MICROTUBULE MANIPULATION BY AN ELECTRIC FIELD IN A FUSED SILICA CHANNEL Tasuku Nakahara 1* , Naoto Isozaki 1 , Suguru Ando 1 , Nagendra K. Kamisetty 1 , Hirofumi Shintaku 1 , Hidetoshi Kotera 1 and Ryuji Yokokawa 1,2 1 Kyoto University, JAPAN, and 2 PRESTO, JST, JAPAN ABSTRACT We propose a fused silica channel to manipulate gliding direction of microtubules with the applied electric field. The fabricated device was examined by injecting a fluorescent dye solution to prove the entire channel has no leakage. Assay sequence to realize microtubule gliding in the channel without losing protein functionalities was optimized to manipulate microtubules by applying an electric field. The nanogold-labeled microtubules showed smaller curvature than non- labeled microtubules in response to the field. It is implied that proposed device has possibility to perform the molecular separation in vitro. KEYWORDS: Motor protein, Microtubule, Fused silica channel INTRODUCTION In vivo, there is kinesin and microtubule system, which transport intracellular cargos by hydrolyzing adenosine triphosphate (ATP). To reconstruct the molecular system in vitro, gliding assay system is widely used, where microtubules glide on kinesin fixed on a substrate. Recently, many methods have been investigated to apply gliding assay system for molecular manipulations [1-4]. Agarwal et al. reported a method to attach molecules to microtubules [5], and van den Heuvel et al. reported microtubule manipulation by an electric field [6][7]. Here, the direction of gliding microtubule is random. However, the microtubules changed their gliding direction to the positive electrode in an electric field because microtubule has originally negative charge. Moreover, there is possibility that an curvature of gliding microtubules is influenced by attached particles. In this report, we integrate the molecular labeling method and the microtubules manipulation method to demonstrate the simultaneous manipulation of two differently labeled microtubules in a single channel. For the labeling, we used nanogold particles to obtain different curvature in the electric field compared with non-labeled microtubules. Figure 1 shows the conceptual diagram of the proposed device. It is composed of introduction channel for controlling a random motion of microtubules, separation channel for changing direction of gliding microtubules by an electric field, and collecting area for accumulating microtubules following the separation. EXPERIMENTAL The proposed device was fabricated by 5 steps (Figure 2): 1) A fused silica substrate was rinsed by piranha solution. Then, the substrate was deposited with a chromium (Cr) thin film (40 nm) by thermal deposition. The substrate is coated with a positive photo resist, s1805, on the Cr-deposited surface by spincoating. 2) The channel pattern is drawn by a laser exposure machine. In the development process, we put the substrate in a developer to remove the exposed photo resist. Then, the chromium layer was etched by a Cr etchant. 3) The exposed glass area was etched to create a 2-m- deep channel by buffered hydrofluoric acid (BHF). 4) The holes for inlet and electrodes were drilled by ultrasonic drilling with an abrasive. 5) To remove glass particles and abrasive produced in the drilling process, the patterned substrate was rinsed by piranha solution for 2 h, and nitric acid with sonication for 30 min. We used the same rinsing condition for another blank fused silica substrate. Then, the surface of blank fused silica substrate was etched by BHF for 2 min. To remove any residual water, the channel patterned substrate and blank substrate were baked on a hotplate at 120°C for 5 min. For glass bonding, we used a sodium silicate solution (10wt%). The blank substrate was coated with the sodium silicate solution by spincoating at 4000 rpm for 30 s along with the first spinning of 500 rpm for 10 s. Finally, the patterned substrate and blank substrate were bonded by a heat press at 90°C for 2 h. We prepared a polydimethylsiloxane (PDMS) sheet with three holes corresponding to inlet and two holes for electrodes. We used this PDMS sheet for reservoirs to prevent drying of channel inside, and contact between electrodes and buffer solution. To examine the fabricated channel, we introduced a fluorescent dye into the channel after bonding process. Moreover, we performed the numerical analysis to confirm the potential for proposed channel. Figure 1: Conceptual diagram of separating device 978-0-9798064-6-9/µTAS 2013/$20©13CBMS-0001 161 17th International Conference on Miniaturized Systems for Chemistry and Life Sciences 27-31 October 2013, Freiburg, Germany