Biomod/2011/Columbia/MotorProTeam:ProjectDevelopment

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Research Topic

Brainstorming for the project began in February 2011. The following ideas were taken into consideration for the competition:

1. Genetic engineering of hemoglobin
2. Microtubule sheets for force multiplication of kinesin
3. Electric power from ATP
4. Smart indicator supermolecule (multi-receptor recognition by multifunctional drug delivery vehicle)
5. Slow protein unfolding by motor proteisn
6. Self-Healing
7. Nanofactory using molecular shuttles
8. Circuit assembly by molecular shuttles

We ultimately decided on an adaptation of the second idea (microtubule sheets for force multiplication of kinesins). This choice was based on interest, feasibility, and the potential application of the prospective projects. We aim to create a universal transport system that can selectively carry nano- and micro- scale cargo over macroscopic distances.

Materials and Methods

To grow the microtubules, 0.5 μL MgCl₂, 0.5μL GTP, 0.6μL DMSO, and 10.9 μL BRB80 are mixed in a small tube. 6.25μL of this growth solution is added to a 20 μg aliquot of rhodamine labeled tubulin. The aliquot is vortexed and heated for 30 min at 37°C.

MT100 (the microtubule stock solution) is made using 490μL BRB80+5μL Taxol+5μL of the prepared microtubules.

BRB80CS is prepared using 58.54μL BRB80 (a buffer) and 1.46μL Casein. This coats the glass surface with casein.

Kinesin motor solution (for a surface density of 2,000kinesin/μm^2) was prepared with 40μL BRB80+0.6μL ATP+1.2μL CS, and two minutes later, 7.44μL Kinesin. This introduces kinesin to the surface.

The motility solution is prepared using 81.6μL BRB80+2.43μL Casein+1μL (Taxol [TX], DG [D-Glucose], Catalase [Cat], Dithiotheritol [DTT], ATP)+10μL MT100. This introduces microtubules to the flow cell.

The antifade solution was prepared with 81.6μL BRB80+2.43μL Casein+1μL (Taxol [TX], DG [D-Glucose], Catalase [Cat], Dithiotheritol [DTT], ATP). This solution flushes away any unattached microtubules.

To make the flow cell, double sided tape (or vacuum grease) is added to each side of a cover slip. A small coverslip placed on top completes the flow cell. This creates a 10-20mm^3 volume for the solutions to flow. A pipet is used to flow solution in from one side, and filter paper collects the solution from the other.

20 μL BRB80CS solution is flowed into the cell. Five minutes later, 30 μL kinesin motor solution is flowed. Five minutes later, 30μL motility solution is flowed. 5 minutes later, 30μL antifade solution is flowed. Then, the flow cell iss imaged under the microscope.

The current method of using molecular shuttles for transport only utilizes the force of an individual microtubule. Micrometer sized cargo can be bound to and transported via the microtubule over millimeter distances. This method, however, is not a very reliable means to transport cargo. Bound cargo would follow the filament rotation of the microtubule and can get stuck on the surface of the flow cell or the cargo can get knocked off the surface of the microtubule. This calls for a new procedure that can optimize cargo transport. By creating a larger structure, microtubules are less likely to be removed from the surface. Moreover, with the use of multiple microtubules, the cargo can stay in place even if it is displaced from one individual microtubule. The force of multiple microtubules should be faster and more reliable than the use of one microtubule to transport nano and micro-spheres.

Structure

Brainstorming

From this point, we met to design the structure of the device.

Initially, we aimed to create a raft-like surface, that is, an array of microtubules that were of one length, pointed in the same direction, and were connected either by biotin-streptavidin bonds. This idea, although elegant, was unfeasible. When multiple microtubules are connected, they form spool-like structures rather than a raft-like structure. Controlling the length of each microtubule also presented a challenge to the idea. We considered using dynein as an alternate to kinesin because of its stiffness. But regardless, the nature of microtubules, when put together, would make this structure impossible.

One potential alternate was a cylindrical system that operated on bead-geometry as opposed to the motility assay. A cylinder would be constructed of microtubules, and cargo would attach to the cylinder. Kinesin molecules on a fixed surface would pass the microtubules along thereby moving the cargo. The benefit of this structure is in the high surface area to volume ratio of the cylinder. This would allow a large amount of cargo to be carried along. Unfortunately, however, the bead geometry is much less effective than the gliding geometry. We therefore continued theorizing and discussing potential structures for the universal transporter.

While waiting for the patterned silicon wafers to arrive, we brainstormed more ideas for the structure. These included constructing a sheet out of biotinlated nano- or microspheres. When the solution containing the biotin spheres dries out, the spheres form an array. Biotin-Streptavidin bonds could then link microtubules to the bottom of the array. Cargo could then load onto the top surface of the array.

The next, and probably most promising idea resembled a nano-truck constructed from microtubules, motor proteins, and a sheet of PDMS as the loading dock. Essentially, we would have a sheet of PDMS (which we could then geometrically manipulate to maximize carrying efficiency) with an oxidized, hydrophilic bottom surface. Microtubules would attach to the bottom surface. Extending the metaphor, these microtubules are the analog wheels of the transport vehicle.

Polymer sheets

Images of possible structure ideas:

First, one sheet of PDMS without any geometric modification would serve as the test structure. This allows us to prevent any steric complications from affecting the results.

(insert image here)

Next, we would work with a topless box-like structure. This allows more space for the particles which we plan on transporting to attach.

(insert image here)

If the topless box works, a surface with a grated pattern (think teeth with gaps between) would be the ideal structure. We can create this by building a mold into which the PDMS is poured.

Image of ridged structure, from Xia and Whitesides, 1998

PDMS, however, could not be made on a small enough scale to fit in the flow cell, so an alternate material to create the structure was polyurethane. We used soft lithography to create a pattern on which we poured PDMS. After the PDMS cured, we poured polyurethane into the patterned side, then scraped the excess off.

Implementing the design

The structure would have to be small enough to fit inside a flow cell. In order to achieve this type of sizing, silicon wafers were patterned with a photoresist mask. Handling such small particles manually would be virtually impossible, so a combination of microtransfer molding and a lift-off technique would be used to acquire small squares of PDMS.

SU-8 photoresist was used to print a pattern on the mask. PDMS was added ontop of the silicon wafer and then removed. This created a PDMS mold. Next, a sacrificial surface was made. A plane of featureless photoresist was added to the top of a silicon wafer. The PDMS mold was micro-transferred onto the photoresist surface. A pre-polymer was used to fill in the grooves of the PDMS mold, and the structure was placed ontop of the photoresist. The prepolymer was cured and the PDMS was peeled off. This results in a layer of photoresist with a pre-polymer pattern.

photolithographic mask

20 micrometer squares on mask

30 micrometer squares on mask

50 micrometer squares on mask

100 micrometer squares on mask

200 micrometer squares on mask

A flow cell was created on the silicon wafer with the photoresist and pre-polymer patterning. The standard flow procedure was used, and microtubules would align and stick to the prepolymer. Next, a diluted ethanol solution was used to dissolve the photoresist, thereby freeing the pre-polymer structures. This would result in a solution with pre-polymers coated with aligned microtubules.

Graphic schematic design of the procedure.

Alignment

After deciding on a structure, we investigated methods to align microtubules. The simplest method appeared to be using magnetic fields to align the microtubules. The procedure was unfeasible to perform within the constraints of the lab because magnetic fields had to be applied in the process of growing the microtubules. This posed a road block (no pun intended).

Another common method of alignment is using channels. Channels can be constructed out of PDMS and force the microtubules to align. However, there is no method to remove the microtubules from the channels without losing the alignment. We considered making the channels on the lower side of the structure and align the microtubules directly onto it. These microtubules, however, would not be functional because microtubules need to protrude from the channel in order to move along the kinesin motors below it. The height of the channel for this should be around 12 nm (half of the diameter of a microtubule), and this detail is impossible to achieve using the standard lithography techniques.

One alternative was using force within the flow cell to align microtubules. AMP-PNP was used to replace ATP in the solutions, and two antifade solutions were prepared (ATP antifade was flowed first, followed by AMP-PNP antifade). A large amount of ATP antifade was prepared, and it was flowed at a rate of 5 to 6 μL per second. Allegedly, this shear flow would align the microtubules, and if applied for a long enough duration (the specific timings are still unclear), the microtubules would eventually align by polarity. In our experiments, however, the microtubules did not align.

A more promising method to align microtubules entailed polymerizing microtubules uniquely from one end and then cross-linking them to the surface. Microtubules polymerize faster on the positive end than on the other. It is thus possible to use this polarity to control their growth. This procedure entails growing the microtubules inside the flow cell. A flow cell was prepared with half of the flor area covered with tape, and casein was flowed through. The tape was removed, and kinesin was flowed through, followed by an antifade solution. The kinesin does not stick to the previously tape-covered portion because there was no casein sticking to that portion. Microtubules were flowed through the cell, and would therefore be forced to line up along the barrier of the kinesin region to the blank region. (insert rest of procedure here).

Cargo

The structure would transport cargo on the nano and micro scale. As a proof of concept, we would use biotinylated nanospheres and microspheres of different sizes in order to test the efficacy of the transport structure. Later, we would try to design our structure so that it could bind to different kinds of biomolecules like proteins and DNA fragments etc by chemically modifying the top surface.

Three types of linkages were investigated for attaching microtubules and cargo to the structure. While microtubules need to bind permanently to our structure, the cargo should be able to load and unload at the respective loading and unloading stations.

• The biotin-streptavidin bond

This bond is well-known for its high strength and specificty. It is it is suited to permanently attach the microtubules to the structure because of its durability. Microtubule movement on kinesins remains unaffected by attaching the microtubules to biotin or streptavidin.

• Antibody-antigen bond

This bond is characterized by high specificity from a combination of non-covalent Hydrogen bonds and Van der Waal forces. These bonds can be easily dissociated by a change in pH or temperature. The antibody-antigen bonds are therefore potential candidates for attaching cargo to the structure.

• DNA Strands

DNA strands have high specificity and allow us to exert precise control on the strength of the bond by adjusting the length of the DNA strand. An increased advantage is that kinesin motors generate enough force to shear the bonds and break them. This property can be used to load and unload the cargo effectively. The bonds between DNA strands can be broken in two ways: the zipper geometry which requires less force and the simultaneous breaking which requires a greater force.

For the purpose of the project, three bond strengths were used on cargo. The weakest bonds bind cargo to the loading station, the intermediate ones bind cargo to the transporter, and the strongest ones bind cargo to the unloading station. The logic behind this is simple: At the loading station, the cargo breaks the weaker bond at the station and make a stronger bond with the transporter. At the unloading station, the cargo should break off the weaker bond with the transporter and attach to the unloading station via a stronger bond. DNA strands allow us to control the bond strength and are therefore good candidates for attaching cargo.