CAREER: Bio-Inspired Transport Assays and Single-Molecule Detection using Micro-and Nanopores

[449088]提议的研究将集中在两个相关的生物启发项目上。在一个项目中，将在硅、玻璃或聚合物衬底上制造体积相当于活细胞（~10 - 12升）的井。这些微孔的开放侧将被生物膜封闭。然后，首席研究员（PI）将运用自然的概念，使用小的、封闭的体积来检测只有少数分子或离子的流入或流出。与细胞一样，拟议的装置将使用多种放大模式来检测少于10,000个分子通过脂质双分子层膜流入后浓度的快速上升。这些装置将非常适合跟踪(i)治疗药物通过脂质双分子层的被动转运，（ii）跨膜受体（如G蛋白偶联受体）的激活，以及（iii）离子通道活性。将微流体与电和单分子荧光测量相结合，可以(i)阐明从荧光配体与离子通道结合到这些蛋白质中孔打开的时间延迟，（ii）在单分子水平上研究临床相关的转运蛋白。在另一个项目中，PI将使用飞秒脉冲激光的纳米制造技术，使生物启发的方法更进一步。生物脂质膜将被一种坚固的合成石英膜取代，这种石英膜支持单个纳米机械孔。与离子通道的门控特性和特异性类似，纳米孔的壁将被修饰以获得对抗体、抗原或病毒的特异性。这些生物分子的结合可能会限制通过纳米孔的电流，从而产生比标准技术更敏感和诊断结论性的临床分析。

0449088MayerThe proposed research will focus on two related, bio-inspired projects. In one project, wells of volumes comparable to living cells (~10 12 liters) will be fabricated in silicon, glass, or polymer substrates. The open side of these microwells will be closed by a biomembrane. The Principal Investigator (PI) will then apply Nature's concept of using small, enclosed volumes to detect the in- or outflux of only a few molecules or ions. Like cells, the proposed setups will use multiple modes of amplification to detect the rapid rise in concentration after influx of less than 10,000 molecules through lipid bilayer membranes. These setups will be ideally suited to follow (i) passive transport of therapeutic drugs through lipid bilayers, (ii) activation of transmembrane receptors such as G protein-coupled receptors, and (iii) ion channel activity. Combining microfluidics with electrical and single-molecule fluorescence measurements might make it possible to (i) elucidate the time delay from binding of a fluorescent ligand to an ion channel to the opening of the pore in these proteins and (ii) investigate clinically relevant transporter proteins on a single molecule level.In the other project, The PI will use nanofabrication by femtosecond-pulsed lasers to take the bio-inspired approach one step further. The biological lipid membrane will be replaced with a robust, synthetic quartz membrane that supports a single nano-machined pore. In analogy to the gating characteristics and specificity of ion channels, the wall of the nanopore will be modified to attain specificity for antibodies, antigens, or viruses. Binding of those biomolecules might gate the current flux through the nanopore and result in more sensitive and diagnostically conclusive clinical assays than standard technology.