Design and Fabrication of Linear-shaped Zero Mode Waveguides for Single Molecule Observation of Kinesin and Fluorescent ATP

Abstract

Kinesin is a motor protein that transport organelles and proteins by repeating 8 nm discrete steps on a microtubule in eukaryotic cells. The mechanism of kinesin motility coupled with the hydrolysis of adenosine triphosphate (ATP) is of great interest to researchers in biophysics. However, it is still poorly understood when kinesin heads make a step during the ATP hydrolysis and when adenosine diphosphate (ADP) is released from kinesin. In this study, we propose a simultaneous measurement of displacements of kinesin and attachment/detachment of fluorescently-labeled ATP (fATP) using Linear-shaped Zero-Mode Waveguides (LZMWs) that enables to use high concentration of fluorescent molecules for single molecule observation. To this end, we designed LZMWs based on FEM simulation and optimized a fabrication process by electron beam lithography and lift-off process. Assay procedure was established by introducing microtubules into LZMWs utilizing gliding motility. Simultaneous observation in LZMWs revealed that motility of kinesin was preserved and single fATP molecules were visualized under the concentration of 250 nM, which is about ten times higher than in conventional Total Internal Reflection Fluorescence Microscopy (TIRFM).