Department of Micro Engineering, Kyoto University, Kyoto-daigaku Katsura, Nishikyo-ku, Kyoto, 615-8540, Japan.
Lab Chip. 2015 May 7;15(9):2055-63. doi: 10.1039/c5lc00148j.
Microtubules driven by kinesin motors have been utilised as "molecular shuttles" in microfluidic environments with potential applications in autonomous nanoscale manipulations such as capturing, separating, and/or concentrating biomolecules. However, the conventional flow cell-based assay has difficulty in separating bound target molecules from free ones even with buffer flushing because molecular manipulations by molecular shuttles take place on a glass surface and molecular binding occurs stochastically; this makes it difficult to determine whether molecules are carried by molecular shuttles or by diffusion. To address this issue, we developed a microtubule-based transport system between two compartments connected by a single-micrometre-scale channel array that forms dynamically via pneumatic actuation of a polydimethylsiloxane membrane. The device comprises three layers-a control channel layer (top), a microfluidic channel layer (middle), and a channel array layer (bottom)-that enable selective injection of assay solutions into a target compartment and dynamic formation of the microchannel array. The pneumatic channel also serves as a nitrogen supply path to the assay area, which reduces photobleaching of fluorescently labelled microtubules and deactivation of kinesin by oxygen radicals. The channel array suppresses cross-contamination of molecules caused by diffusion or pressure-driven flow between compartments, facilitating unidirectional transport of molecular shuttles from one compartment to another. The method demonstrates, for the first time, efficient and unidirectional microtubule transport by eliminating diffusion of target molecules on a chip and thus may constitute one of the key aspects of motor-driven nanosystems.
驱动蛋白马达驱动的微管已被用作微流控环境中的“分子梭”，在自主纳米操纵中具有潜在应用，如捕获、分离和/或浓缩生物分子。然而，传统的基于流控池的检测方法难以分离结合的靶分子和游离的靶分子，即使使用缓冲液冲洗也是如此，因为分子梭的分子操纵是在玻璃表面上进行的，并且分子结合是随机发生的；这使得难以确定分子是由分子梭携带还是由扩散携带。为了解决这个问题，我们开发了一种基于微管的运输系统，该系统在由单个微尺度通道阵列连接的两个隔室之间进行运输，该通道阵列通过气动致动聚二甲基硅氧烷膜动态形成。该装置包括三层-控制通道层（顶部）、微流道层（中间）和通道阵列层（底部）-可将测定溶液选择性注入靶隔室并动态形成微通道阵列。气动通道还可用作测定区域的氮气供应路径，从而减少荧光标记的微管的光漂白和氧自由基对驱动蛋白的失活。通道阵列抑制了隔室之间扩散或压力驱动流引起的分子交叉污染，促进了分子梭从一个隔室单向运输到另一个隔室。该方法首次证明了通过消除芯片上靶分子的扩散，可以有效地进行单向微管运输，这可能是马达驱动纳米系统的关键方面之一。
