Amphiphobic Septa Enhance the Mechanical Stability of Free-Standing Bilayer Lipid Membranes
Amphiphobic Septa Enhance the Mechanical Stability of Free-Standing Bilayer Lipid Membranes
复制标题
双疏隔膜增强独立式双层脂质膜的机械稳定性
DOI：
10.1021/acs.langmuir.8b00747
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发表时间：
2018
期刊：
影响因子：
3.9
通讯作者：
Ayumi Hirano-Iwata
中科院分区：
文献类型：
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作者：
Daichi Yamaura;Daisuke Tadaki;Shun Araki;Miyu Yoshida;Kohei Arata;Takeshi Ohori;Ken-ichi Ishibashi;Miki Kato;Teng Ma;Ryusuke Miyata;Hideaki Yamamoto;Ryugo Tero;Masao Sakuraba;Toshio Ogino;Michio Niwano;Ayumi Hirano-Iwata
人工双层脂质膜 (BLM) 提供了明确的系统，用于研究膜蛋白（包括离子通道）的基本特性，以及筛选作用于它们的药物的效果。然而，由于工艺稳定性差、重构效率低，该技术的应用受到限制。我们之前报道过基于在 SiO2/Si3N4 隔膜中制造具有锥形边缘的微孔和基于离心力加速的囊泡融合的有效离子通道掺入来提高 BLM 的稳定性。尽管使用这些方法时 BLM 稳定性和掺入概率显着提高，但一些 BLM 在受到离心力时会破裂。为了进一步提高 BLM 的稳定性，我们研究了 SiO2/Si3N4 隔垫表面的改性对悬浮在隔垫中的 BLM 稳定性的影响。改性表面的特征在于疏水性、疏油性和表面粗糙度。还测定了在改性表面上形成的脂质单层的扩散系数。在用长链全氟化碳修饰的两疏基材上形成了高流动性的脂质单层。与用短烷基链修饰的隔膜中形成的那些相比，在双疏隔膜中形成的独立式 BLM 显示出更高的机械稳定性，包括对水运动的耐受性以及在有或没有蛋白脂质体的情况下施加的离心力。这些结果表明，当隔膜表面具有两疏特性时，就会形成高度稳定的 BLM。由于隔膜上形成的高流动性脂质单层与独立区域中的 BLM 无缝连接，因此脂质的高流动性有助于减少施加机械应力时对 BLM 的潜在损害。这种提高 BLM 稳定性的方法提高了 BLM 系统的实验效率，并将有助于开发用于离子通道蛋白功能测定的高通量平台。
Artificial bilayer lipid membranes (BLMs) provide well-defined systems for investigating the fundamental properties of membrane proteins, including ion channels, and for screening the effect of drugs that act on them. However, the application of this technique is limited due to the low stability and low reconstitution efficiency of the process. We previously reported on improving the stability of BLM based on the fabrication of microapertures having a tapered edge in SiO2/Si3N4septa and efficient ion channel incorporation based on vesicle fusion accelerated by a centrifugal force. Although the BLM stability and incorporation probability were dramatically improved when these approaches were used, some BLMs were ruptured when subjected to a centrifugal force. To further improve the BLM stability, we investigated the effect of modifying the surface of the SiO2/Si3N4septa on the stability of BLM suspended in the septa. The modified surfaces were characterized in terms of hydrophobicity, lipophobicity, and surface roughness. Diffusion coefficients of the lipid monolayers formed on the modified surfaces were also determined. Highly fluidic lipid monolayers were formed on the amphiphobic substrates that had been modified with long-chain perfluorocarbons. Free-standing BLMs formed in amphiphobic septa showed a much higher mechanical stability, including tolerance to water movement and applied centrifugal forces with and without proteoliposomes, than those formed in the septa that had been modified with a short alkyl chain. These results demonstrate that highly stable BLMs are formed when the surface of the septa has amphiphobic properties. Because highly fluidic lipid monolayers that are formed on the septa seamlessly connect with BLMs in a free-standing region, the high fluidity of the lipids contributes to decreasing potential damage to BLMs when mechanical stresses are applied. This approach to improve the BLM stability increases the experimental efficiency of the BLM systems and will contribute to the development of high-throughput platforms for functional assays of ion channel proteins.