH 6 MES buffer (morpholineethanesulfonic acid, Acros Organics). Nominal bead diameters were 20, 40, and 100 nm. Bead solutions were freshly prepared each time and sonicated for 3 min before use. Clusters were removed from solutions of smaller beads (40 nm or less) by centrifuging the solution at 10000g for 5 min and then using only the beads still suspended in solution. If templates were to be repatterned for attachment of another set of materials, excess amines were quenched by immersion for 1 h in a 10 mM aqueous solution of sulfo-NHS-acetate (Molecular BioSciences) before the next exposure step to avoid attaching subsequent beads to unreacted areas in the previous pattern. Linking Materials to Surface Amines with Biotin. Biotinterminated regions were created on the amine surfaces using an amine-reactive NHS ester linked to biotin by a carbon chain. Solutions consisted of 5 mg/mL succinimidyl-6-(biotinamido)hexanoate (LC-NHS-biotin, Figure 1g, Molecular Biosciences) in dimethyl sulfoxide (DMSO) or 10 mg/mL sulfosuccinimidyl6-(biotinamido)hexanoate (Ez-Link sulfo-LC-NHS-biotin, Figure 1h, Pierce) in water. The water-based solution was necessary for multiply exposed samples containing previously patterned polystyrene spheres, which dissolve in DMSO. Samples were immersed in the biotin solution for 20 min, rinsed in DMSO and water (for DMSO-based biotin solution) or water (for aqueous biotin solution), and were not allowed to dry before reacting with the next solution. For attaching fluorescent polystyrene beads to the biotin regions, 5 µL of 40-nm diameter NeutrAvidin beads (1% solids, from Molecular Probes, Inc.) was mixed with 1 mL BlockAid buffer (Molecular Probes, Inc). A 30-µL drop was left on the biotin pattern for 1 h without covering, after which the liquid was viscous but not dry. The surface was then rinsed with large amounts of water and dried with nitrogen gas. Alternatively, fluorescent mouse anti-biotin IgG1 (Molecular Probes, Inc.) was attached. A 20 µg/mL anti-biotin solution was prepared with 150 mM phosphate-buffered saline (PBS, pH 7.2, from Sigma Diagnostics), 0.1% bovine serum albumin (BSA, 10% solution in PBS from Pierce), and 0.01% Tween 20 (Lancaster Synthesis). A 30-µL drop was left on the biotin pattern for 1 h with a cover and then rinsed in the PBS/BSA/Tween solution, water rinsed, and dried with nitrogen gas. Fluorescence Microscopy. Fluorescent bead and anti-biotin patterns were observed in a Zeiss Axiotron optical microscope equipped with a mercury arc lamp and filter sets for red and green fluorescent dyes (Omega Optical). Images were collected by a Spot CCD camera (Diagnostic Instruments, Inc.)
Harnett et al.
Figure 2. Fluorescence image of 250 nm wide single-pass lines in MHDA on gold, backfilled with cysteamine and immersed in 20-nm green aldehyde-functionalized polystyrene beads. Exposure conditions: 1 kV accelerating voltage; 1.5 nC/cm electron dose.
cysteamine, and immersed in a solution of aldehyde-coated 20 nm fluorescent polystyrene beads which adhere to amines on the surface (Figure 2). No contrast was visible in similar samples that were not backfilled with cysteamine. Exposure conditions for the sample in Figure 2 were 1 kV accelerating voltage and a dose of 1.5 nC/cm. The measured line width of 300 nm, which approaches the diffraction limit of our optical microscope, is comparable to atomic force microscopy (AFM) measurements of 250 nm on similar samples. Factors affecting the line width are primarily electron beam spot size at 1 kV and also secondary electron exposure at the edges of exposed areas and the grain size of attached materials. Attaching 500nm diameter aldehyde-coated spheres to a similar sample generated enough surface height variation to produce a diffraction pattern from a 10 mW helium-neon laser beam. Because we will show that the backfilled amine regions can be functionalized with biotin, which can bind antibodies that interact with specific types of cells,20 the polystyrene spheres in this experiment may be considered a model for cell patterning. Applications of cell adhesion to single pass lines include diffraction-based cell sensors21 and adhesive/nonadhesive regions of varying separation to study cell mobility across barriers22 for cells on the scale of 1 µm, such as bacteria. Variable Density Patterns. Another type of pattern possible with electron beam lithography is a gradient or other controlled-density pattern. Figure 3a shows a gradient pattern produced by exposing 1-µm pixels in a MHDA monolayer on gold, backfilling with cysteamine, and attaching 40-nm red carboxylic acid beads to the amine-terminated regions. The pattern contains a high density of pixels where a high bead density is desired and few pixels in low-density areas. All exposed pixels were written at 1 kV accelerating voltage with a dose of 200 µC/cm2. This “binary” method for producing a gradient can also be accomplished with mask-based UV photolithography, but the higher ultimate resolution of the electron beam can produce a more continuous pattern composed of smaller pixels. By use of alignment marks, the sample may be reexposed and other chemicals attached
Results and Discussion Single Pass Lines. A pattern of single pass lines was written in a MHDA monolayer on gold, backfilled with (19) Dulcey, C. S.; Georger, J. H.; Krauthammer, V.; Stenger, D. A.; Fare, T. L.; Calvert, J. M. Science 1991, 252, 551-554.
(20) Lahiri, J.; Ostuni, E.; Whitesides, G. M. Langmuir 1999, 15, 2055-2060. (21) St. John, P. M.; Davis, R.; Cady, N.; Czajka, J.; Batt, C. A.; Craighead, H. G. Anal. Chem. 1998, 70, 1108-1111. (22) Bailly, M.; Yan, L.; Whitesides, G. M.; Condeelis, J. S.; Segall, J. E. Exp. Cell Res. 1998, 241, 285-299.
Bioactive Templates
Langmuir, Vol. 17, No. 1, 2001 181
Figure 4. Red and green fluorescent carboxylic acid coated polystyrene spheres attached to cysteamine-filled exposed regions of a ODT/Au monolayer. Red beads were patterned and then sent back to the electron beam where a green pattern was aligned and written. Excess fluorescent beads were deactivated by writing a third time in the black areas.
Figure 3. (a) Gradient pattern created with 40 nm diameter carboxylic acid coated spheres attached to cysteamine-backfilled areas on a MHDA/Au monolayer. Pattern consists of a dithered arrangement of 1-µm square pixels. Exposure conditions: 1 kV, 200 µC/cm2. (b) Study of variation of sphere attachment density with exposure dose. 100 nm diameter carboxylic acid coated spheres were attached to cysteamine-backfilled areas on a MHDA/Au monolayer. Exposure conditions: 1 kV, 5-500 µC/cm2. (c) Surface density of 100 nm spheres on cysteamine patterns in MHDA/Au vs exposure dose.
to the unfunctionalized pixels of the first pattern, creating a gradation from one chemical to another. Electron beam lithography differs from conventional UV lithography in that it is maskless and the dose may be varied throughout the pattern. The closest light-based analogue is scanned excimer laser lithography with a spot size of approximately 200 nm, or about 10 times that of an optimized low-energy electron beam lithography system. The behavior of attached materials on electron beam patterns exposed at different doses suggests an alternative to binary patterning. Figure 3b shows a series of dose bars patterned in a MHDA monolayer and then backfilled with cysteamine and 100-nm green carboxylic acid beads. Exposure conditions were 1 kV accelerating voltage and doses of 5 500 µC/cm2. Figure 3c plots the bead surface density vs electron dose for these exposures. Bead surface density rises steeply for doses between 50 and 200 µC/cm2 and then decreases slowly. Similar exposure characteristics were noted for OTS and ODT monolayers. The steep increase with dose occurs around the critical dose of approximately 100 µC/cm2 for removal of the MHDA monolayer, while the slow dropoff may be due to monolayer cross-linking or carbon contamination
of the surface at higher doses, which prevents cysteamine adsorption. Translating desired surface particle densities into electron-beam doses will allow-density gradients to be produced with a much simpler datafile and more continuous surface density than the binary pixel technique. Applications of surface gradients include chemoattractantpatterned surfaces for studying chemotaxis and cell immobilization22,23 and surfaces with precisely defined wetting characteristics for control of fluids on microscopic scales.24 Multiple Adhesion Cycles. A cycling process was developed for patterning two or more sets of polystyrene beads. The first pattern was aligned with marks on a ODT/ gold sample, written at 1 kV and 200 µC/cm2, backfilled with cysteamine, and immersed in 40 nm diameter red carboxylic acid coated beads. Excess amine groups remaining in the red areas were quenched with sulfo-NHSacetate, and the sample was realigned in the electron beam system. The second pattern was processed similarly and reacted with green carboxylic acid coated beads. In the fluorescence microscope, the red and green pattern appeared along with a background glow from beads sparsely distributed in the unexposed, “inert” ODT regions. One solution to this problem would be to use a different inert monolayer such as the carboxylic acid-terminated MHDA, which does not adhere carboxylic acid beads well, as seen in Figure 3b. However, during previous experiments, electron beam exposure of fluorescent spheres was observed to permanently quench their fluorescence. The sample was aligned a third time, and the areas surrounding the patterned beads were exposed at the same electron energy and dose as the first two patterns, decreasing the background signal significantly. This deactivation strategy is also likely to work with biological molecules that adhere nonspecifically to templates. Figure 4 is a superposition of red- and green-filtered images of this sample. Patterns consisting of multiple materials have wide-ranging applications in multiplexed chemical and cell-based sensors. However, materials must be robust to survive the cycling process described here, which does not involve harsh solvents as in typical resist-based processes but does (23) Herbert, C. B.; McLernon, T. L.; Hypolite, C. L.; Adams, D. N.; Pikus, L.; Huang, C.-C.; Fields, G. B.; Letorneau, P. C.; Distefano, M. D.; Hu, W.-S.; Chem. Biol. 1997, 4, 731-737. (24) Chaudhury, M. K.; Whitesides, G. M. Science 1992, 256, 15391541.
182
Langmuir, Vol. 17, No. 1, 2001
Harnett et al.
1 kV accelerating voltage and a dose of 1.5 nC/cm. The fluorescent beads formed lines of submicrometer width on the exposed regions. Without biotin, the avidin-coated spheres exhibited some preference for the amine-filled areas, but pattern edges were poorly defined and adhesion was sparse. Pattern tone was observed to be dependent upon contact time with the beads. For short durations (
