Biology and engineering come together as scientists aim to create miniature, autonomous analytical devices.
Molecular Motors Meet Microfluidic Systems Rajendrani Mukhopadhyay
© 2005 AMERICAN CHEMICAL SOCIETY
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magine a microfluidic device that could isolate and detect the contents of a single red blood cell within minutes. In the device, the cell components would be picked up by “molecular motors” and carried toward an onboard detector, like crumbs hauled from a picnic table to a nest by ants. These motors would only require simple molecules, like adenosine triphosphate (ATP), as fuel. The fuel would be loaded into the device at the time of manufacture so that the instrument would be self-contained and ready to use. Once the analysis of the red blood cell was completed, the device would be thrown away and replaced with a new one at an inconsequential cost. J U LY 1 , 2 0 0 5 / A N A LY T I C A L C H E M I S T R Y
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Researchers are working toward making these autonomous, miniaturized instruments a reality. The challenges are considerable, but experts are willing to tackle them because the devices promise to fill a niche in analytical instrumentation. “The ability to use pressure-driven flow becomes prohibitive as the size of fluidic channels gets below 5–10 µm, similar to the size of the capillaries in our bodies. Electrokinetic flow works well at smaller dimensions (e.g., ~100 nm) but requires integrated electronics,” explains George Bachand of Sandia National Laboratories. “Molecular motors offer the ability to create true ‘nanofluidic’ systems where all the features are sub-100 nm.”
Why biomolecular motors? Molecular motors fall into two broad categories: synthetic and biological. Examples of synthetic motors include rotaxanes, which are relatively simple motors of ~100 atoms (1). Biological motors are sophisticated proteins with motion that are ubiquitous in cells. They include the rotary motor F0F1-ATPase and the cytoskeleton motor proteins that walk along linear tracks to transport a myriad of cargo (2, 3). Currently, the emphasis is on integrating the biological motors, rather than synthetic ones, into microfluidic devices. In particular, effort is concentrated on the cytoskeleton motor proteins. Henry Hess of the University of Washington explains that cytoskeleton motor proteins “convert their chemical energy with >50% efficiency into mechanical energy. They are already evolved to bind to cargo in a very specific way. One could almost say that the synthetic motors are at this point a few hundred million years behind!” Inspired by these proteins that transport cargo over distances as long as several micrometers inside cells, researchers want to recreate the biological railroad system inside analytical devices. Cytoskeleton motor proteins are classified according to the type of track they use. Myosin motors work on flexible filamentous tracks called F-actin that are 10 nm in diameter. Motors like kinesins walk along hollow, 25-nm-diam pipes called microtubules. The properties of the tracks have a greater influence on which type of motors is used in microfluidic devices than the motors themselves. “Microtubules are like tubes,” says Hess. “It’s much easier to define the path of one of these tubes” than it is to control the path of the spaghetti-like actin filaments, he explains. For this reason, microtubules and kinesins are the more intensely studied cytoskeleton proteins in microfluidic devices. Kinesins come in a variety of forms, but conventional kinesin is the motor of choice because, for the time being, its behavior is the best understood. Conventional kinesin is a processive motor—it can walk as far as a micrometer in a single shot, consuming ATP, before disengaging from a microtubule (4, 5). Microtubules are structurally polar tubes—one end is called the plus end and the other is the minus end. Conventional kinesin only walks toward the plus end of a microtubule. Two 250 A
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head regions of kinesin do the walking in a hand-over-hand fashion. The tail end of the protein binds cargo that can be many times the actual size of the kinesin itself. Kinesin motors are optimized for transport across multiple length scales. Bachand explains, “For example, at the nanometer and micrometer scale, molecular motors are very efficient for transport. Although their transport rates are quite similar to rates of diffusion at this scale, molecular motors also have an intrinsic directionality to transport at this scale, which is absent from diffusional transport. Thus, molecular motors are excellent at transporting cargo from point A to point B along the shortest possible track. A good example is their involvement in chromosomal movement during cell division.” Kinesins also can move cargo over distances of millimeters. The motors can transport vesicles inside axons, which are long extensions protruding out of neuronal cells. These axons, which connect one neuron to the next in our nervous system, can be several millimeters long.
Laying down kinesin and microtubules Kinesins and microtubules can be integrated in one of two configurations in a microfluidic system. The first is the “bead assay”, which mimics the in vivo configuration. Microtubules are put down as tracks in a channel and kinesins step along the microtubules carrying cargo, just the way nature does it. A bead is often attached to a motor so that the movement of the motors can be visualized. The second is the inverted gliding assay configuration. Here, the motor and track switch places. Kinesins are laid down in the channel with the head regions sticking up in the air. Microtubules are modified to carry cargo and are pushed around by the surface-bound kinesins as the heads do the walking motion. According to its proponents, the inverted gliding assay configuration offers more benefits than the bead assay configuration. Viola Vogel of the Swiss Federal Institute of Technology explains: “If you think about creating a transport system as complex as a train, nature’s approach of transporting the cargo along linear rods, where the cargo drops off when reaching the end of the rod, is insufficient. The inverted assay allows us to create complex railroad networks at will where the tracks are functionalized with kinesin. Such networks can be shaped in any manner [with] curved segments and intersections. . . . It allows us to be inspired by active transport, as provided by nature, but then take it to another dimension where we can take control over cargo transport.” Bachand says that another reason the inverted gliding assay configuration is preferred is because long-distance transport can be achieved. The biological geometry, in which kinesin is walking along the microtubule, is a stochastic process, he says. “Even though [kinesin] maintains contact with the microtubule, generally, after 100 steps or so, it has a high probability of falling off. If you tether [kinesin] to a surface and you have 100 kinesins moving a microtubule, that microtubule can move distances of several millimeters or greater. Since [the microtubule] is in contact with several motors, it will never fall off its track and float away,” he explains.
Inverted gliding assay configuration Microtubule
Direction of transport
It’s not easy
using bovine serum albumin or self-assemThe use of biological bled monolayers. motors inside devices How to start and brings together the disstop the kinesin motor parate disciplines of biaction in a controlled ology and engineering, Casein Kinesin manner is also an issue. so it’s understandable Glass So far, researchers have that numerous commanipulated the availplicating factors have ability of ATP, for exemerged. For example, Bead assay configuration ample, by using a caged cantankerous problems Cargo version of ATP which interfacing the device’s can’t be used by kimaterial with the bioKinesin Direction of nesins. When a pulse of molecules have cropped transport UV light irradiates the up. Microtubule device, ATP is freed PDMS, usually the from the cage and the material of choice in motors walk. Once the making microfluidic defree ATP is used up, the vices, is incompatible motors stop. with the fluorescently Edgar Meyhöfer of labeled microtubules the University of Michused for visualization igan says that although purposes in the inverted Silanized glass the caged-ATP strategy gliding assay configuraworks, it doesn’t seem tion. PDMS is highly applicable to the longpermeable to oxygen. The inverted gliding assay configuration and the bead assay configuration are the term goal of developWhen UV light is shone two ways kinesins and microtubules can be integrated into microfluidic devices. ing autonomous microon the PDMS, reac- (Adapted with permission from Ref. 3. Copyright 2001 Elsevier Ltd.) fluidic analytical instrutive oxygen species are thought to be released from the material. These cause the mi- ments. “You really want to load into your devices a sufficient crotubules to fall to pieces within seconds. An oxygen-scav- amount of fuel that can complete the entire job,” he says. “You enging system can be included to mop up the oxygen species. know that from your car. I think we’d consider it as a pretty However, under constant UV illumination, the kinesins and bad strategy to decide to put in only 1 oz of fuel for you to go microtubules only survive for ~10 min because the scavenging to work.” Other strategies could control movement, but the problem is system can’t keep up with the influx of oxygen. William Hancock at the Pennsylvania State University says the lack of a thorough understanding of in vivo kinesin regulathat if an impermeable material, under a thin layer of PDMS, is tion. “In theory, you can use caged magnesium chelators or used to make the channels, then the kinesins and microtubules something like that to stop [the motors]. But in fact, the regulacan carry out their functions. Alternatively, he points out that tion is the difficult part. We don’t understand motor regulation other materials, such as SU8 and PMMA, can be used to con- very well,” says Hancock. Factors such as phosphorylation and steric inhibition are thought to modify kinesin behavior in vivo. struct the devices. Another difficulty is with kinesin. If it is deposited onto a Once researchers have a firmer grasp on how kinesin naturally bare surface, it denatures. This problem can be circumvented if functions, more innovative ways of manipulating motor action in a layer of the protein casein is put down first at saturating con- vitro may be developed. Getting analytes pointed in the right direction inside a device centrations. Once a generous carpet of casein has been placed on the surface, kinesins can be introduced at low concentra- is another problem. The devices are only useful if cargo can be tions. The kinesin motors in- carried along a defined trajectory in a given direction. Retersperse within the casein searchers are attempting to direct all analytes to a central point carpet by a mechanism that is so that the location of the analytes will be known for detection not fully understood and bind purposes. “The problem has never been you don’t have enough to the surface, with the head molecules to detect. You can detect single molecules. The probregions poking out to shuttle lem is you don’t know where they are,” says Ernest F. “Charlie” microtubules around. Besides Hasselbrink at the University of Michigan. “The problem is just casein, other options of ma- an issue of being able to put the molecules exactly where you nipulating the surface proper- want them, and the idea here is to get the motor molecules to do ties of the channels include that for you.” J U LY 1 , 2 0 0 5 / A N A LY T I C A L C H E M I S T R Y
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But it looks like even the mind-set of engineering
field anticipate that synthetic motors The biological interaction bewill soon catch up to biological motween kinesins and microtubules imwill have to change if tors. Vogel says, “I hope, now looking poses some directionality because kiforward to the next 10 years, that nesin only walks toward the plus end some synthetic motors will be powerof microtubules. But how do you get microfluidic devices with ful enough to do strokes that can all the microtubules to go in the transport cargo.” Bachand adds, same direction, from point A to point molecular motors are to “One of the features of the chemical B, without some going from B to A? motors [is that] since they are synthetIn the inverted gliding assay conically made, they most likely have a figuration, rectifiers that look like arbecome the staple analytical much better chance of robustness and rowheads are etched into the walls of stability than the biological motors.” the channel (6). “When the microinstruments of the future. Researchers say the greatest potentubules go in the wrong direction, tial both types of motors hold is the they have a higher probability of runcapability of being produced en ning into a flat wall of the arrowhead masse. The large-scale production of and then turning around and going motors is predicted to drive down the manufacturing costs of the the other direction,” explains Bachand. In the bead assay configuration, researchers have used fluid microfluidic devices. But mass fabrication of devices with nano- and microscale diflow or electric fields to align microtubules in channels. But some experts argue that using external methods for alignment defeats mensions does raise questions about how these devices are engithe purpose of molecular motors in the first place if the device neered. For one, Meyhöfer explains, the materials that are used in research laboratories to build devices may not translate well into can’t be independent of external sources. Longevity of the proteins is also a factor with which re- industrial-scale manufacturing processes. “This is a sort of growsearchers must contend. Proteins will eventually denature, so the ing pain that a field like this will go through. What might be an inevitable has to be delayed by controlling factors like tempera- efficient tool for doing research in evaluating the potential of deture. Bachand says that temperature “can greatly fluctuate due to vice structure may not be an efficient way of mass producing these the small size of the devices and high rate of heat transfer. In devices for the future,” he says. But it looks like even the mind-set of engineering will have to most cases, the operating range is limited to ~45 ºC with kinesin, which is similar to the temperature stability of microtubules.” He change if microfluidic devices with molecular motors are to becontinues, “On the other end of the temperature scale, micro- come the staple analytical instruments of the future. “The typical tubules tend to depolymerize at temperatures
