Development of a Microfluidics-Based Approach for Investigating Microtubule Polymer Mechanics

Microtubules are cytoskeletal polymers that play essential roles in cell division and intracellular transport. In this study, we adopt microfluidics to study microtubule mechanics in vitro. This work addresses two specific limitations for studying microtubules in microfluidic devices, the potential for air bubbles, which can denature proteins and the lack of use of high throughput assays. Our microfluidic device and protocol allow for a range of experimental setups with more robust high throughput testing capabilities than our previous flow cell assays.

[Instructor] To begin, plasma clean a three inch silicon wafer under vacuum for five minutes using either oxygen or clean dry air plasma. Ensure that the vacuum pressure is below five times ten to the power of minus five torr. Center the clean silicon wafer onto the spin coder for photo resist deposition and deposit one to two milliliters of SPR 227.0 photo resist onto the center of the silicon wafer. Spin coat the photoresist to achieve a 13 micrometer thick layer at 1000 revolutions per minute for 30 seconds. While minimizing contact with the coated surface, transfer the silicon wafer to a hot plate set to 70 degrees Celsius. Incubate the silicon wafer on the hot plate, increasing the temperature by 10 degrees Celsius every three to five minutes until the temperature reaches 115 degrees Celsius. Then turn off the hot plate and allow the silicon wafer to cool until its temperature is below 65 degrees Celsius. Using forceps, transfer the cooled wafer to the mask aligner. Load both the silicon wafer and the appropriate photo mask into the aligner according to the manufacturer's or site specific protocols, now expose the wafer to ultraviolet radiation with energy of approximately 400 millijoules per square centimeter. Calculate the required exposure time using the formula. After rehydration and heat treatment, submerge the wafer in the appropriate developer. Then gently rinse both sides of the wafer with deionized water for 30 seconds. After drying the developed wafer using nitrogen gas, transfer it into a desiccator. Place a small aluminum container in the desiccator and add one drop of silane into the aluminum container. After desiccation, pour the mixed and degassed Polydimethylsiloxane onto the master mold inside a Petri dish. Incubate the dish at 65 degrees Celsius overnight to allow PDMS to fully cure. Around the device features, use a scalpel or razor blade to cut out rectangular pieces of PDMS from the master layer. Ensure each piece includes adequate flanking space to allow proper bonding contact and fits a 22 by 22 millimeter glass cover slip. Place the PDMS on a spare sacrificial PDMS layer, avoiding hard surfaces. Then using a clean 1.5 millimeter hole punch make inlet and outlet holes in each PDMS piece. Now retrieve a 22 by 22 millimeter glass cover slip and clean it using a wipe wedded with isopropyl alcohol. Then plasma clean the glass cover slip under vacuum for five minutes using clean, dry air plasma. Wipe both the glass cover slip and the feature side of the PDMS with isopropyl alcohol wedded wipes before placing both into the plasma cleaner and simultaneously plasma clean them for 30 seconds under vacuum using clean, dry air plasma. After cleaning, invert the PDMS so its feature side faces downward. Place the PDMS onto the glass cover slip and press lightly to encourage bonding. Stabilized microtubule extensions were bent by flowing buffer solution perpendicular to their growth direction, demonstrating the capacity to apply directional force within the device. Near surface flow velocity experienced by microtubules was calculated as 92 micrometers per second using simulation and analytical modeling based on the Navier-Stokes equation. Computational simulations demonstrated the establishment of stable gradients across the device confirmed experimentally by a fluorescent dye showing predictable concentration patterns. Dual labeled microtubule extensions, confirmed gradient based partitioning with different fluorescent proteins dominating at distinct spatial zones along the device.