Anal. Chem. 2004, 76, 3861-3863
Plastic Tip Arrays for Force Spectroscopy Peter T. Lillehei, Mark A. Poggi, Brian J. Polk, J. Anthony Smith, and Lawrence A. Bottomley*
School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, Georgia 30332-0400
The mechanical stability and viability of molecules investigated with the atomic force microscope (AFM) continue to be limiting factors in the duration of force spectroscopy measurements. In an effort to circumvent this problem, we have fabricated an all-plastic array of over 30 000 tips with dimensions similar to common AFM probes using silicon micromolding techniques. This approach enables rapid fabrication of tip arrays with improved properties, as compared to tip arrays made entirely of silicon. The invention of the atomic force microscope1 (AFM) has led to a revolution in imaging and characterization of biomolecules. Submolecular-scale images of biomolecules and biomolecular assemblies can now be obtained in air, in a vacuum, or in liquid. For example, Bustamante and co-workers2 obtained high-resolution images of the diffusion of Escherichia coli RNA polymerase along DNA. Their results provided new insight into the mechanism by which RNA polymerase searches for the promoter, a key step in transcription. In force spectroscopy, an AFM is used as a surface forces apparatus, and mechanical measurements on single molecules are made. In these measurements, a single molecule is attached to both the probe tip and an opposing surface. A tensile force is applied to the molecule by retraction of the surface by the piezo scanner. Simultaneous monitoring of cantilever deflection affords measurement of the molecule’s response to the applied load. The mechanical properties of titin and other proteins,3-6 DNA,7-9 polysaccharides,10-12 and several polymers13,14 have been determined using this approach. * Corresponding author. Phone: 404-894-4014. Fax: 404-894-7452. E-mail:
[email protected]. (1) Binnig, G.; Rohrer, H.; Gerber, C. Phys. Rev. Lett. 1982, 49, 57. (2) Bustamante, C.; Guthold, M.; Zhu, X.; Yang, G. J. Biol. Chem 1999, 274, 16665-16668. (3) Best, R. B.; Clarke, J. Chem. Commun. (Cambridge) 2002, 183-192. (4) Fotiadis, D.; Scheuring, S.; Muller, S. A.; Engel, A.; Muller, D. J. Micron 2002, 33, 385-397. (5) Becker, N.; Oroudjev, E.; Mutz, S.; Cleveland, J. P.; Hansma, P. K.; Hayashi, C. Y.; Makarov, D. E.; Hansma, H. G. Nat. Mat. 2003, 2, 278-283. (6) Hugel, T.; Seitz, M. Macromol. Rapid Commun. 2001, 22, 989-1016. (7) Krautbauer, R.; Pope, L. H.; Schrader, T. E.; Allen, S.; Gaub, H. E. FEBS Lett. 2002, 510, 154-158. (8) Krautbauer, R.; Rief, M.; Gaub, H. E. Nano Lett. 2003, 3, 493-496. (9) Williams, M. C.; Rouzina, I. Curr. Opin. Struct. Biol. 2002, 12, 330-336. (10) Zhang, W.; Zhang, X. Prog. Polym. Sci. 2003, 28, 1271-1295. (11) Rief, M.; Grubmuller, H. ChemPhysChem 2002, 3, 255-261. (12) Seog, J.; Dean, D.; Plaas, A. H. K.; Wong-Palms, S.; Grodzinsky, A. J.; Ortiz, C. Macromolecules 2002, 35, 5601-5615. (13) Merkel, R. Phys. Rep. 2001, 346, 343-385. (14) Hugel, T.; Holland, N. B.; Cattani, A.; Moroder, L.; Seitz, M.; Gaub, H. E. Science (Washington, D.C.) 2002, 296, 1103-1106. 10.1021/ac035226+ CCC: $27.50 Published on Web 05/07/2004
© 2004 American Chemical Society
Performing repetitive measurements with conventional probes remains problematic. Oftentimes, force spectroscopic experiments are cut short because immobilized molecules are damaged or lose activity.15 Mechanical or chemical degradation of molecules on the tip requires the exchange of the AFM probe and, therefore, exposure of the substrate to the environment. Green and coworkers16 utilized tip arrays in conjunction with tipless cantilevers to circumvent this problem. When a tip loses functionality, a simple translation of the cantilever to a new tip in the array permits experiments to continue. Since the contact area of the tip is small, the number of possible fresh interaction sites with a tipless cantilever is very large. The tip array fabrication method used by Green et al. was subtractive.16 Briefly, a 200-250-nm-thick protective layer of oxide was grown on a silicon (100) wafer and lithographically patterned. The tips were created with an anisotropic etch that undercut the oxide caps. Repetitive microscopic examination of tips during processing was required to attain the desired shape. In this Technical Note, we present a method for fabricating tip arrays out of the photodefinable epoxy SU-8. Our method, schematically illustrated in Figure 1, utilizes silicon micromolding techniques to define the shape of the tip; arrays of sharp tips are produced in a single step. SU-8 when cured is chemically inert; resistant to solvents, acids, and bases; mechanically robust; and optically transparent. These properties render SU-8 a suitable material for tip array applications. When compared with tip arrays made from silicon, arrays fabricated from SU-8 require fewer processing steps and cost less per array. In addition, literally hundreds of tip arrays can be produced from a single mold. EXPERIMENTAL SECTION The fabrication procedure is as follows: SiO2 or Si3N4 was thermally grown (over 1 µm thick) onto the polished surface of a single crystal Si (100) wafer. A thin film of negative tone photoresist (Shipley 1827) was applied, and an array of squares was lithographically patterned onto the resist (Karl Suss MA-6 Mask Aligner). Following development, anisotropic etching preferentially along the {111} planes of the exposed Si surface in aqueous KOH (Fisher Scientific, ACS Grade) (10 wt % in water, 65 °C, 30 min) yielded square pyramidal voids in the silicon wafer. These voids served as the mold for defining the shape of the plastic tips. Nano SU-8 photoresist (Formulation 25, MicroChem (15) Green, J. B. D.; Novoradovsky, A.; Lee, G. U. Langmuir 1999, 15, 238243. (16) Green, J.-B. D.; Novoradovsky, A.; Lee, G. U.; Park, D. Appl. Phys. Lett. 1999, 74, 1489-1491.
Analytical Chemistry, Vol. 76, No. 13, July 1, 2004 3861
Figure 1. Fabrication scheme for plastic tip array by silicon micromolding.
Corp.) was then applied over the wafer by spin-coating and heated to facilitate migration into the mold. Two or more applications of the SU-8 were necessary to build up the structure of the polymer (1-1.5 mm in total thickness). The sample was then prebaked at 95 °C for 24 h (Blue M Gravity Oven). The SU-8 was then exposed to UV light at 365 nm (total dosage of 12 000 mJ/cm2) followed by a prebake for 30 min at 95 °C and then hard bake/cure for 30 min at 150 °C to further cross-link the polymer. The SU-8 tip array was released by placing the wafer into a Si etching solution of HNO3/CH3COOH/HF (volume ratio 2/1/1) to dissolve the mold. This procedure yields the sharpest tips but also destroys the mold. Alternatively, a thin film of aluminum (∼50 nm) can be evaporated onto the mold prior to the application of SU-8. Following UV exposure and thermal curing of the photodefinable epoxy, release of the tip array from the mold is readily achieved by dissolving the aluminum layer in H3PO4/CH3COOH/HNO3/H2O (volume ratio: 19/1/1/2). While this alternative method enables reuse of the mold, tip sharpness is diminished. Gold-coated tips were prepared by e-beam evaporation (CVC Products Inc., Rochester, NY). Chromium or titanium underlayers were used to promote strong adhesion of the gold to the SU-8. Typical coatings were 30 nm of either chromium or titanium, followed by 100 nm of gold; both metal layers were deposited at 3 Å s-1 at a processing pressure below 2 × 10-6 Torr. RESULTS AND DISCUSSION Tip arrays were characterized by optical and scanning electron microscopy. In 1 cm2, we have fabricated more than 30 000 tips, which is sufficient for the most demanding combinatorial experiments.17 Figure 2 presents optical micrographs of the mold prior 3862 Analytical Chemistry, Vol. 76, No. 13, July 1, 2004
Figure 2. Optical micrograph of the tip array mold prior to deposition of SU-8. Scale bar is (a) 10 µm and (b) 200 µm.
to deposition of the SU-8. The edges of the pyramidal voids are clearly visible in Figure 2a. An indication of the sheer number of tips is given in Figure 2b. Both images are in a false color to provide better contrast. Figure 3 presents scanning electron micrographs of the tip array after release. These tips are coated with ∼100 nm of gold to enable stable imaging in the SEM. The radius of curvature of gold-coated tips ranged from 50 to 100 nm. Although this range is somewhat larger than conventional AFM probes, they are suitable for force spectroscopy measurements. The average height of each tip was 7.0 um. Figure 4 displays sequential force measurements made on a single tip. In this experiment, a mixed monolayer of 99:1 of 6-mercaptohexanol/11-dithio-bis(succinimidylundecanoate) (DSU) was self-assembled onto the surface of both a gold-coated tip array and a gold-coated tipless cantilever. A single-stranded DNA molecule (100 nts) with the 5′ terminus labeled with a hexylamine group and the 3′ terminus labeled with biotin was covalently anchored onto the tip array through a peptide linkage involving the NHS group on DSU. Streptavidin was anchored to the cantilever through exposed amino groups (lysine) on its periphery in a similar manner as ssDNA. When the two surfaces immersed (17) Henderson, E. R.; Mosher, C. L.; Jones, V. W.; Green, J. D.; Porter, M. D. U.S. Patent 5763768; 1998.
Figure 4. Overlay of 57 force curves acquired on a single tip in the array. Yellow traces indicate approach of the tip array to the cantilever; black traces indicate retraction of the tip array.
the restoring force of the cantilever exceeded the binding force between biotin and streptavidin. Sequential rupture events acquired on the same tip suggest that streptavidin rapidly refolds after force-induced unfolding and that the refolded form retains its ability to bind biotin. These data also suggest that repetitive, force-induced separation measurements are facilitated using the tip array. CONCLUSION The fabrication of a tip array entirely of photodefined epoxy allows for the rapid manufacture of the tip arrays for force spectroscopy measurements. The array presented herein consists of highly periodic, sharp, optical semitransparent tips that are mechanically robust and chemically inert. The micromolding technique used herein produced transparent tip arrays that are suitable for use in AFMs mounted on optical microscopes. This technique could be used to fabricate tips arrays of unique dimensions and shapes, if desired. Similarly, the surface chemistry of the tip can be varied by either oxidation of the SU-8 or evaporation of a coating layer to achieve desired reactivity. For example, transparent oxide surfaces can be obtained by overcoating with a thin layer of chemical vapor deposited SiO2.
Figure 3. Scanning electron micrographs of gold-coated SU-8 tip array. Scale bars: (a) 100 µm; (b) 2.5 µm.
in aqueous buffer were brought into contact, the biotin inserted into the streptavidin-binding pocket effectively anchoring the ssDNA molecule to both surfaces. Retraction of the tip array from the cantilever stretched both the DNA and the streptavidin until
ACKNOWLEDGMENT We thank Drs. Alexey Novarodovsky (Stratagene, Inc.) and John-Bruce Green (University of Alberta) for helpful discussions and acknowledge financial support by NIH (Grant No. 5 R21 EB 737-02). Received for review October 16, 2003. Accepted March 26, 2004. AC035226+
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