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Atomic Force Microscopy with Patterned Cantilevers and Tip Arrays: Force Measurements with Chemical Arrays John-Bruce D. Green Department of Chemistry and Biochemistry, Southern Illinois University, Carbondale, Illinois 62901-4409
Gil U Lee* Chemistry Division, Code 6177, Naval Research Laboratory, Washington, D.C. 20375-5342 Received November 23, 1999 Interactions between molecules and at interfaces are vital to many scientific and technological fields. Techniques such as atomic force microscopy (AFM) have been used to measure forces and force gradients associated with interactions between individual molecules as well as interactions between interfaces. A recent alteration of the AFM configuration combined a tipless cantilever with an array of substrate supported tips. Herein, we present a further extension of AFM force measurement capabilities, by chemically patterning both the cantilever and the tip array. In a proof-of-concept experiment a gold-coated cantilever and tip array were patterning with alkylthiolate monolayers, and the interfacial forces were measured for the various combinations. This patterning allows many different interactions to be rapidly measured in situ, under identical conditions, thereby improving reliability and opening the door to combinatorial applications. Future developments are discussed including the means to measure hundreds of different interactions.
I. Introduction Many scientific and technological developments depend on intermolecular interactions, often at interfaces. In an effort to understand and control the underlying processes, scientists pose questions concerned with these interactions and the participating molecules. How strong is the interaction? What is its range? Is it highly specific or not? What is the ideal guest for this host? How selective is this host? One way to gain insight into the interactions of molecules is to directly measure the interaction force as a function of separation producing direct insight into the nature of the active forces. The atomic force microscope1 has been used to measure interfacial2-15 and intermolecular16-30 forces, as well as other physical properties of * To whom correspondence should be addressed. E-mail: Glee@ ccs.nrl.navy.mil. (1) Bottomley, L. A. Anal. Chem. 1998, 70, 425R. (2) Burnham, N. A.; Dominguez, D. D.; Mowery, R. L.; Colton, R. J. Phys. Rev. Lett. 1990, 64, 1931. (3) Overney, R. M.; Meyer, E.; Frommer, J.; Brodbeck, D.; Lu¨thi, R.; Howald, L.; Gu¨ntherodt, H.-J.; Fujihira, M.; Takano, H.; Gotoh, Y. Nature 1992, 359, 133. (4) Salmeron, M.; Neubauer, G.; Folch, A.; Tomitori, M.; Ogletree, D. F.; Sautet, P. Langmuir 1993, 9, 3600. (5) Frisbie, C. D.; Rozsnyai, L. F.; Noy, A.; Wrighton, M. S.; Lieber, C. M. Science 1994, 265, 2071. (6) Green, J.-B. D.; McDermott, M. T.; Porter, M. D.; Siperko, L. M. J. Phys. Chem. 1995, 99, 10965. (7) Noy, A.; Sanders, C. H.; Vezenov, D. H.; Wong, S. S.; Lieber, C. M. Langmuir 1998, 14, 1508. (8) Nakagawa, T.; Soga, M. Jpn. J. Appl. Phys. 1997, 36, 5226. (9) Williams, J. M.; Taejoon, H.; Beebe, T. P., Jr. Langmuir 1996, 12, 1291. (10) Van der vegte, E. W.; Hadziioannou, G. Langmuir 1997, 16, 4357. (11) Tsukruk, V. V.; Bliznyuk, V. N. Langmuir 1998, 14, 446. (12) Han, T.; Williams, J. M.; Beebe, T. P., Jr. Anal. Chim. Acta 1995, 307, 365. (13) Wenzler, L. A.; Moyes, G. L.; Raiker, G. N.; Hansen, R. L.; Harris, J. M.; Beebe, T. P., Jr. Langmuir 1997, 13, 3761. (14) Thomas, R. C.; Tangyunyong, P.; Houston, J. E.; Michalske, T. A.; Crooks, R. M. J. Phys. Chem. 1994, 98, 4493. (15) Noy, A.; Frisbie, C. D.; Rozsnyai, L. F.; Wrighton, M. S.; Lieber, C. M. J. Am. Chem. Soc. 1995, 117, 7943.
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the interface.31-34 In many of the initial studies, the number of molecules contributing to the measured interaction was quite large;2-4 however, recent chemical and biomolecular studies were able to measure interactions between two individual molecules.16-23,28,29 Natural and technological systems are composed of a tremendous number of molecules, which interact with each other often in a complex heterogeneous environment. In addition to the myriad of traditional experimental characterization techniques and theoretical models, scientists have recently employed combinatorial35 approaches to (16) Lee, G. U; Kidwell, D. A.; Colton, R. J. Langmuir 1994, 10, 354. (17) Moy, V. T.; Florin, E.-L.; Gaub, H. E. Science 1994, 266, 257. (18) Lee, G. U; Chrisey, L. A.; Colton, R. J. Science 1994, 266, 771. (19) Dammer, U.; Popescu, O.; Wagner, P.; Anselmetti, D.; Guthernodt, H. J.; Misevic, G. N. Science 1995, 267, 1173. (20) Chilkoti, A.; Boland, T.; Ratner, B. D.; Stayton, P. S. Biophys. J. 1995, 69, 2125. (21) Hinterdorfer, P.; Baumgartner, W.; Gruber, H. J.; Schlicher, K.; Schindler, H. P. Natl. Acad. Sci. U.S.A. 1996, 93, 3477. (22) Rief, M.; Oesterhelt, F.; Heymann, B.; Gaub, H. E. Science 1997, 275, 1295. (23) Allen, S.; Chen, X.; Davies, M. C.; Dawkes, A. C.; Edwards, J. C.; Roberts, C. J.; Sefton, J.; Tendler, S. J. B.; Williams, P. M. Biochemistry 1997, 36, 7457. (24) Lo, Y.-S.; Huefner, N. D.; Chan, W. S.; Stevens, F.; Harris, J. M.; Beebe, T. P., Jr. Langmuir 1999, 15, 1373. (25) Dammer, U.; Hegner, M.; Anselmetti, D.; Wagner, P.; Dreier, M.; Huber, W.; Guntherodt, H.-J. Biophys. J. 1996, 70, 2437. (26) Stuart, J. K.; Hlady, V. Langmuir 1995, 11, 1368. (27) Rief, M.; Gautel, M.; Oesterhelt, F.; Fernandez, J. M.; Gaub, H. E. Science 1997, 276, 1109. (28) Fritz, J.; Katopodis, A. G.; Lolbinger, F.; Anselmetti, D. P. Natl. Acad. Sci. U.S.A. 1998, 95, 12283. (29) Ros, R.; Schwesinger, F.; Anselmetti, D. P. Natl. Acad. Sci. U.S.A. 1998, 95, 7402. (30) Wong, S. S.; Joselevich, E.; Woolley, A. T.; Cheung, C. L.; Lieber, C. M. Nature 1998, 394, 52. (31) Ducker, W. A.; Senden, T. J. Nature 1991, 353, 239. (32) Overney, R. M.; Bonner, T.; Meyer, E.; Ruetschi, R.; Lu¨thi, R.; Howald, L.; Frommer, J.; Gu¨ntherodt, H. J.; Fujihira, M.; Takano, H. J. Vac. Sci. Technol., B 1994, 12, 1973. (33) Finot, M. O.; McDermott, M. T. J. Am. Chem. Soc. 1997, 119, 8564. (34) Noy, A.; Sanders, C. H.; Vezenov, D. V.; Wong, S. S.; Lieber, C. M. Langmuir 1998, 14, 1508.
This article not subject to U.S. Copyright. Published 2000 by the American Chemical Society Published on Web 03/18/2000
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each of the patterned surfaces. Further, the capability exists to measure these combinations of interactions under a wide range of environmental conditions. As a proof of concept, both a cantilever and a tip array were patterned with a small set of alkylthiols, and the corresponding set of interaction forces were measured. By patterning the tip array we produce tips with different surface chemistries, and by patterning the cantilever we produce a chemically heterogeneous surface, which may be imaged with the various tips of the tip array. Due to the nature of the monolayers, this measurement is an interfacial measurement, which likely involves tens to hundreds of molecules, and will be highly sensitive to surface geometry. The extension of this technique to single macromolecule interactions is readily attainable, via routes discussed below. Figure 1. Schematic diagram of the array of chemical interactions that can be measured with a chemically functionalized tip array and cantilever. The tips from a tip array modified with a library of n compounds (n ) 3 here; A, B and C) can be used to measure interactions with a cantilever patterned with m distinct chemical species (m ) 3 here; X, Y, and Z). This will produce n × m (in this case 9) measurable interactions.
discover new materials. Often combinatorial approaches can provide insight into subtle molecular differences, which would have been difficult to predict. We propose and demonstrate a force-measuring technique that can be used to examine interactions between a large number of substances that can be assembled using combinatorial synthesis. This technique promises to not only make it possible to screen a large number of surface or intermolecular interactions but also to quickly gain direct physical insight into the nature of these interaction. In a conventional AFM, there are a number of measurement and repeatability obstacles associated with the controlled chemical modification at the apex of the tip, as well as with the continued integrity of those molecules throughout the experiment. In a recent application of microfabricated tip arrays with tipless cantilevers, we demonstrated that we could circumvent many of the obstacles inherent to the conventional design.36 By patterning the surfaces of both the cantilever and the tip array, we may not only circumvent the restrictions associated with conventional AFM, but also extend force measurements to previously unattainable experiments. Figure 1 illustrates the essential features of our approach, which are summarized below. The substrate is formed from an array of tips, each which can be chemically modified with a unique chemical species, e.g., A, B, and C. The cantilever is patterned with an array of different chemical species, e.g., X, Y, and Z. The tips of the patterned tip array are used to inspect the patterned cantilever, thus permitting measurement of all possible combinations of interactions, e.g., AX, AY, AZ; BX, BY, BZ; CX, CY, CZ. The tip arrays allow us to use the AFM in all the conventional modes of operation, e.g., contact normal deflection, lateral force, oscillating in contact, force curves, or oscillating while not in contact. Thus, all the information that can be gathered with a conventional AFM may be acquired with the tip arrays, e.g., topography, friction, adhesion, elasticity, or long-range forces. Using existing patterning technologies, it is possible to use this platform to measure thousands of distinct interactions within minutes, by simply having tens of distinct molecules on (35) Wendler, A. Frontiers in Organic Synthesis. Acc. Chem. Res. 1996, 29 (3). (36) Green, J.-B. D.; Novoradovsky, A.; Lee, G. U.; Park, D. Appl. Phys. Lett. 1999, 74, 1489.
II. Experimental Section Metalization. The silicon tip arrays were prepared by microfabrication techniques as previously described,36 and the silicon oxynitride tipless cantilevers were purchased from Park Scientific (now Thermomicroscopes, Santa Clara, CA). Using an electron beam vacuum deposition system, we coated both the cantilevers and the tip arrays with gold for subsequent reaction with alkylthiols. The tip arrays were first primed with ∼5 nm of chromium as an adhesive, and then coated with ∼200 nm of gold. Both sides of the cantilevers were coated with ∼2 nm of chromium and then ∼30 nm of gold. Stress-induced bending of the cantilever was avoided by rotating the cantilevers about their long axis at ∼20 Hz during deposition. The chromium and the gold were both deposited at 1-2 Å/s. The surfaces were cleaned for 10 min in an ozone cleaner (Model No. 42, Jelight Co. Inc., Irvine CA) immediately prior to the deposition of the monolayers. Physical Characterization. To estimate the force constant of the cantilever, we assume that the cantilever can be approximated as a harmonic oscillator which is in thermal equilibrium with its surroundings.37 According to the equipartition theorem, the average of the harmonic oscillator potential energy, (1/2)kx2, is equal to a unit of thermal energy (1/2)kBT. Spectral analysis of the detector noise was fit by the theoretical damped harmonic oscillator spectrum. For the cantilever used in this study, a 200 × 20 × 0.6 µm rectangular cantilever, the force constant k ) 0.085 N/m. The radii of the tips of the tip array used in this experiment were determined by scanning electron microscopy (SEM) using a Leica 360-FE (LEO Electron Microscopy Inc., Thornwood, NY). Most of the tip radii were approximately 40 nm. However, some tips were significantly sharper with radii in the range of 5-10 nm. Chemical Modification. Chemical modification of both the cantilever and tip array was achieved with the contact printing of thiols onto gold, developed by Kumar and Whitesides.38 Three different alkylthiols were used for the experiment: 16-mercaptohexadecan-1-oic acid (MHA), 16-mercaptohexadecan-1-ol (MHO), and n-mercaptohexadecane (MHD). In this way, we could impart the chemical functionality of either carboxylic acid, alcohol, or methyl groups, respectively, to different regions of the surface. The stamping procedure is illustrated in Figure 2 for the cantilever and Figure 3 for the tip array. Immediately prior to patterning, both the tip array and the cantilever were cleaned with ozone for 10 min. In the case of the cantilever, a patterned poly(dimethylsiloxane) (PDMS) stamp was first cleaned by sonication for 10 min in anhydrous ethanol and dried under a stream of N2 (Figure 2A). The ethanol used for all solutions and for all rinsing was anhydrous ethanol (USP grade) supplied from the Warner Graham Company (Cockeysville, MD), and the N2 used for drying was liquid nitrogen boil off passed through a 200 µm particulate filter. The stamp was then immersed in a 1 mM ethanolic solution of MHA. This acid stamp was then dried with a stream of N2, placed onto a microscope slide, and inserted into an optical (37) Hutter, J. L.; Bechhoefer, J. Rev. Sci. Instrum. 1993, 64, 1868. (38) Kumar, A.; Whitesides, G. M. Appl. Phys. Lett. 1993, 63 (14), 2002.
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Figure 2. Schematic diagram and image of the experimental approach used to patterning the cantilever. (A) A bare gold cantilever is aligned above a microscopically patterned stamp that has been inked with MHA. (B) The cantilever is placed in contact with the surface of the stamp, and thiols are transferred to the cantilever from the raised regions of the stamp. (C) The alignment is shown in the optical micrograph for future comparison with images. The bar on this optical micrograph corresponds to 100 µm, with 20 µm gradations. (D) The patterned cantilever is removed from solution. (E) Cantilever is immersed into a solution of MHD. microscope (Zeiss Axiotech, Carl Zeiss, Inc., Thornwood, NY) fitted with a CCD camera and a video capture board. A cantilever chip was carefully aligned and placed onto the stamp, so that the cantilever surface was in direct contact with the stamp, as shown schematically in Figure 2B and in the optical micrograph in Figure 2C. Some additional load was applied to the back of the cantilever with a microposition controlled glass fiber. The cantilever and stamp were in contact for ∼5 min, following which the cantilever was carefully removed (Figure 2D). Finally, the cantilever was immersed in a 1 mM ethanolic solution of MHD, producing a chemically patterned cantilever as illustrated in Figure 2E. Two separate unpatterned slabs of PDMS were used to modify the tips of the tip array. Since the tip array is a square with 1 cm on a side, the stamps were cut into smaller 3 mm squares. Following immersion into 1mM ethanolic solutions of MHA and MHO, the acid and alcohol stamps were dried with a stream of N2 (Figure 3A). Both stamps were then placed into contact with the tip array in such a manner that the edges of the stamps were
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Figure 3. Schematic diagram and image of the approach used to pattern the tip array. (A) Two featureless stamps inked with MHA and MHO are aligned with tips of the tip array, leaving ∼1 mm separation between the stamps. (B) The stamps are placed in contact with the tip array. (C) Several optical micrographs similar to the one shown were taken to verify the registry of the tips with respect to the two stamps. The bar on this optical micrograph corresponds to 500 µm, with 100 µm gradations. (D) The tips are removed from solution. (E) The tip array is immersed into a solution of MHD. separated by ∼1 mm leaving unstamped tips near the center of the tip array. As with the cantilever, reflection optical microscopy was used to determine exactly which tips were modified with which thiol, as seen schematically in Figure 3B and in the optical micrograph in Figure 3C. The tip array was in contact with the stamps for ∼10 min, at which time the stamps were removed (Figure 3D). Finally, the tip array was placed into a 1 mM ethanolic solution of MHD, producing a chemically modified tip array as illustrated in Figure 3E. Instrument Assembly. These experiments used a NanoscopeIIIa controller equipped with a multimode scanning probe microscope and a scanner capable of a maximum scan size of 140 µm (Digital Instruments, Santa Barbara, CA). The measurements were performed under pure water, using a fluid cell cantilever holder. The pure water used throughout this experiment was produced by running distilled water through a Milli-Q gradient system (Bedford, MA) and had a nominal pH of 5.5. Due to the importance of contamination to force measurements, we consistently clean the cantilever holder with peroxysulfuric acid (a 3:1 ratio of concentrated H2SO4 and 30% H2O2) according to the
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Figure 4. Optical micrograph showing the cantilever alignment above a specific tip of the tip array. The cantilever is the dark rectangular structure on the right side of the image, and each dark square is a pyramidal tip. The tips are separated by 25 µm, and the bar corresponds to 200 µm, with 100 µm gradations. The white line on the image has been artificially added to illustrate the boundary between the tips modified with MHD and those modified with MHA. following procedure. This procedure requires extreme care for both the user and the cell. A glass syringe filled with fresh peroxysulfuric acid was used to inject acid through both fluid ports of the holder and to fill the O-ring recess. This was allowed to sit for ∼2 min, and then the process was repeated with more acid. The acid was then purged with several syringe volumes of pure water. Finally the cell was vigorously rinsed with a stream of pure water and then dried under a stream of N2. Immediately prior to use, the cantilever and the tip array were removed from the ethanolic MHD solutions, rinsed with copious amounts of ethanol, and blown dry with N2. The patterned cantilever was mounted into the clean cantilever holder, which was then fixed into the AFM laser-detector head. When working with tip arrays in solution, we avoid using O-rings, as these damage the surface and limit spatial accessibility. Several milliliters of pure water were slowly injected through the ports of the holder and emerged as a stream of drops from openings near the cantilever. The patterned tip array was mounted onto the scanner, and it was primed with a single droplet of pure water. The cantilever was approached toward the tip array by course approach, and these two droplets merged. In this captured droplet the cantilever was positioned above a specific tip of known chemical functionality, as shown in Figure 4. The optical micrograph recorded during stamping allowed explicit location of tips with known chemical functionality. Force Measurement. Force curves were measured by monitoring the deflection of the cantilever while sweeping the vertical position of the tip array toward and away from the cantilever. The tip array traversed 2.4 µm moving toward and away from the cantilever at a speed of 45 µm/s, for each force curve. The vertical position of the tip array was detected with the deflection signal of the cantilever. When the cantilever deflection equaled a trigger value of 50 nm, the motion of the tip array was changed from approach to retract. For the cantilever used in this study, this corresponds to fixing the maximum cantilever applied load at 4.25 nN. Force curves were acquired in either a high-resolution or force volume imaging mode. In the high-resolution mode, a single force curve was measured at a specific position with 1024 data points per force curve. In the force volume mode, a force curve was measured at each point of a 64 × 64 grid of defined size. Each of these points corresponds to a relative xy-position of the cantilever measured with respect to the tip array. At each point, a single force curve is measured with 128 data points per force curve. Thus, the imaging mode produces an array of 4096 force curves where each force curve is related to a specific xy-location. Following the acquisition of a force volume image, each force curve was analyzed to determine the rupture force at each xy-
III. Results and Discussion To demonstrate surface interaction screening, force measurements were made in water between cantilevers and tip arrays patterned with alkylthiolate monolayers. These monolayers form ideal surfaces for studying surface forces because of their well-defined physical and chemical properties. Figure 5A shows a 25 × 25 µm force map of the end of the cantilever patterned with carboxyl- and methyl-terminated monolayers and a methyl tip. Each of the pixels in the image corresponds to a measured rupture force at that xy-coordinate, with the larger rupture forces having brighter shades. The rupture event, as seen in Figure 5C, occurs at the point at which the spring constant of the cantilever exceeds the gradient of the tip-cantilever interaction force, and thus is determined by the specific cantilever, tip, and point on the cantilever. The force map shows a strong correlation between the pattern of the rupture force on the cantilever and the chemistry transferred to the cantilever by the PDMS stamp, as illustrated in the schematic of Figure 5A. Interaction forces depend on the chemical, geometric, and mechanical properties of both the tip and cantilever surface, as well as properties of the intervening medium. The high-resolution force curves, shown in Figure 5B,C, were recorded on specific areas of the cantilever identified with the arrows. The force curve in Figure 5B was measured over a MHD area of the cantilever and shows such a strong attractive interaction that rupture takes place outside the range of the measured cantilever deflection. The strong adhesive interaction measured between the hydrophobic tip and the hydrophobic region of the cantilever is consistent with the hydrophobic effect,39-41 although the magnitude of the MHD attractive forces is larger than that measured for mercaptododecane surface in water.42 We have confirmed that the difference in the magnitude of the force does not result from the inversion of the tip-cantilever geometry or the process of patterning. If the tip is located beneath the hydrophilic MHA region of the cantilever, then the force-distance data exhibit a significantly smaller rupture force, which is consistent with previous measurements of hydrophilichydrophobic interactions.43 The force map shown in Figure 5A clearly reflects the physical properties of the surface chemistry but also shows point-to-point variation in the rupture force. We attribute this variation to changes in the tip-surface contact area produced by the nanometerscale roughness inherent to the gold crystallites formed on room temperature evaporated films. As is evidenced by these images and by examination of the data, the measured adhesion is not significantly dependent upon the lateral location of the contact on the cantilever. (39) Kauzmann, W. Adv. Protein Chem. 1959, 14, 1. (40) Tanford, C. The Hydrophobic Effect; Wiley: New York, 1980. (41) Israelachvili, J. N.; Pahsley, R. M. Nature 1982, 300, 341. (42) Sinniah, S. K.; Steel, A. B.; Miller, C. J.; Reutt-Robey, J. E. J. Am. Chem. Soc. 1996, 118, 8925. (43) Dufreˆne, Y. F.; Boland, T.; Schneider, J. S.; Barger, W. R., Lee, G. U. Faraday Discuss. 1999, 111, 79.
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Figure 5. Force map of a MHA and MHD patterned cantilever with a MHD tip. (A) A 25 µm force volume image of the cantilever taken with the methyl tip. The image is a map of the rupture force between the methyl tip and the various locations on the cantilever, as seen in the inset schematic. Brighter corresponds to a higher rupture force. Areas that are completely white correspond to locations where the cantilever and the tip do not make contact. (B) Force curve taken within the portion of the cantilever that was modified with MHD, corresponding to a methyl-methyl interaction in water. (C) Force curve taken within the portion of the cantilever that was modified with MHA, corresponding to a methyl-acid interaction in water.
The tip array was chemically modified with three different alkylthiolate monolayers, MHD, MHO, and MHA. Force maps corresponding to each of the three different chemical groups are shown in Figure 6 A-C. These 25 × 25 µm force maps were produced by imaging the same patterned cantilever with three different tips, each with a different surface chemistry. The cantilever supports two surface chemistries, and the tip array supports three, resulting in five different chemical combinations. The average rupture force is plotted beneath each image in Figure 6D-F. These traces are the average of 20 scan lines, corresponding to the enclosed rectangles. The rupture forces can be ranked for each tip-surface pair: methyl-methyl > methyl-carboxyl, alcohol-methyl > alcohol-carboxyl, and carboxyl-methyl > carboxylcarboxyl. For contact area dependent force measurements, the rupture force must be scaled by the radius of the probe to identify the chemical and mechanical contributions of the interaction.44 Direct comparison of rupture forces
between the tips in this study is limited by the variations in the tip radii. While the tips nominally have 40 nm radii, as determined by SEM, the exact radius contacting the cantilever is determined by the exact orientation of the cantilever with the respect to the tip. Patterning the cantilever provides several potential sources of internal calibration. For example, the methyl-carboxyl interaction is measured in two tip-surface configurations in this experiment which allows us to rank the rupture forces across two tips, i.e., methyl-methyl > methyl-carboxyl > carboxyl-carboxyl. Alternatively, a possible internal calibration to determine the tip radii in situ involves placing colloidal spheres in one of the patterned regions of the cantilever; in this way they could be imaged to determine the radius of that particular tip.45 For single macromolecule force measurements, which we believe will be the primary application of tip arrays, contact area effects can be minimized by placing the interacting molecules on a polymer tether which minimizes surface effects.
(44) Schneider, J.; Dufreˆne, Y. F.; Barger, W. R.; Brady, R., Lee, G. U. Manuscript in preparation.
(45) Vesenka, J.; Manne, S.; Giberson, R.; Marsh, T.; Henderson, E. Biophys. J. 1993, 65, 992.
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Figure 6. Force maps of MHA and MHD patterned cantilever taken with three different tips. These three 25 × 25 µm force maps are rupture force images of the same patterned cantilever taken with (A) MHD, (B) MHO, and (C) MHA functionalized tips. The rupture force averaged over 20 scan lines is presented for the (D) MHD, (E) MHO, and (F) MHA functionalized tips.
Figure 7. A force map obtained with multiple tips. This 140 × 140 µm force map corresponds to rupture forces between 25 different alcohol modified tips and a single patterned cantilever in pure water.
Thus far the images have always consisted of a single surface-bound tip imaging a single cantilever; however, Figure 7 was obtained by imaging a single cantilever with 25 different tips. Figure 7 is a 140 × 140 µm force map of the patterned cantilever taken by 25 separate alcohol tips. The image is composed of 25 features. Each of these small features is actually an image of the cantilever, obtained by measuring rupture forces with a single tip. All of these tips in this image were modified with MHO, and thus the chemical contrast of all 25 features should be identical. The minor variations in contrast clearly demonstrate the role of tip-tip radii variations. For example, there is a single feature at the bottom of Figure 7, which is extremely dark. Comparison with SEM images of tip radii verified that tips producing extremely low rupture forces were extremely sharp, on the order of 5-10 nm. It is important to note that within a single image, over the course of minutes, we can obtain statistically relevant data for hundreds of possible interactions. The data in Figure 7 demonstrate that 25 tips can be imaged within a few minutes. If each of these 25 tips were modified with a different type of molecule, and if the cantilever were patterned with an array of 5 × 5 different types of molecules, then we would rapidly measure 625 different
interactions. The capability of this technique to examine large numbers of interactions is impressive. Limitations. This technique is not without its limitations: (1) Variation in tip geometry from tip to tip creates a distribution in contact area that will act to broaden the spread in the data associated with contact mechanics correlated measurements. (2) For statistically relevant data sets, large bodies of data must be processed, and depending upon the nature of the measurement, this may require complex interpretive analysis, i.e., pattern recognition. (3) Techniques exist for patterning the cantilever and tips with libraries of certain chemicals, such as polypeptides, oligonucleotides, or alkylthiols; however, patterning with arbitrary libraries of chemicals will certainly contain a wide range of obstacles specific to each system. (4) Although the possibility exists for parallel arrays of cantilevers,46 the characterization of interactions for the library contained within the bounds of a single cantilever is still serial, and as such is limited in throughput. (5) The system is rugged with respect to the inactivation of a few tips; nonetheless, the system is still sensitive to time-dependent degradation of both surfaces, such as adsorption of trace contaminants from solution with time. This is of course a problem for any surfacebased sensor system. While these limitations may be corrected in the research setting, they may pose serious obstacles for commercialization. Many of the above limitations can also be considered as strengths, when and if the associated difficulties are overcome. For example, the complex interpretation for some systems is required because of the information contained within the measurement, and proper extraction of that information could be of added value. Possibilities. The amount of molecular diversity found in both natural and technological systems is staggering. From the time that a molecule is created, it may interact with many other substances, and with each encounter the possibility exists that the molecule will be changed by the interaction. Combinatorial techniques have been used to create a vast number of new molecules as well as to (46) Minne, S. C.; Yaralioglu, G.; Manalis, S. R.; Adams, J. D.; Zesch, J.; Atalar, A.; Quate, C. F. Appl. Phys. Lett. 1998, 72, 2340.
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probe the interactions between different substances. As an example, a library of polypeptides may be created by combinatorial techniques. Then each element of this library may be exposed to a new substance, such as a protein. By examining the resulting solution, researchers gain insight into which kinds of polypeptides are most likely to interact most strongly with that protein. Combinatorial imaging with the AFM is not restricted to any specific mode of AFM operation, imaging of topography, viscoelastic properties (with amplitude or phase), high-frequency techniques (amplitude or phase), and rupture force. With current patterning techniques achieving resolution limits in the tens of nanometers,47 and with the vast number of material and biological systems, the possibilities of this platform are far-reaching. Theoretically, the cantilever could be loaded with hundreds of different materials (10s × 10s). If the tips of the tip array can be patterned with tens of thousands of different substances (100s × 100s), then millions of distinct interactions could theoretically be measured. A realistic expectation of the next demonstration would involve using existing and commercial products and techniques. Patterning of a 20 µm cantilever, to produce a 5 × 5 array of 25 different molecules, has each region 4 µm on a side. Similar patterning of the tip array at that resolution would allow each tip within a 125 µm field of view to be modified with a different molecule. Within a few minutes we could generate an interaction map of the cantilever with 25 different tips, each tip imaging its interactions with the 25 different regions of the cantilever, and producing in a single image 625 distinct measurements. The only requirement for this next step is controlled patterning at this higher resolution, which is well within the state of the art. Future experiments might include patterning the tip array and the cantilever with biologically relevant systems such as key ligand-receptor couples. Feasible applications range from the effects certain drugs have on interactions (47) Piner, R. D.; Zhu, J.; Xu, F.; Hong, S.; Mirkin, C. A. Science 1999, 283, 661.
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between antigens and immune regulatory receptors to the mapping of interaction forces across the surfaces of complex real-world surfaces. When combined with existing platforms of parallel, independently scanning, cantilevers this offers the capability for both enhanced numbers of interactions and elevated throughput. IV. Conclusions We have proposed a technique that can theoretically be used to measure millions of distinct interactions between thousands of different types of molecules, and with existing technology we should be able to easily examine hundreds of interactions. The technique is based on conventional atomic force microscopy where the sample has been replaced by a chemically patterned microfabricated array of tips, and the tip-supporting cantilever has been replaced with a chemically patterned tipless cantilever. This is demonstrated using a small number of well-defined alkylthiolate monolayers at gold-coated cantilevers and tip arrays. This paper demonstrates that patterned cantilevers and patterned tip arrays can be used to probe interactions between a small set of chemically modified interfaces, thus identifying a new tool for gathering detailed intermolecular force information on large libraries of chemicals. Further studies are planned to examine the principal factors that affect the performance of this platform, as well as to deconvolute the material, chemical, and solution effects from measured interactions. Acknowledgment. The authors gratefully acknowledge Doewon Park for fabrication of the tip arrays, Loretta Shirey for the SEM of our tip arrays, Ken Lee for the data analysis program, and Marc Porter for the supply of both the alkylthiols and the PDMS stamp. We acknowledge both the American Society for Engineering Education and the Office of Naval Research for financial support of J.B.G.’s postdoctoral fellowship. Additionally we thank Larry Bottomley, Jonah Harley, Thomas Boland, and Jim Schneider for many valuable discussions. LA991533S
