PAPER www.rsc.org/loc | Lab on a Chip A nano-needle/microtubule composite gliding on a kinesin-coated surface for target molecule transport. Mehmet C. Tarhan,*a Ryuji Yokokawa,bc Celine Bottier,d Dominique Collardd and Hiroyuki Fujitaa a Center for International Research on MicroMechatronics, Institute of Industrial Science, The University of Tokyo. b Dept. of Microengineering, Kyoto University. c PRESTO, JST, 4-1-8, Hon-chou, Kawaguchi, Saitama. d Laboratory for Integrated Micro Mechatronic Systems/CNRS-IIS, The University of Tokyo.

Introduction: Bead assay Gliding assay

*Previous Work.

Due to the tubular geometry of microtubules, cylindrical structures might be better suited as linkers for cargo transport.

Materials and Methods: Proteins: Materials and Methods: Proteins: *Kinesin 1 (Homo sapiens kinesin family member 5B (KIF5B)). *Tubulin with a concentration of about 4 mg/ml was polymerized into microtubules in BRB80 buffer. Needles: *Needles were obtained by micromachining a 3 inch silicon wafer. *Silica beads with a diameter of 0.33 micro meter. *The silicon wafer was etched in a DRIE chamber.

Average needle height- 4.05±0.13 micro m.

Functionalization of needles: Functionalization was achieved on the gold coating of the needles in three layers: self-assembled monolayer (SAM), biotin layer and streptavidin layer.

Collection of needles: An ultrasonic bath was used to break the needles from wafer.

Attachment of microtubules to needles 100-fold diluted microtubule solution was mixed with needle suspension with a ratio of 1 : 1 (v/v) and incubated for 20 min for attachment. Visualization setup For visualization of the experiments, an inverted microscope with oil immersion lens was used with a photomatrix camera. Transport experiments Transport experiments were performed in a flow cell made from two cover slips connected with greased thin paper slices forming a channel of about 15 ml.

Due to the viscous　drag caused by the buffer flow, the direction　of motion of the biotinylated microtubule–needle composite　became aligned with the flow direction. This result validates the integration of the “fluid flow” method for controlling the direction of motion with proposed transport system.

The average gliding velocity was measured to be 0. 35±0 The average gliding velocity was measured to be 0.35±0.07 micro m/s (mean± SD) in 43 events. 　　　　　　In previous research, the average gliding velocities of biotinylated microtubules without any fabricated structure were 0.37±0.03 micro m/s 0.48±0.13 micro m/s 0.54±0.14 micro m/s. Avidin-biotin. The average velocity of non biotinylated-needle composite was 0.36±0.07 micro m/s. Without needle- 0.36±0.04 micro m/s.

Conclusion: 1- The easy fabrication process of needles, allows mass production of needles with the desired dimensions providing a tool for bioassay applications. 2- Using cylindrical carriers such as needles offers better geometrical matching with microtubules and superior visualization and attachment properties when compared to spherical beads. 3- This strategy will have a significant impact on future devices by enabling the use of complex carriers for various bioengineering applications. THANK YOU