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Selective Assembly and Guiding of Actomyosin Using Carbon Nanotube Network Monolayer Patterns Kyung-Eun Byun,† Min-Gon Kim,‡ P. Bryant Chase,§ and Seunghun Hong*,† Physics and Astronomy, Seoul National UniVersity, Shilim-Dong, Kwanak-Gu, Seoul, Korea, BioNanotechnology Research Center, KRIBB, Daejeon, Korea, and Biological Science, Program in Molecular Biophysics, and Chemical & Biomedical Engineering, Florida State UniVersity, Tallahassee, Florida 32306 ReceiVed June 29, 2007. In Final Form: July 30, 2007 We report a new method for the selective assembly and guiding of actomyosin using carbon nanotube patterns. In this method, monolayer patterns of the single-walled carbon nanotube (swCNT) network were prepared via the self-limiting mechanism during the directed assembly process, and they were used to block the adsorption of both myosin and actin filaments on specific substrate regions. The swCNT network patterns were also used as an efficient barrier for the guiding experiments of actomyosin. This is the first result showing that inorganic nanostructures such as carbon nanotubes can be used to control the adsorption and activity of actomyosin. This strategy is advantageous over previous methods because it does not require complicated biomolecular linking processes and nonbiological nanostructures are usually more stable than biomolecular linkers.
Motor protein has great potential for the application of nanomechanical systems because of its small dimensions and high fuel efficiency.1-13 One example is actomyosin composed of actin and myosin. Myosin walks on an actin filament by consuming ATP as fuel, which is the basic mechanism of muscle contraction in our body. The key technology for protein motorbased nanomechanical systems is a method that can be used to assemble motor protein selectively onto desired locations on solid substrates and control its motility. Previous reports showed the successful guiding of protein motor motions using physical walls. However, these methods required significant modification of surface morphology. However, some researchers reported the selective assembly of motor protein utilizing biomolecular linkers or an organic self-assembled monolayer (SAM).14-19 However, * To whom correspondence should be addressed. E-mail: seunghun@ snu.ac.kr. Phone: 82-2-880-1343. Fax: 82-2-884-3002. † Seoul National University. ‡ BioNanotechnology Research Center. § Florida State University. (1) Vale, R. D.; Milligan, R. A. Science 2000, 288, 88. (2) Schliwa, M.; Woehlke, G. Nature 2003, 422, 759. (3) Ishii, Y.; Esaki, S.; Yanagida, T. Appl. Phys. A 2002, 75, 325. (4) Forkey, J. N.; Quinlan, M. E.; Shaw, M. A.; Corrie, J. E. T.; Goldman, Y. E. Nature 2003, 422, 399. (5) Clemmens, J.; Hess, H.; Lipscomb, R.; Hanein, Y.; Bohringer, K. F.; Matzke, C. M.; Bachand, G. D.; Bunker, B. C.; Vogel, V. Langmuir 2003, 19, 10967. (6) Romet-Lemonne, G.; VanDuijin, M.; Dogteron, M. Nano Lett. 2005, 5, 2350. (7) Asokan, S. B.; Jawerth, L.; Carroll, R. L.; Cheney, R. E.; Washburn, S.; Superfine, R. Nano Lett. 2003, 3, 431. (8) Suda, H.; Sasaki, N.; Sasaki, Y. C.; Goto, K. Eur. Biophys. J. 2004, 33, 469. (9) Giffard, R. G.; Weeds, A. G.; Spudich, J. A. J. Cell Biol. 1984, 98, 1796. (10) Niemeyer, C. M. Angew. Chem., Int. Ed. 2001, 40, 4128. (11) Soong, R. K.; Bachand, G. D.; Neves, H. P.; Olkhovets, A. G.; Craighead, H. G.; Montemagno, C. D. Science 2000, 290, 1555. (12) Gooding, J. J.; Wibowo, R.; Liu, J.; Yang, W.; Losic, D.; Orbons, S.; Mearns, F. J.; Shapter, J. G.; Hibbert, D. B. J. Am. Chem. Soc. 2003, 125, 9006. (13) Zanello, L. P.; Zhao, B.; Hu, H.; Haddon, R. C. Nano Lett. 2006, 6, 562. (14) Sundberg, M.; Rosengren, J. P.; Bunk, R.; Lindahl, J.; Nicholls, I. A.; Tagerud, S.; Omling, P.; Montelius, L.; Mansson, A. Anal. Biochem. 2003, 323, 127. (15) Jaber, J. A.; Chase, P. B.; Schlenoff, J. B. Nano Lett. 2003, 3, 1505. (16) van den Heuvel, M. G. L.; Butcher, C. T.; Lemay, S. G.; Diez, S.; Dekker, C. Nano Lett. 2005, 5, 235. (17) Suzuki, H.; Yamada, A.; Oiwa, K.; Nakayama, H.; Mashiko, S. Biophys. J. 1997, 72, 1997. (18) Manandhar, P.; Huang, L.; Grubich, J. R.; Hutchinson, J. W.; Chase, P. B.; Hong, S. Langmuir 2005, 21, 3213.
organic molecular linkers were usually unstable, and previous methods based on biomolecular linkers required complicated processing steps that often included the modification of the motor protein itself. Herein, we report a simple, efficient method for the selective assembly and guiding of actomyosin on solid substrates. In this process, monolayer patterns of single-walled carbon nanotube (swCNT) networks were prepared on a solid substrate via the self-limiting mechanism during the directed assembly processes,20-26 and they were used to block the adsorption of biomotors in specific surface regions on the substrate. We successfully demonstrated the selective adsorption and guiding experiments of actomyosin using swCNT network patterns. This is the first report showing that a layer of inorganic nanostructures such as an swCNT network monolayer can be an efficient means of controlling the adsorption and motility of actomyosin. Because this method does not require complicated biomolecular processing steps or significant modification of surface morphology, it can be an ideal strategy for the future applications of biomotor-based nanomechanical systems. Figure 1 shows the schematic diagram depicting the basic concepts of our experiments. At first, SAM patterns composed of polar and nonpolar regions were prepared for the directed assembly of swCNTs (Figure 1a).20-26 The details of SAM patterning methods can be found elsewhere.23-35 Dip-pen (19) Clemmens, J.; Hess, H.; Howard, J.; Vogel, V. Langmuir 2003, 19, 1738. (20) Wang, Y.; Maspoch, D.; Zou, S.; Schatz, G. C.; Smalley, R.; Mirkin, C. A. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 2026. (21) Oh, S. J.; Cheng, Y.; Zhang, J.; Shimoda, H.; Zhou, O. Appl. Phys. Lett. 2003, 82, 2521. (22) Rao, S. G.; Huang, L.; Setyawan, W.; Hong, S. Nature 2003, 425, 36. (23) Myung, S.; Lee, M.; Kim, G. T.; Ha, J. S.; Hong, S. AdV. Mater. 2005, 17, 2361. (24) Im, J.; Huang, L.; Kang, J.; Lee, M. B.; Lee, D. J.; Rao, S.; Lee, N. K.; Hong, S. J. Chem. Phys. 2006, 124, 224707. (25) Lee, M.; Im, J.; Lee, B. Y.; Myung, S.; Kang, J.; Huang, L.; Kwon, Y.-K.; Hong, S. Nat. Nanotechnol. 2006, 1, 66. (26) Hannon, J. B.; Afzali, A.; Klinke, Ch; Avouris, Ph. Langmuir 2005, 21, 8569. (27) Piner, R. D.; Zhu, J.; Xu, F.; Hong, S.; Mirkin, C. A. Science 1999, 283, 661. (28) Maynor, B. W.; Li, Y.; Liu, J. Langmuir 2001, 17, 2575. (29) Manandhar, P.; Jang, J.; Schatz, G. C.; Ratner, M. A.; Hong, S. Phys. ReV. Lett. 2003, 90, 115505. (30) Cho, N.; Ryu, S.; Kim, B.; Schatz, G. C.; Hong, S. J. Chem. Phys. 2006, 124, 24714. (31) Nyamjav, D.; Ivanisevic, A. AdV. Mater. 2003, 15, 1805.
10.1021/la7019318 CCC: $37.00 © 2007 American Chemical Society Published on Web 08/18/2007
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Figure 1. Schematic diagram depicting the basic concepts of our experiment. (a) SAM patterning to create polar and nonpolar regions on solid substrates. (b) Selective adsorption and alignment of swCNTs on polar regions in swCNT solution. After the adsorption of swCNTs onto bare surface regions, the exposed bare surface was backfilled with nonpolar SAMs to create swCNT patterns on the nonpolar surface. (c) Selective adsorption of HMM on regions without swCNT coatings. (d) Addition of actin filaments and motility assay.
nanolithography (DPN) was utilized to generate nanoscale SAM patterns in the early stages of our process development.27-34 However, microcontact printing (µCP)35 and photolithography23,25 were utilized to pattern SAMs over a large area on Au and SiOx substrates, respectively. In a common process, nonpolar molecules such as 1-octadecanethiol (ODT) and octadecyltrichlorosilane (OTS) were first patterned on Au and SiOx, respectively. The remaining bare surface region with polar properties was used without further modification, or it was backfilled with polar SAMs to prepare a polar surface with well-controlled properties. On Au surfaces, cysteamine and 16-mercaptohexadecanoic acid (MHA) were used to create positively and negatively charged polar surfaces, respectively. A 3-aminopropyltriethoxysilane (APTES) SAM was utilized to prepare positively charged polar surfaces on SiO2 and glass substrates. When the patterned substrate was placed in swCNT suspensions (0.2 mg/mL swCNT in o-dichlorobenzene) for 30 s, swCNTs were adsorbed selectively onto polar regions (Figure 1b). The substrate was then rinsed with o-dichlorobenzene to remove any extra swCNTs. These steps allowed us to prepare swCNT patterns on polar surfaces. swCNT patterns on nonpolar surfaces were prepared by first adsorbing swCNTs directly onto bare surfaces and then backfilling the exposed bare surface area with nonpolar SAM molecules (Supporting Information). After patterning swCNTs, we followed the procedure of conventional protein motor assay.18 Actin and myosin were extracted from rabbit skeletal muscle, and heavy meromyosin (HMM) was prepared from myosin by the method as described by Kron et al.36 Actin filaments were labeled with rhodaminephalloidin (RhPh). Flow cells for motility assay were constructed using vacuum grease and coverslip spacers.18 In the flow cell, HMM was found to adsorb selectively on the regions without swCNT coatings (Figure 1c). Then, the region without adsorbed HMM was passivated with bovine serum albumin (BSA) to block nonspecific actin adsorption. Finally, the RhPh-labeled actin filament solution was applied to the surface (Figure 1d) so that (32) Jaschke, M.; Butt, H. J. Langmuir 1995, 11, 1061. (33) Sheehan, P. E.; Whitman, L. J. Phys. ReV. Lett. 2002, 88, 156104. (34) Coffey, D. C.; Ginger, D. S. J. Am. Chem. Soc. 2005, 127, 4564. (35) Xia, Y.; Whitesides, G. M. J. Am. Chem. Soc. 1995, 117, 3274. (36) Kron, S. J.; Toyoshima, Y. Y.; Uyeda, T. Q. P.; Spudich, J. A. Methods Enzymol. 1991, 196, 399.
we could observe the translocation of actin by HMM via a fluorescence microscope (Nikon TE2000U microscope equipped with an intensified CCD VE-1000 SIT). We used Metamorph analysis software (purchased from Molecular Devices) for the motility analysis of actin translocation. Figure 2a shows swCNTs selectively adsorbed onto a polar cysteamine SAM on the Au substrate. It should be noted that swCNTs did not cross the boundary, and the patterns exhibited well-defined edges.22,25 The graph in Figure 2b shows the effective thickness of adsorbed swCNT layers depending on the dipping time of the substrate in the swCNT suspensions. The effective thickness of the swCNT network film was obtained by first measuring the volume of adsorbed swCNTs using AFM topography and then dividing it by the pattern area.25 Unlike conventional thin films (e.g., Au, SiO2, etc.) that often exhibited atomically flat surfaces, swCNT films were composed of rough swCNT networks, which resulted in relatively large error bars in the effective thickness graph. An important aspect of this swCNT assembly process is the self-limiting mechanism. In the early stage of the adsorption, the thickness of the swCNT film increased quickly. As more and more swCNTs, which have nonpolar surfaces, were adsorbed onto the polar surface regions, the surface became less polar, and additional swCNT adsorption was reduced until the saturation thickness was reached.24 This has similar behavior to the Langmuir isotherm, and we could fit our data using the Langmuir isotherm model. The saturation number of adsorbed swCNTs per unit area is ∼6.9/µm2.24 We utilized swCNT patterns with saturated swCNT density for all actomyosin experiments in this letter. It is significant that this self-limiting mechanism of the swCNT adsorption process allowed us to prepare monolayer coatings of swCNT network with a uniform number density of swCNTs in different regions, which can be ideal for the control of motor protein adsorption without significant modifications of surface morphology. This simple strategy was utilized to prepare nano- and microscale swCNT network patterns over a large surface area (∼1 cm2) on commonly used substrates such as SiO2 (Figure 2c), glass (Figure 2d), and Au (Figure 2e). It should be noted that, unlike self-assembled monolayer films, swCNTs formed random networks, and they did not form a densely close-packed monolayer. However, because of the self-limited mechanism,
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Figure 2. Patterning of the swCNT network over a large surface area (∼1 cm2) on various substrates. (a) Atomic force microscope (AFM) topography image of swCNTs (white line) adsorbed on cysteamine SAM regions (dark regions) on the Au surface. ODT SAM (brighter area) was patterned via the microcontact printing method and used for passivation. (b) Effective thickness of swCNT films adsorbed onto bare Au surfaces depending on the dipping time of the substrate in the swCNT suspensions. The graph shows the averaged values measured from 10 samples. The data were fitted using the Langmuir isotherm model.24 The R2 value is ∼0.9908. (c) AFM topography image of swCNT adsorbed onto bare SiO2 regions. The remaining area was passivated with OTS. (d) AFM topography image of swCNT’s adsorbed onto bare glass regions. The OTS SAM was patterned via photolithography and used for passivation. (e) AFM topography image of swCNT’s adsorbed onto nanoscale bare Au regions. The ODT SAM was patterned via microcontact printing and was used for passivation.
swCNTs formed a single layer of swCNT networks with a saturated swCNT number density, which was somewhat analogous to the maximum density monolayer of molecular films. We examined the effect of swCNT coating on HMM adsorption via conventional motility assay (Figure 3). In this experiment, HMM was first adsorbed onto various substrates with or without swCNT coatings, and the motility of actin filaments on the substrate was measured using fluorescence microscopy. The movie frames in Figure 3a show the typical motion of actin filaments on swCNT-coated cysteamine SAM surfaces. We could rarely find moving actin filaments on swCNT-coated substrates. Even a few moving actin filaments soon diffuse into the solution and detach from the substrate (Figure 3a). We confirmed via fluorescence microscopy that the diffusing actin filaments were floating in the solution. Presumably, active HMM density on swCNT-coated substrates was extremely low, and the substrates could not support actomyosin motility. We found actin filaments moving continuously without diffusion events on cysteamine SAM surfaces without swCNT coatings.18 We performed a systematic study of HMM adsorption onto swCNT-coated surfaces using three typical substrates, Au, cysteamine SAM, and ODT SAM, which were reported to support actomyosin motility (Figure 3b).18 Cysteamine SAM is terminated with amine groups that are positively charged in physiological buffer solution, whereas methyl-terminated ODT SAM remains neutral. Previous reports showed that both positively charged and neutral surfaces supported actomyosin motility well whereas negatively charged surfaces such as MHA SAM did not.18 A nitrocellulose-coated coverslip was used as a reference substrate for the in vitro motility assay. Our experiment showed that bare Au, cysteamine SAM, and ODT SAM surfaces without swCNT coatings supported motor activity very well, whereas we observed no or very little motility after swCNT coating (Figure 3b). These
results imply that the swCNT network monolayer significantly reduced the adsorption of active HMM. One important question might be whether the swCNT layer blocked the adsorption of HMM or simply denatured the adsorbed HMM. Figure 3c shows the fluorescence micrograph image of the HMM (labeled with Alexa 555) adsorbed onto patterns composed of ODT SAM regions and swCNT-coated ODT SAM regions.37 Here, the regions without swCNT coatings were much brighter than the swCNT-coated regions. The results clearly show that swCNT network patterns actually blocked the adsorption of HMM. The immunofluorescence image for HMM adhesion using the antibody is also provided as Supporting Information. We also studied the effect of swCNTs on actin filament adsorption (Figure 4). Here, we applied an actin filament solution (concentration ∼0.033 mg/mL) to various substrates for 30 min and observed the number of adsorbed actin filaments. Figure 4a shows that actin filaments exhibited a strong affinity for MHA SAMs. We found that the swCNT coating on MHA SAMs dramatically reduced the actin filament adsorption (Figure 4b). Even the adsorbed actin filaments anchored to the surface by only a small portion so that the remaining parts showed Brownian motion in solution. We observed similar behaviors on various surfaces (Figure 4c). For efficient guiding of actomyosin using swCNT network patterns as a barrier, it is crucial that swCNT patterns can block the adsorption of actin filaments as well as HMM. Our results show that the swCNT network monolayer effectively blocked the adhesion of both HMM and actin filaments and can be an ideal barrier for the guiding experiments of actomyosin. One advantage of swCNT film as a blocking reagent compared with biological reagents such as BSA might be its (37) Byon, H. R.; Hong, B. J.; Gho, Y. S.; Park, J. W.; Choi, H. C. ChemBioChem 2005, 6, 1331.
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Figure 3. (a) Movie frames of a fluorescence micrograph showing the diffusion event of RhPh-labled actin filaments on an swCNT-covered surface. The directions of actin filament motion are marked by arrows, and the actin filaments that are about to diffuse into the solution are marked by dotted circles. (b) Motility of HMM adsorbed on conventional surfaces (nitrocellulose) and other typical surfaces with or without an swCNT coating. We observed a significant reduction of actin motility on swCNT-coated surfaces implying the reduced adsorption of HMM on swCNT-coated surfaces. (c) Fluorescence image of Alexa 555-labeled HMM adsorbed onto the patterns composed of ODT SAM regions and swCNT-coated ODT SAM regions.
Figure 4. Fluorescence micrograph images showing the adsorption behaviors of actin filaments on (a) an MHA SAM and (b) an swCNTcovered MHA SAM. (c) Number of actin filaments adsorbed onto various substrates when the substrates were kept in the solution of actin filaments for 30 min.
stability. The blocking result by BSA was provided as Supporting Information for comparison. However, swCNTs formed stable film structures, and we could continue additional process (e.g.,
photolithography) after swCNT adsorption. However, because swCNTs were not adsorbed onto hydrophobic surfaces, they could not be used on nonpolar surfaces. We successfully performed guiding experiments of actomyosin utilizing swCNT network patterns as a barrier (Figure 5). In this experiment, we studied the motility of actomyosin on the patterns composed of ODT SAM regions and swCNT-coated cysteamine SAM regions. Actin filaments exhibited active motion on ODT SAM, and they were floating in the solution without motion on the swCNT-coated cysteamine SAM. The movie file showing this motion is provided as Supporting Information. It should be noted that the blurred white lines on the swCNT regions were actin filaments in the solution away from the substrate. Presumably, they were released from the swCNT-coated surfaces after initial physisorption for a short time period (Figure 3). When an actin filament moving in the ODT region encountered the swCNT region, it exhibited one of two different behaviors depending on the angle of incidence. When the angle of incidence was relatively small, the actin filament diffused away from the surface (Figure 5a). However, if it encountered the boundary with a large angle of incidence, it was reflected by the swCNT patterns, and as a result, its translocation was guided (Figure 5b). The guiding probability was measured by calculating the ratio of guiding events to total actin filaments that encountered the boundary at a certain angle of incidence θ (Figure 5c). We observed no actin filament crossing the swCNT-coated regions, implying that the swCNT coating was an efficient barrier for the guiding experiments of actomyosin. We also performed the motility experiments on ODT and cysteamine patterns without swCNTs. Because both ODT and cysteamine SAM supported the actomyosin mobility very well, SAM patterns without the swCNT coating did not guide the actomyosin.18
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Figure 5. Motion of actin filaments on HMM adsorbed onto patterns composed of ODT and swCNT-coated cysteamine SAMs. Only actin filaments on the ODT SAM exhibited high motility. On the swCNT-coated cysteamine SAM, actin filaments were floating in the solution without motion. (a) Movie frames of a fluorescence micrograph showing the diffusion event of an actin filament on the boundary. The actin filament diffused into the solution and detached from the substrate. (b) Movie frames of fluorescence micrograph showing a guiding event on the boundary. (c) Guiding probability as a function of the angle of incidence θ. The guiding probability represents the number of guiding events divided by total number of actin filaments that hit the boundary with a certain angle of incidence θ. We measured a total of 149 events showing actin translocation from movie frames.
In summary, we developed a new method for the selective adsorption and guiding experiments utilizing swCNT network patterns. In this strategy, swCNT network patterns with uniform number density were prepared via the self-limiting mechanism during the directed assembly process and were used to selectively block the adsorption of both HMM and actin filaments on the substrates. This is the first successful demonstration that inorganic nanostructured surfaces such as swCNT network patterns can be utilized for the selective adsorption and guiding of protein motors. Significantly, because our method does not require complicated biomolecular linking processes and the swCNT coating is only a few nanometers thick, it can be an ideal strategy for future applications of biomotor-based nanomechanical systems.
Acknowledgment. We thank Lori McFadden, Brenda Schoffstall, Nicolas Brunet, and Victor Miller in the Department of Biological Science, Florida State University, for their help in preparing proteins. This project was supported by the NANOSystem Institute National Core Research Center (NSI-NCRC) program of KOSEF and in part by MOCIE. S.H. acknowledges financial support from the National Research Laboratory program. Supporting Information Available: Protocol to assemble swCNTs onto nonpolar surfaces, immunofluorescence assay for HMM adhesion, blocking experiment results by BSA, and movie files showing guiding experiments on swCNT network patterns. This material is available free of charge via the Internet at http://pubs.acs.org. LA7019318
