Nanomechanical analysis of microtubule translocation by biomolecular motors in the presence of electric and flow fields.

Abstract

Demands for compact, self-contained Micro Total Analysis Systems (muTAS) that do not require external power sources have recently led to the concept of powering these microfluidic devices with biomolecular motors derived from nature. Our research group has demonstrated a molecular sorter that specifically selects biomolecules from a sample and concentrates them in a collector by employing biomolecular motors and microtubules as transport systems [4]. Based on this work, we identified that the development of active control over the direction of microtubule translocation on kinesin-coated surfaces is a high priority research area. While some passive methods for controlling the trajectory of microtubules in microdevices had been established at the start of this research, active control methods had not been established. This dissertation explores electric and flow fields to actively control the direction of microtubule translocation. To investigate the engineering science principles underlining these active control mechanisms, so-called <italic>in vitro</italic> motility assays are used, in which kinesin is bound to the device surface such that microtubules are translocated in the presence of adenosine triphosphate (ATP). The general strategy is to apply external forces to the translocating microtubules to bend the leading ends of the microtubules towards the direction of the applied forces, with the remainders of them following behind. This study successfully demonstrates that microtubules are aligned to the parallel direction of external fields, and statistical analysis of the experimental observations strongly suggests that the surface density of kinesin, the strength of external forces, and the velocity of microtubule translocation are major factors governing how rapidly and completely microtubules align in the presence of external fields. A beam-theory based model is developed that further supports the statistical results, agrees well with observed transient behaviors, and suggests an optimal strength of an external field for efficient active directional control. Inputs to this model include the charge density and flexural rigidity of microtubules, as well as low Reynolds number hydrodynamics in the presence of electrokinetic forces. Where literature values were widely scattered or unavailable, independent experiments and theoretical calculations were performed to obtain model input parameters. The results and the model have been used in improving the performance and reliability of the molecular sorter. It is hoped that the external-field-based active control methods developed and characterized in this dissertation can expand the realm of possibilities for micro-/nano-devices that integrate molecular motors into their design.