Dynamic formation of a microchannel array enables kinesin-driven microtubule transport between separate compartments on a chip
Dynamic formation of a microchannel array enables kinesin-driven microtubule transport between separate compartments on a chip
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微通道阵列的动态形成使得驱动蛋白驱动的微管在芯片上的不同隔室之间传输
DOI：
10.1039/c5lc00148j
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发表时间：
2015
期刊：
影响因子：
6.1
通讯作者：
R. Yokokawa
中科院分区：
文献类型：
--
作者：
K. Fujimoto;M. Nagai;H. Shintaku;H. Kotera;R. Yokokawa
由运动蛋白马达驱动的微管已被用作微流体环境中的“分子穿梭”，在自主纳米级操作中具有潜在的应用，如捕获、分离和/或浓缩生物分子。然而，传统的基于流动细胞的分析方法即使用缓冲冲洗法也很难分离结合的目标分子和游离的分子，因为分子穿梭的分子操作发生在玻璃表面，分子结合是随机发生的，这使得很难确定分子是通过分子穿梭还是通过扩散携带的。为了解决这个问题，我们开发了一种基于微管的运输系统，通过单微米尺度的通道阵列连接在两个隔室之间，该通道阵列通过气动驱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)、微流控通道层(中间)和通道阵列层(底部)，其能够选择性地将分析溶液注入靶室并动态形成微通道阵列。气动通道也是通往分析区域的氮气供应路径，这减少了荧光标记的微管的光漂白和氧自由基对激动素的失活。通道阵列抑制了由隔室之间的扩散或压力驱动的流动引起的分子的交叉污染，促进了分子穿梭从一个隔室到另一个隔室的单向运输。该方法首次证明，通过消除目标分子在芯片上的扩散，可以有效和单向地传输微管，因此可能构成电机驱动纳米系统的关键方面之一。
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.