NANO LETTERS
Molecular Shuttles Operating Undercover: A New Photolithographic Approach for the Fabrication of Structured Surfaces Supporting Directed Motility
2003 Vol. 3, No. 12 1651-1655
Henry Hess,*,† Carolyn M. Matzke,‡ Robert K. Doot,† John Clemmens,† George D. Bachand,‡ Bruce C. Bunker,‡ and Viola Vogel† Department of Bioengineering, UniVersity of Washington, Seattle, Washington 98195, and Sandia National Laboratories, Albuquerque, New Mexico Received September 5, 2003; Revised Manuscript Received October 15, 2003
ABSTRACT The integration of active transport into nanodevices greatly expands the scope of their applications. Molecular shuttles represent a nanoscale transport system driven by biomolecular motors that permits the transport of molecular cargo under user-control and along predefined paths. Specifically, we utilize functionalized microtubules as shuttles, which may be transported by kinesin motor proteins along photolithographically defined tracks on a surface. While it was thought that efficient guiding along these tracks requires a combination of surface chemistry and topography, we show here that channel-like tracks with a particular wall geometry can be created to efficiently guide microtubules in the absence of selectively adsorbed motor proteins. This new wall geometry consists of an undercut 200 nm high at the bottom of the channel wall fabricated by image reversal photolithography using AZ5214 photoresist. Microtubules move unencumbered in the undercut, suggesting applications for nanofluidic systems and for in vitro motility assays mimicking the restricted environment characteristic of intracellular transport. Because adsorbed kinesin supports motility on top and bottom surfaces of the guiding channels, this guiding mechanism may serve as a first step toward the development of three-dimensional architectures.
Biomolecular motors, such as the motor proteins kinesin and myosin, are highly efficient nanoscale engines that have proven their usefulness in a wide range of biological systems.1 The ability to produce and isolate these motors using standard methods of biotechnology permits the design of hybrid devices, where biomolecular motors serve as forcegenerating modules in an artificial environment.2 One concept of such a device is the molecular shuttle,3,4 a nanoscale transport system designed for the controlled manipulation of molecules and supramolecular structures in a liquid environment. The potential applications for such a system include molecular assembly, nanoscale sensors,5 and singlemolecule studies.6 In our molecular shuttle system, kinesin motor proteins are adsorbed to a structured surface and transport microtubules, which are hollow filaments with an outer diameter of 30 nm assembled from the protein tubulin (Figure 1). Functionalization of the microtubules7 with fluorescent dyes * Corresponding author. Phone (206) 616-4194. Fax (206) 685-4434. E-mail
[email protected] † University of Washington. ‡ Sandia National Laboratories. 10.1021/nl0347435 CCC: $25.00 Published on Web 11/01/2003
© 2003 American Chemical Society
and biotin linkers permits the observation and selective loading of these “shuttles”, while managing the supply of ATP, which serves as fuel for the motor proteins, is a means to control motor activity.8 In addition to the loading of cargo and controlling the speed of such a shuttle, guiding shuttles along predetermined tracks is a critical issue in developing biomolecular motorbased systems. Strategies for defining such tracks on planar surfaces have evolved considerably in the past few years. While initial studies relied on the deposition of poly(tetrafluoroethylene) films with parallel nanoscale grooves to guide the movement of microtubules3 or actin filaments,9 current methodologies rely on fabrication of complex track patterns using electron-beam lithography,10-12 photolithography,13,14 or soft-lithography techniques.15 Three approaches to designing a track have been discussed previously:16 (1) the creation of motor protein-adsorbing tracks surrounded by non-fouling regions with the goal of restricting the binding sites for the microtubule or actin filament (Figure 2A), (2) the fabrication of guiding channels with steep side-walls, which guide microtubules moving on the bottom surface of the channel (Figure 2B), and (3) the combination of both
Figure 3. Novel wall geometry for efficient guiding of microtubules on motor protein-coated surfaces imaged by scanning electron microscopy. The undercut prevents microtubules moving in the channel from climbing the sidewall, even if all surfaces are coated with motor proteins.
Figure 1. Molecular shuttle system envisioned to load, transport, sort, and assemble nanoscale building blocks (top). A hybrid design approach, combining synthetic environments and biomolecular motors, utilizes surface-bound kinesin motor proteins to transport functionalized microtubules along fabricated tracks.
Figure 2. Previous approaches to guide the movement of microtubules and actin filaments on engineered surfaces functionalized with motor proteins.
techniques, where only the bottom of a guiding channel adsorbs motor proteins and supports shuttle movement (Figure 2C). The first approach (Figure 2A) is of limited utility for guiding stiff filaments16,17 such as microtubules, which have a persistence length of 5.2 mm.18 Microtubules fail to reorient themselves on chemical tracks when crossing the boundary to the nonadhesive surface and eventually detach. The second approach so far has been hampered by the ability of the microtubules to climb the motor protein-coated side-walls and subsequently escape from the track.15 The implementation of the third approach by Hiratsuka et al.13 demonstrated, for the first time, highly efficient guiding. The technique relies on selective adsorption of motor proteins to glass exposed at the bottom surface of a guiding channel patterned in photoresist. It has been further studied for an 1652
actin-myosin system,12 and its general applicability for enzyme patterning has been realized.14 Here we demonstrate that guiding channels (Figure 2B) with uniformly adsorbed motor proteins and a specific wall geometry can efficiently guide microtubules (Figure 3). Our previous work16 has demonstrated the importance of a steep wall for guiding microtubules efficiently. Taking the idea of “steep” walls to its logical conclusion, we have photolithographically prepared 1 µm high walls with a 200 nm high and 1 µm deep undercut at the bottom. Both the resist and glass surface will adsorb kinesin motor proteins and support microtubule binding if the photoresist surface is rendered hydrophilic by oxygen plasma treatment. However, microtubules moving on the bottom surface are unable to climb the sidewall and remain on the bottom surface, preferentially moving in the undercut section of the channel. This result is significant in several ways: (1) it facilitates the fabrication of tracks for molecular shuttles by offering an alternative to non-fouling surfaces; (2) it demonstrates that microtubules can move in vitro in very narrow channels, resembling the restricted environment of axons, which typically have a diameter of less than 1 µm;27,28 and (3) it is the first step toward three-dimensional architectures, because the bottom of the channel and the top surfaces can support different functionalities. Materials and Methods. Experiments were performed in flow cells assembled from a slide (Fisher Scientific, Fisherfinest premium slides), two spacers (Scotch double-coated tape, 3M, St. Paul, MN), and a transparent 0211 glass substrate (Precision Glass & Optics, Santa Ana, CA) with patterned AZ5214 photoresist (Shipley Company, L. L. C., Marlborough, MA) on one side. AZ5214 can be processed to provide either a positive tone or negative tone (image reversal) pattern of the photo mask. We used the image reversal process, which is known to produce re-entrant profiles and is commonly used as a metallization lift-off technique. Details of our image reversal process are as follows. Glass wafers, 175-200 µm in thickness, were cleaned in oxygen plasma at 215 W for 5 Nano Lett., Vol. 3, No. 12, 2003
min. Wafers were dehydrated at 110 °C for 2 min and HMDS adhesion promoter was spun on the surface at 5000 rpm. AZ5214 photoresist was spun on at 5000 rpm and then was soft baked to drive out solvent at 110 °C for 115 s. Exposure to 400 nm UV light for 2.3 s was followed by a 110 °C, 50 s post bake to cross-link the exposed resist. A final aggressive flood exposure for 45 s was used to expose resist that had not been cross-linked. Photoresist was developed in a 1:1.4 ratio MIF 312 developer to deionized water. Oxygen plasma treatment for 5 min at 17 W is used to clean the glass surfaces and oxidize the photoresist. We believe that the large undercut profiles are a result of processing image reversal photoresist on a transparent substrate. For typical image reversal processing, exposed photoresist areas remain in place at the end of the process. However, if the photoresist is not fully exposed, the photoresist will be softer and soluble to some degree in developer, resulting in the large undercut regions. Since we used exposure times typical for reflective semiconductor surfaces, the photoresist is exposed to lower dose levels on the transparent wafers. We found that the size of the undercut area was a function of development time, indicating that the resist was indeed soluble in developer. To minimize interfering autofluorescence of the photoresist, Oregon-green labeled tubulin (4 mg/mL, gift from J. Howard) was polymerized in 4 mM MgCl2, 1 mM GTP, 5% DMSO, in BRB80, and stabilized by 100-fold dilution into BRB80 with 10 µM paclitaxel.19 A kinesin construct (generously provided by J. Howard) consisting of the wildtype, full-length Drosophila melanogaster kinesin heavy chain and a C-terminal His-tag was expressed in Escherichia coli and purified using a Ni-NTA column.20 The eluate contained functional motors with a concentration of ∼0.1 mM and was stored as stock solution after adding 5% sucrose at -80 °C. Two procedures for the motility assay were used: (A) a standard procedure,21 which consists of precoating the surface with casein, adsorbing kinesin, adsorbing microtubules, and finally introducing an antifade solution with ATP,19 and (B) a modified procedure22 where a detergent was added to the buffer as suggested by Hiratsuka et al.13 The modified procedure (B) was designed to enhance a potential contrast in the capability to adsorb motors between the glass surface at the bottom of the channel and the photoresist sidewalls hydrophilized by the plasma treatment. However, no differences in microtubule motility could be observed between the two procedures, which provides additional evidence for the assumption that no difference in motor adsorption exists between top and bottom surfaces. Gliding motility of microtubules was imaged with an epifluorescence microscope (Leica DMIRBE) equipped with a 100× oil objective (N. A. 1.33) and a cooled CCD camera (Hamamatsu Orca II). Results and Discussion. While the photoresist AZ5214 has been previously tested for microtubule guiding,13 its bright autofluorescence prevented its application for the observation of rhodamine-labeled microtubules;13,14 the use of Oregon green-labeled microtubules overcame this limitaNano Lett., Vol. 3, No. 12, 2003
Figure 4. Microtubules approaching an isolated wall with undercut on the lower surface are efficiently guided along the wall (10 s between frames). After moving uninhibited in the undercut, the microtubules are able to leave the undercut region.
tion. The faint green autofluorescence of AZ5214 helps to confirm the presence and uniformity of the undercut, since the fluorescence of the photoresist layer drops to 80% at the undercut, before increasing directly at the edge. Untreated, AZ5214 shows selective adsorption of motor proteins under appropriate buffer conditions.13 However, an oxygen plasma etch, which is commonly used to clean monolayers of photoresist residue from open areas of the photoresist pattern, rendered the AZ5214 surface hydrophilic, thus conferring a similar affinity to motor protein adsorption compared to the exposed glass at the bottom of the channel. Consequently, microtubules adsorbed and moved on the bottom surface of the channel as well as on the top surfaces. This behavior was observed, with (procedure B) and without (procedure A) detergent added during the kinesin adsorption step, as would be expected if photoresist and glass are hydrophilic. While imaging 43 microtubules approaching isolated walls (Figure 4), we did not observe an unsuccessful guiding event where a microtubule approaching from the bottom surface climbs up to the top surface. In contrast, microtubules approaching the boundary from the top surface routinely descend to the bottom surface. This is roughly in agreement with the observation of Stracke et al. that microtubules cannot climb steps higher than 300 nm.23 Since our undercut has a height of 200 nm, the microtubules are probably unable to contact the upper region of the sidewall before entering the undercut. After entering the undercut region the microtubules move to the sidewall, are redirected, and continue their movement 1653
Figure 5. Multiple microtubules crowding the undercut region of a channel (200 nm × 1000 nm) create a situation reminiscent of the interior of an axon (D < 1 µm).
while remaining in the undercut region. However, microtubules also frequently leave the undercut and continue moving on the lower surface, which prevents an accumulation of microtubules in the undercut region. While moving in the undercut region, the microtubules experience an environment drastically different from the planar regions of the surface. Previous measurements by Stracke et al.23 on the microtubule motility between two coverslips with varying distance have shown that microtubules glide in clefts as low as 100 nm. However, the gliding velocity drops slowly to approximately one-half of the maximum gliding velocity as the distance between the coverslips decreases to 100 nm. This velocity decrease would be a serious impediment for the design of nanofluidic devices utilizing active transport based on motor proteins. However, we did not observe a change in velocity as microtubules entered the undercut region (V ) 615(60 nm) from the open surface (V ) 595(60 nm/s - 14 microtubules sampled, mean(SD quoted). While Stracke et al. suggested that ATP may be depleted in the long narrow cleft between the coverslips, causing the decrease in velocity, in our case ATP can diffuse efficiently into the undercut, explaining the difference in our observations. The small height (200 nm) of the undercut also increases the viscous drag on the moving microtubule by roughly one-third.24 This increase should not affect the velocity according to previous measurements of gliding velocity as function of solution viscosity.25 The small height potentially allows the microtubule to bind simultaneously to upper and lower surfaces of the undercut along its length. If multiple microtubules encounter each other while moving in the same or opposing directions, the space in the undercut gets rapidly crowded (Figure 5), resembling the situation in an axon where the microtubule density is on the order of 20 µm-2.26 For guiding channels with a width smaller than the length of our microtubules, we frequently observe that a microtubule approaching the channel on the top surface will not descend into the channel but bridge the channel and continue its movement on the top surface on the opposing side (Figure 6). The high stiffness of microtubules (persistence length 5.2 1654
Figure 6. The large stiffness of the microtubules allows the bridging of guiding channels (focus on top surface, 10 s between frames). This decouples the movement on the top and bottom plane and is a first step toward three-dimensional architectures.
mm) prevents the tip of the microtubule from binding to motors on the bottom surface of the channel, provided the channel is not too wide, deep enough, and the approach angle is steep enough. If the channel is deeper than it is wide, the microtubules are always more likely to bridge the channel than to descend into it but can detach from the surface completely for small approach angles. If, in addition, the channel is narrow enough that microtubules either bridge the channel or are able to rebind to the side of the top surface they are approaching from, the transfer of microtubules between top and bottom surface could be almost entirely prevented. Clemmens et al.16 have presented data and a model on the approach angle dependence of microtubule guiding on tracks of motors, which show that microtubules rebind to the motor track if they approach the boundary between a motor-rich and motorfree region under a slight angle of less than ∼5 degrees. The situation here is similar in the sense that the top surface is motor-rich, and the guiding channel constitutes a motorfree region in the plane of the top surface. Therefore, we can estimate that for 5 µm long microtubules, a channel with a width of 0.5 µm and a depth of 1 µm does not permit microtubules to descend into the channel independent of the approach angle, since the microtubule will either bridge the channel or return to the surface from which it approaches. A possible application of independent planes is to use the large surface of the top plane to efficiently adsorb microtubules and cargo from the solution, and the narrow tracks on the bottom plane as a structured delivery system. Conclusion. The new geometry for microfabricated channels serving as tracks for molecular shuttles successfully directs microtubule movement on kinesin-coated surfaces. It removes the requirement for adsorption resistant surfaces, which was found previously to be essential for effective guiding using vertical sidewalls. Motor adsorption to bottom and top surfaces not only drastically simplifies the experimental procedure but also permits a multilevel architecture with two functionally independent planes. While motordriven microtubule movement has been previously confined to micrometer-wide open channels, we succeeded in creating an environment with submicron dimensions, which apNano Lett., Vol. 3, No. 12, 2003
proximates the dimensions of biological structures such as axons more closely. Acknowledgment. We thank Jonathon Howard for providing tubulin and kinesin constructs, Michael Wagenbach for kinesin expression, Sheila Luna for the artwork, as well as Yuichi Hiratsuka and Taro Uyeda for helpful discussions and advice on their fabrication methods. Financial support was provided by DOE/BES grant DE-FG03-03ER46024. Sandia is a multiprogram laboratory operated by Sandia Corporation, a Lockheed Martin Company, for the United States Department of Energy under contract DE-AC0494AL85000. References (1) Vale, R. D. Cell 2003, 112, 467-480. (2) Hess, H.; Vogel, V. ReV. Mol. Biotechnol. 2001, 82, 67-85. (3) Dennis, J. R.; Howard, J.; Vogel, V. Nanotechnology 1999, 10, 232236. (4) Hess, H.; Clemmens, J.; Qin, D.; Howard, J.; Vogel, V. Nano Lett. 2001, 1, 235-239. (5) Hess, H.; Clemmens, J.; Howard, J.; Vogel, V. Nano Lett. 2002, 2, 113-116. (6) Hess, H.; Howard, J.; Vogel, V. Nano Lett. 2002, 2, 1113-1115. (7) Hyman, A. A.; Drechsel, D. N.; Kellog, D.; Salser, S.; Sawin, K.; Steffen, P.; Wordeman, L.; Mitchison, T. J. Methods Enzymol. 1991, 196, 478-485. (8) Bohm, K. J.; Stracke, R.; Unger, E. Cell Biol. Int. 2000, 24, 335341. (9) Suzuki, H.; Oiwa, K.; Yamada, A.; Sakakibara, H.; Nakayama, H.; Mashiko, S. Jpn. J. Appl. Phys. Part 1 1995, 34, 3937-3941. (10) Suzuki, H.; Yamada, A.; Oiwa, K.; Nakayama, H.; Mashiko, S. Biophys. J. 1997, 72, 1997-2001. (11) Nicolau, D. V.; Suzuki, H.; Mashiko, S.; Taguchi, T.; Yoshikawa, S. Biophys. J. 1999, 77, 1126-1134. (12) Bunk, R.; Klinth, J.; Montelius, L.; Nicholls, I. A.; Omling, P.; Tagerud, S.; Mansson, A. Biochem. Biophys. Res. Commun. 2003, 301, 783-788. (13) Hiratsuka, Y.; Tada, T.; Oiwa, K.; Kanayama, T.; Uyeda, T. Q. Biophys. J. 2001, 81, 1555-1561. (14) Moorjani, S. G.; Jia, L.; Jackson, T. N.; Hancock, W. O. Nano Lett. 2003, 3, 633-637. (15) Clemmens, J.; Hess, H.; Howard, J.; Vogel, V. Langmuir 2003, 19, 1738-1744. (16) Clemmens, J.; Hess, H.; Lipscomb, R.; Hanein, Y.; Boehringer, K. F.; Matzke, C. M.; Bachand, G. D.; Bunker, B. C.; Vogel, V., Langmuir, in press. (17) Hess, H.; Clemmens, J.; Matzke, C. M.; Bachand, G. D.; Bunker, B. C.; Vogel, V. Appl. Phys. A 2002, 75, 309-313.
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(18) Gittes, F.; Mickey, B.; Nettleton, J.; Howard, J. J. Cell Biol. 1993, 120, 923-934. (19) Howard, J.; Hunt, A. J.; Baek, S. Methods Cell Biol. 1993, 39, 137147. (20) Coy, D. L.; Wagenbach, M.; Howard, J. J. Biol. Chem. 1999, 274, 3667-3671. (21) Standard procedure. After assembly, flow cells were filled for 5 min with a 0.5 mg/mL casein solution to precoat the surfaces in order to reduce kinesin denaturation. The casein solution was exchanged against a solution containing 10% kinesin stock solution, 0.1 mM ATP, and 0.02 mg/mL casein in BRB80 buffer. After 5 min, a microtubule solution together with an antifade system (20 mM DTT, 0.02 mg/mL glucose oxidase, 0.008 mg/mL catalase, 20 mM D-glucose) and 0.15 mg/mL casein and 10 µM paclitaxel and 1 mM ATP was introduced for 15 min. To reduce background fluorescence from the microtubule solution, we perfused the flow cell with a wash solution that was identical to the microtubule solution with the exception that the new solution had no microtubules. (22) Modified procedure. After assembly, flow cells were filled with kinesin stock solution diluted 10-fold in dilution buffer (0.05% Triton ×100; 10 mM Tris acetate, pH 7.5; 50 mM potassium acetate; 4 mM MgSO4; 1 mM EGTA), 10 µM MgATP, and 0.02 mg/mL casein. After 3 min, unbound kinesin was washed out by perfusing with 20 µL dilution buffer plus 10 µM MgATP. A third perfusion with 20 µL dilution buffer plus 10 µM MgATP, and 0.2 mg/mL casein was then added for 3 min. Finally, a microtubule solution (20 µM tubulin) based on dilution buffer together with an antifade system (20 mM DTT, 0.02 mg/mL glucose oxidase, 0.008 mg/mL catalase, 20 mM D-glucose), 0.02 mg/mL casein, 10 µM paclitaxel, and 1 mM ATP was introduced. To reduce background fluorescence from the solution, we perfused the flow cell with dilution buffer together with the antifade system, 0.02 mg/mL casein, 10 µM paclitaxel, and 1 mM ATP for the experiments shown in Figures 5 and 6. (23) Stracke, P.; Bohm, K. J.; Burgold, J.; Schacht, H. J.; Unger, E. Nanotechnology 2000, 11, 52-56. (24) The parallel drag coefficient per unit length of a cylinder near a plane surface is given by c(h) ) 2πη/arcosh(h/r), with η ) solution viscosity, h ) 25 nm - height of microtubule axis above the surface when tethered by kinesin, and r ) 15 nm - radius of microtubule. We approximate the drag coefficient per unit length of a tethered microtubule between two parallel planes c2 as a function of distance D by c2(D) ) c(h) + c(D - h), based on the insight that the dissipation is concentrated on the region between microtubule and surface (see also ref 25). (25) Hunt, A. J.; Gittes, F.; Howard, J. Biophys. J. 1994, 67, 766-781. (26) Caselli, U.; Bertoni-Freddari, C.; Paoloni, R.; Fattoretti, P.; Casoli, T.; Meier-Ruge, W. Gerontology 1999, 45, 307-311. (27) Graf von Keyserlink, D.; Schramm, U. Anat. Anz. 1984, 157, 97111. (28) Mikelberg, F. S.; Drance, S. M.; Schulzer, M.; Yidegiligne, H. M.; Weis, M. M. Ophthalmology 1989, 96, 1325-1328.
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