Design Goals

  1. Achieve attachment of biotinylated microtubules to streptavidin coated beads in a flow cell

  2. Achieve bridging and networking between beads with microtubules as linkers

  3. Optimize parameters of bead attachment to maximize distance and network size.

  4. Deposit metal on the microtubules-microsphere networks.

Methods

Self-Assembly of Tubulin into Microtubules

To aid the polymerization of the microtubules, we suspended them in a growth buffer composed of BRB80, Mg, GTP and DMSO, and then incubated in a 37 degree water bath to allow the microtubules to polymerize [4]. During this process, the alpha and beta tubulin dimers bound to GTP and assembled onto the (+) end of growing microtubules [5]. The Mg was used to stabilize the negative charge of the phosphate groups in GTP and facilitate the cleavage of GTP into GDP and Pi to release energy for microtubule assembly. DMSO is a polar aprotic solvent that was used to stabilize the salts in the growth buffer. The length of the microtubules formed was directly proportional to the incubation time.

 

After the microtubules were polymerized, the solution was diluted 100-fold and paclitaxel (taxol) was added. Taxol is an organic compound which interferes with the breakdown of microtubules and stabilizes their structures [6].

 

Self-Assembly in the Flow cell

Our flow cell was created using a 35 mm x 55 mm glass slide, a 22 mm x 22 mm coverslip and double-sided tape. Double-sided tape (100 μm thick) was placed along the lengths of the glass slide and the coverslip was placed on top, creating a ~15 μL chamber through which liquid could be flowed in from one side and out the other.

 

A casein solution was first flowed into the cell so that it covered all interior surfaces of the cell. This ensured that other proteins, such as the microtubules would not adhere very much to the glass surface. After 5 minutes, a bead solution of 1.7% Streptavidin Coated Nile Red Beads (Spherotech, Lake Forest, IL), was added to the flow cell. Once the the beads settled, the microtubule solution was flowed into the flow cell. After this, the flow cell was allowed to sit for five minutes in order for attachments to form. Next, GFP-labeled streptavidin was added in to coat the remaining open biotin sites on the microtubules, in order to visualize them on the microscope. Finally, a taxol solution was flowed into the cell to wash out the excess GFP-streptavidin, remove the unattached microtubules, and stabilize the attached beads in the flow cell.

 

Imaging

Flow cells were imaged using an epifluorescence microscope (Nikon TE2000) equipped with an X-cite 120 lamp (EXFO, Ontario, Canada) and an iXON DU885LC EMCCD camera (Andor Technology, South Windsor, CT). Images were taken using a 40X air objective (N.A. 0.75). Data analysis was conducted using ImageJ imaging soware (available at http://rsbweb.nih.gov/ij/). The imaging occurred at room temperature (T = 21° C)