Biomolecular Catalysis - American Chemical Society

Chapter 21

Surface Patterning and Functionalization for Biomolecular Motor Nanotechnology

Downloaded by PRINCETON UNIV on November 11, 2014 | http://pubs.acs.org Publication Date: June 23, 2008 | doi: 10.1021/bk-2008-0986.ch021

WenLiang He, Thorsten Fischer, and H e n r y Hess* Department of Materials Science and Engineering, University of Florida, Gainesville, FL 32611-6400

The successful integration of biomolecular motors into synthetic environments enables the design of active nanostructures and nanodevices, such as the "molecular shuttle". Over the past few years, continuous efforts have been devoted to guide the movement of such shuttles consisting of surface-adhered motors transporting functionalized filaments. Both, controlled placement of motors and guiding obstacles, have been achieved, employing diverse surface modification techniques including microcontact printing, laser ablation, plasma deposition, photolithography, E-beam and nanoimprint lithography. This paper reviews recent developments in this area, with emphasis on techniques and the corresponding materials used to achieve this goal.

Introduction Organizing transport on the nanoscale is a challenge for nanotechnology. Transport by diffusion is effective on small scales but requires concentration gradients as driving forces. A "molecular shuttle" offers an alternative means to transport nanoscale cargos under user control (1). The idea of the molecular motor shuttle is inspired by nature, where nanoscale transport systems are already realized (2). In cells, motor proteins powered by hydrolysis of adenosine triphosphate (ATP) transport cellular cargos

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© 2008 American Chemical Society

In Biomolecular Catalysis; Kim, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2008.

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throughout the cytoplasm by moving along stationary parts of the cytoskeleton, in particular actin filaments and microtubules (3,4). Molecular shuttle systems often utilize the inverted geometry, where motors are adsorbed to the surface and transport the corresponding filaments, and have found applications as nanoscale sensors and probes (5), in single-molecule studies (6,7), molecular assembly (8), lab-on-a-chip systems (9,10), and nanofluidics (11,12). The primary topic of this paper is to review various means of achieving directed shuttle movement along predetermined tracks. Motor Proteins and Their Related Filaments Kinesin and myosin are two families of molecular motors which have found application in such hybrid systems. Kinesin proteins are eukaryotic microtubuleassociated motor proteins that are typically composed of three parts: (i) two large N-terminal globular "heads" (head region) that allow it to attach to and move along microtubules (MTs), (ii) a central coiled-coil region, and (iii) a C-terminal tail region termed light-chains, which bind kinesins to the cargo (13). Kinesin molecules are about 80 nm in length, and move unidirectional along MTs towards the fast-growing plus end in a hand-over-hand fashion with 8 nm per step (14). Individual kinesin can generate force up to 6 pN (15), and can be adsorbed on surfaces at 10 motors/^m , theoretically producing forces on the order of nN per |im . Microtubules are hollow dimeric tubular protein filaments, self-assembled from a and p tubulins. They are 25 nm in diameter, and range in length from 1 to 100 nm, depending on polymerization conditions (16). Skeletal myosin, termed Myosin II, also contain two globular motor heads, a neck, and a tail domain. Myosin II binds to actin filaments, which are composed of two strands of actin monomer wound around each other in a helical arrangement with a repeat distance of 72 nm are 8 nm in diameter and typically 1 to 10 \xm in length. (16) 3

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Motility Assays Motility assays have been used extensively to determine the mechanisms of molecular motor movements and interactions (17-19). Two distinct configurations are commonly used: the bead motility assay and the gliding motility assay, also known as the "inverted assay", as shown in Figure 1. The bead assay mimics the way motor proteins and filaments interact in nature: the filaments act as static components while motor proteins step along them. In the inverted gliding assay a carpet of motor proteins coats the surface while the head groups propel the filaments. The inverted gliding assay is more popular, due to the possibility of producing extended networks and complex patterns of tracks.



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Figure 1. Illustration of the gliding motility assayfor the kinesin-MT system: casein is pre-coated on glass to support kinesin function, and MT is transported towards its minus end as kinesin moves towards the plus end.

Confinement and Motility Regulation In early studies of both kinesin-MT (20) and myosin-actin systems (18), filaments moved over smooth glass surfaces with no preferred orientation. However, meaningful work cannot be extracted from such systems unless filament motion can be controlled. For the rest of this paper, the topic of filament control will be termed "confinement". Confinement can be achieved via chemical or topographic methods, or by a combination of the two. Chemical confinement is achieved by selectively coating a surface, resulting in regions with high functional motor density and regions with no motor functionality, as seen in Figure 2A. Filaments are confined to move only in regions where motors are available to bind to and transport them. Since a single kinesin motor can move a MT (21), high motility contrast among surfaces cannot be obtained unless the active motor density on the motility blocking regions is decreased to a degree that the average distance between two available motors is smaller than the average length of the filaments being transported. Physical confinement is obtained by physically imposing barriers to confine filament motility, as shown in Figure 2B. If the walls are made of non-fouling materials, filaments will travel only along the bottom motor protein channel, which gives rise to the combined confinement as shown in Figure 2C. Within the channels, filaments are transported in both directions. In order to achieve unidirectional transportation, patterned structures can be added to regulate filament directionality, as shown by Figure 3. For both chemical and combined confinements, protein adsorption is the primary concern and established strategies to prevent it are employed (26). An ethylene glycol based polymer (-CH CH 0-) is commonly used to create a nonfouling surface. This material is also called poly(ethylene glycol) (PEG) when n 2

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Figure 2. Three basic ways to confinefilamentmotility: A. chemical confinement - motility contrast is obtained by contrast in active surface motor density among surfaces; B. physical confinement - filaments can move on both upper and lower surfaces; C. combined confinement - filaments only move within channels. Reproduced with permission from Nano Lett. 2003, 3, 1651-1655.

Copyright 2003 Am. Chem. Soc.

Figure 3. Structures to regulate gliding direction: A. filaments moving upwards are unaffected in their direction of motion; B. Filaments going downwards are redirected upwards (22,23); C. a ratchet pattern used to obtain counter clockwise motion (24,25); D. a structure that can concentrate gliding filaments (25). In Biomolecular Catalysis; Kim, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2008.

358 is around 15-3500, while the term poly(ethylene oxide) (PEO) is designated when n is greater than 3500. Two main classes of PEG-based materials have been utilized in this field: plasma polymerized tetraglyme (pp4G) coating (27,28), and various kinds of poly(ethylene glycol) self-assembled monolayers (PEG-SAM) (23,29,30).


C o m m o n Confinement Strategies Kinesin-MT System overview In general, chemical confinement is of very limited use for the kinesin-MT system (24), due to the high rigidity of the MTs. Successful guidance can only occur at shallow approach angles (defined as the angle between a MT and the sidewall when they were first in contact), since the guiding probability decreases exponentially with approach angle (28), indicating that only narrow tracks can assure desirable filament transportation (27). Since chemical confinement relies on the Brownian motion of the swiveling tip of a gliding microtubule to bind to an off-axis motor, the minimum radius of a curving track is on the order of the persistence length of a trajectory of an unconfined microtubule (0.1 mm (31)). For the actin-myosin system, however, chemical confinement is much more successful due to the more than hundred-fold higher flexibility of actin filaments compared to microtubules (32). Physical barriers can be used to transform the pushing forces of kinesins into bending forces, which makes open channels an attractive means to direct MTs on smaller scales. But confinement by open channels (33,34) also has its own problems (28,35): motor proteins are uniformly adsorbed on all the surfaces, and thus filaments are often observed climbing up the walls, since active motors attached on sidewalk can transport them to the upper surfaces. In fact, for MTs moving parallel to the edges of the walls, the probability of successful confinement is only about 60%, thus the probabilities for climbing up walls or continuing to travel along the channels are almost equal. The guiding probability of physical confinement decreases linearly from about 60% at low angles to almost 0% at 90° angles (28,35). Unlike for chemical confinement, "the narrower the better" does not apply for the width of the channel, but an intermediate value is optimal for directed transport (35). This is explained as a compromise between the favorable reduction of approach angles and the unfavorable increase in the number of collision per distance covered. However, with the introduction of semi-enclosed (36) and totally enclosed channels (37), 100% confinement can be easily achieved, since microtubules are prevented from climbing the overhanging sidewall. But for such restricted structures, two problems may occur: (i) the supply of reagents, e.g. ATP, is restricted and (ii) the introduction of solutions without creating trapped air bubbles is challenging.


359 Combined confinement is a very successful method to control filament movement (22,28,29,38), since by creating non-fouling sidewalls microtubules cannot bind to motors and escape. The probability of successful confinement is typically over 90%.


Myosin-Actin System overview For the myosin-actin system, it has been shown that only cationic or hydrophobic surfaces can adsorb and support myosin motor function (12,39-43), while negatively charged or hydrophilic surfaces are usually used to bring motility contrast to the system. Among the photoresists, MRL-6000 l.XP (11,12,44) is identified as a superior material supporting motility, while treatment of glass surfaces with hexamethyldisilazane (HMDS) (45), trimethylchlorosilane (TMCS) (41) and positively charged poly(allylamine hydrochloride) (PAH) (42) also supported myosin-induced actin motility. Ebeam treated tBuMA & MM A (4:1) (39) and PMMA 950 (11,12,44) are photoresists that disable myosin motor function and this effect can be further enhanced by 0 plasma etching (12). Bovine serum albumin (BSA) (43,45) and negatively charged PEBSS (42) as well as poly(styrene sulfonic acid) (PSS) are reagents that help to block motor protein adsorption onto surfaces. Compared to the kinesin-MT system, actin filaments move much faster (typically more than 4 \im/s; MTs typically move less than 1 |xm/s) and can pass through smaller curvatures (through circles 2 nm in diameter), which enables fast transportation and miniaturization of devices. However, much narrower channels (usually down to 100 - 500 nm) are also required to effectively confine actin filament motility and to prevent it from taking U-turns. The requirement of a hydrophobic surface to support myosin motor function may further impose some difficulty with respect to solution introduction - air bubbles can be trapped in the channels due to the unfavorable interactions between the hydrophobic surface and the aqueous buffer solutions. In the first motility assays (18), nitrocellulose was spread on a glass slide to produce a hydrophobic surface that supported myosin function. However, nitrocellulose lacks the ability to be lithographically patterned, and it does not facilitate production of films with controlled uniform thickness. Later studies focused on finding effective methods to confine and regulate actin filament motility. 2

Methods to Achieve Chemical Confinement Thermal Stretching ofPTFE Through mechanical deposition of PTFE on glass a layer of polymer with ridges and grooves along the sheer-axis can be produced for both kinesin-MT (1) In Biomolecular Catalysis; Kim, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2008.

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and myosin-actin systems (46). The dimensions of these ridges are randomly distributed; they are 10 - 20 nm in height, 10 -100 nm in width and 10 - 100 nm apart. Although MTs were observed to become oriented toward the shear-axis after several directional changes the oriented motion was maintained only at moderate motor densities. For lower motor density, MTs tended to detach, while for higher motor density, MTs began moving randomly as they switched among adjacent lines. Similarly, at high motor density areas, orientated actin filament motility would be lost, and actin filaments tended to switch among lines and to take U-turns.

E-beam Treatment of tBuMA.MMA (4:1) Copolymer Under E-beam exposure, the copolymer of tert-butyl-methacrylate (tBuMA) and methyl methacrylate (MMA) (4:1) tends to become more hydrophilic as the contact angle is dropped from 87° to 82° (40). Thus, contrast in protein adsorption can be produced by selectively exposing the photoresist, which creates hydrophilic regions surrounded by hydrophobic regions and discourages adsorption of functional myosin. Unfortunately, the mobility confinement obtained by this method was not very effective; both the exposed hydrophilic regions and the unexposed hydrophobic regions absorbed motor proteins. Filaments moving on myosin-rich regions tended to be either immediately reflected or gradually deflected when they encountered the edges, while filaments on myosin-poor regions were not guided by the edges (40).

Glow Charge Plasma Deposition of pp4G Tetraglyme, C H 3 - 0 - ( C H C H 0 ) 4 - C H 3 , also known as tetraethylene glycol dimethyl ether, can be used to deposit a "PEG-like" coating by glow charge plasma deposition. This coating strongly exhibits the hydrophilic character of PEG, and thus it discourages kinesin motor adsorption effectively. As shown in Figure 4, selective coating of polymerized tetraglyme on glass can be obtained by a succession of UV lithography, plasma charge deposition and final lift-off process (27). Through this method, Lipscomb et al. (27) have demonstrated that narrow and smooth tetraglyme tracks can significantly improve MT motility performance under chemical confinement. When MTs moved along rough and wide (125 pm) tracks, they couldn't trace the edges but tended to detach when they reached the edges. On the other hand, MTs could be reflected by the edges and several of them traveled as long as 250 pm along smooth and narrow (2 pm) lines. Clemmens et al. (28) also showed that about 88% of MTs would detach as they encountered the edges, and chemical confinement was effective only at very shallow approach angles (< 10°, almost 100%), while it became almost useless at larger angles (> 30°, almost 0%). 2

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Biomolecular Catalysis - American Chemical Society

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