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Molecular Shuttles OperatingUndercover: A New PhotolithographicApproach for the Fabrication of

Structured Surfaces Supporting DirectedMotility

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 force-

generating 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 single-

molecule studies.6

In our molecular shuttle system, kinesin motor proteins

are adsorbed to a structured surface and transport micro-

tubules, 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

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 motor-

based 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 microtubules

3

or actin filaments,

9

current methodologies rely on fabrication of complex track

patterns using electron-beam lithography,10-12 photolithog-

raphy,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

* Corresponding author. Phone (206) 616-4194. Fax (206) 685-4434.E-mail hhess@u.washington.edu

University of Washington. Sandia National Laboratories.

NANO

LETTERS

2003Vol. 3, No. 12

1651-1655

10.1021/nl0347435 CCC: $25.00 2003 American Chemical SocietyPublished on Web 11/01/2003

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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

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 photo-

lithographically prepared 1 m high walls with a 200 nm

high and 1 m 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 m;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, Fisher-

finest premium slides), two spacers (Scotch double-coated

tape, 3M, St. Paul, MN), and a transparent 0211 glasssubstrate (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 m in

thickness, were cleaned in oxygen plasma at 215 W for 5

Figure 1. Molecular shuttle system envisioned to load, transport,sort, and assemble nanoscale building blocks (top). A hybrid designapproach, combining synthetic environments and biomolecularmotors, utilizes surface-bound kinesin motor proteins to transportfunctionalized microtubules along fabricated tracks.

Figure 2. Previous approaches to guide the movement of micro-tubules and actin filaments on engineered surfaces functionalizedwith motor proteins.

Figure 3. Novel wall geometry for efficient guiding of micro-tubules on motor protein-coated surfaces imaged by scanningelectron microscopy. The undercut prevents microtubules movingin the channel from climbing the sidewall, even if all surfaces arecoated with motor proteins.

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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 surfacesand 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 photo-

resist, 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 M paclitaxel.19 A kinesin construct

(generously provided by J. Howard) consisting of the wild-

type, 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 differ-

ences in microtubule motility could be observed between

the two procedures, which provides additional evidence for

the assumption that no difference in motor adsorption existsbetween 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 limita-

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 cleanmonolayers 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

Figure 4. Microtubules approaching an isolated wall with undercuton the lower surface are efficiently guided along the wall (10 sbetween frames). After moving uninhibited in the undercut, themicrotubules are able to leave the undercut region.

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while remaining in the undercut region. However, microtu-

bules 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 microtu-

bules 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 opensurface (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 m-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

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 descendinto 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 motor-

free region under a slight angle of less than 5 degrees.

The situation here is similar in the sense that the top surfaceis motor-rich, and the guiding channel constitutes a motor-

free region in the plane of the top surface. Therefore, we

can estimate that for 5 m long microtubules, a channel with

a width of 0.5 m and a depth of 1 m 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 microtu-

bules and cargo from the solution, and the narrow tracks on

the bottom plane as a structured delivery system.

Conclusion. The new geometry for microfabricated chan-

nels serving as tracks for molecular shuttles successfullydirects 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 experi-

mental procedure but also permits a multilevel architecture

with two functionally independent planes. While motor-

driven microtubule movement has been previously confined

to micrometer-wide open channels, we succeeded in creating

an environment with submicron dimensions, which ap-

Figure 5. Multiple microtubules crowding the undercut region ofa channel (200 nm 1000 nm) create a situation reminiscent ofthe interior of an axon (D < 1 m).

Figure 6. The large stiffness of the microtubules allows thebridging of guiding channels (focus on top surface, 10 s betweenframes). This decouples the movement on the top and bottom planeand is a first step toward three-dimensional architectures.

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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 SandiaCorporation, a Lockheed Martin Company, for the United

States Department of Energy under contract DE-AC04-

94AL85000.

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, 232-

236.(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, 335-341.

(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.

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(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.

(18) Gittes, F.; Mickey, B.; Nettleton, J.; Howard, J. J. Cell Biol. 1993,

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(21) Standard procedure. After assembly, flow cells were filled for 5 minwith a 0.5 mg/mL casein solution to precoat the surfaces in order to

reduce kinesin denaturation. The casein solution was exchangedagainst 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 mMD-glucose) and 0.15 mg/mL casein and 10 M paclitaxel and 1 mMATP was introduced for 15 min. To reduce background fluorescence

from the microtubule solution, we perfused the flow cell with a washsolution 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 withkinesin stock solution diluted 10-fold in dilution buffer (0.05% Triton

100; 10 mM Tris acetate, pH 7.5; 50 mM potassium acetate; 4mM MgSO4; 1 mM EGTA), 10 M MgATP, and 0.02 mg/mL casein.After 3 min, unbound kinesin was washed out by perfusing with 20

L dilution buffer plus 10 M MgATP. A third perfusion with 20L dilution buffer plus 10 M MgATP, and 0.2 mg/mL casein was

then added for 3 min. Finally, a microtubule solution (20 M 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 mMD-glucose), 0.02 mg/mL casein, 10 M paclitaxel, and 1 mM ATPwas introduced. To reduce background fluorescence from the solution,

we perfused the flow cell with dilution buffer together with theantifade system, 0.02 mg/mL casein, 10 M 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 planesurface is given by c(h) ) 2/arcosh(h/r), with ) 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 distanceD by c2(D) ) c(h) + c(D - h), based on the insight that the

dissipation is concentrated on the region between microtubule andsurface (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, 97-

111.(28) Mikelberg, F. S.; Drance, S. M.; Schulzer, M.; Yidegiligne, H. M.;

Weis, M. M. Ophthalmology 1989, 96, 1325-1328.

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