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Microtubule Alignment

Early experiments focused on using flow to align microtubules. The group performed motility assays, then flowed an antifade solution (a solution that removes unattached microtubules from the surface) containing AMP-PNP as a method of securing the microtubules in their positions for imaging. This method proved unreliable. While flow occasionally aligned a small proportion of the microtubules, in general this procedure did not work.
Image of flow test. Angle mean = 2.85°, sd = 55.145°‎

Currently, the team then began investigation into using a protein coated / blank surface boundary as a method to align microtubules. Microtubules that align themselves into the blank region of the flow cell (held at the positive end by a single kinesin motor protein) do not move out of alignment because the majority of their length is not acted upon by any motor proteins.

The team successfully created a barrier in a flow cell between an area with microtubules and an area without. This was performed by coating an isolated area (created using tape) with casein, and then removing the tape, attaching the top coverslip, and flowing kinesin through. When the motility solution and antifade were prepared with casein, the boundary was less defined when the solutions were prepared without.

Image of boundary with casein in solutions. Angle mean = -5.47°, sd = 50.65°
Image of boundary without casein in solutions. Angle mean on boundary = .14°, sd = 42.60°

Unfortunately, there was little alignment at the boundaries. The average angle of had a standard deviation of 48 degrees. The experiment was repeated with a higher concentration of kinesin and microtubules in order to make more microtubules at the boundary and to see if the concentration has an affect on the alignment of microtubules. There was no difference in the alignment patterns between the highly concentrated microtubules and the lower concentrated ones.

Alignment ultimately went unresolved, however there is hope for the future as our lab is investigating the use of micropumps to maintain a stead flow over long periods of time, which should increase the potential for alignment. Currently flow is delivered in intermittent bursts by hand using a micropipette, so obviously there remains room for improvement in the future.

Structure Formation

The formation of the polyurethane beds by microtransfer molding was generally successful. Adhesive forces usually pulled some amount of the liquid prepolymer out of the mold, but we were still able to form defined squares of polyurethane that the microtubules successfully adhered to. Using glutaraldehyde as a binding agent irreversibly bound the microtubules to the squares as desired, preventing them from moving relative to the squares, while theoretically maintaining their ability to move on a kinesin coated surface upon flipping the entire structure.

Image of microtubules on polyurethane squares.

Photoresist AZ 5214 was used as the sacrificial layer underneath the polyurethane squares. This photoresist was chosen because it can form a very thin layer of 1.6 μm. The photoresist did not dissolve as readily as we had hoped when using a 1:4 acetone to BRB80 solution, nor did it dissolve with a 1:4 solution of photoresist developing solution:BRB80. The ratios of acetone and developing solution were limited because the 1:2 acetone to BRB80 and 1:2 photoresist to BRB80 solutions denatured the microtubules while the 1:4 solutions did not. Other methods of force were applied to the surface in order to acquire the microtubule coated polyurethane squares such as sonicating the wafer in the 1:4 solutions, applying pressure with the solution, or by applying the force of the tip of a pipette. None of these methods proved particularly successful however. Progress on the project concluded at this step.

Future Endeavors

We plan to continue research towards the creation of our molecular transport system. The first step is to investigate different photoresists and/or developer solutions to find a combination that will readily release the squares from the surface. Caution must be taken in the latter section because solutions that easily dissolve the photoresist also depolymerize the microtubules. Once the structures are isolated, it is necessary to attach cargo to the bare side of the polymer square. After this is achieved, it would logically follow that the methods of cargo loading and unloading be applied to the device.