Fabrication of electron tunneling probes for measuring single-protein conductance
Fabrication of electron tunneling probes for measuring single-protein conductance
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DOI：
10.1038/s41596-023-00846-3
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发表时间：
2023-07
期刊：
影响因子：
14.8
通讯作者：
Tao Jiang;Long Yi;Xu Liu;A. Ivanov;J. Edel;Long-Jun Tang
中科院分区：
文献类型：
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作者：
Tao Jiang;Long Yi;Xu Liu;A. Ivanov;J. Edel;Long-Jun Tang
研究单个蛋白质的电学性质是生物电子学领域的一个重要研究领域。电子隧道或量子力学隧道(QMT)探针可以作为研究蛋白质电学性质的强大工具。然而，目前制备这些探针的方法往往具有重复性有限、接触不可靠或蛋白质与电极结合不充分等问题，因此需要更好的解决方案。在这里，我们详细介绍了一套可概括和直接的说明，用于制造简单的、基于纳米管的隧道探针，适用于测量单个蛋白质的电导。我们的QMT探测器是基于一个高纵横比的双通道纳米管，它集成了一对间隙小于5 nm的金隧道电极，通过热解沉积碳和电化学沉积金来制备。黄金隧道电极可以使用大量可用的表面修饰来功能化，以实现单一蛋白质-电极接触。我们使用生物素化的硫醇修饰，其中生物素-链霉亲和素-生物素桥被用来形成单个蛋白质连接。由此产生的蛋白质偶联QMT探针能够对溶液中的同一单一蛋白质进行长达数小时的稳定电测量。我们还描述了用于解释与时间相关的单个蛋白质电导测量的分析方法，这可以为理解电子传递和探索蛋白质动力学提供必要的信息。完成协议的总时间为~33h，用户培训不到24小时即可完成协议。
Studying the electrical properties of individual proteins is a prominent research area in the field of bioelectronics. Electron tunnelling or quantum mechanical tunnelling (QMT) probes can act as powerful tools for investigating the electrical properties of proteins. However, current fabrication methods for these probes often have limited reproducibility, unreliable contact or inadequate binding of proteins onto the electrodes, so better solutions are required. Here, we detail a generalizable and straightforward set of instructions for fabricating simple, nanopipette-based, tunnelling probes, suitable for measuring conductance in single proteins. Our QMT probe is based on a high-aspect-ratio dual-channel nanopipette that integrates a pair of gold tunneling electrodes with a gap of less than 5 nm, fabricated via the pyrolytic deposition of carbon followed by the electrochemical deposition of gold. The gold tunneling electrodes can be functionalized using an extensive library of available surface modifications to achieve single-protein–electrode contact. We use a biotinylated thiol modification, in which a biotin–streptavidin–biotin bridge is used to form the single-protein junction. The resulting protein-coupled QMT probes enable the stable electrical measurement of the same single protein in solution for up to several hours. We also describe the analysis method used to interpret time-dependent single-protein conductance measurements, which can provide essential information for understanding electron transport and exploring protein dynamics. The total time required to complete the protocol is ~33 h and it can be carried out by users trained in less than 24 h.