Carbon Nanopipette ElectroProbe in Cell-Electrophysiology Applications

描述(由申请人提供)：该第一阶段SBIR项目的主要目标是优化制造工艺，证明使用新型碳基纳米管(CNP电探针)以最小的细胞侵入穿透单个细胞的可行性，并允许测量跨细胞膜的电位或离子电流的变化。CNP的制造不需要任何繁琐的组装。CNP由一根拉出的玻璃毛细管组成，末端是一根暴露的碳管，直径从几十到数百纳米不等。玻璃毛细管的整个内腔覆盖有碳膜，该碳膜提供了从毛细管远端到暴露的碳尖的导电路径。导电膜可以在玻璃毛细管的外表面形成图案化，以形成对电极/参比电极。这些设备可以将化学物质和大分子注入单细胞，从细胞内部提取液体和特定蛋白质，并在细胞穿透时产生电信号。 与常用的拉拔玻璃移液管电极相比，CNP电极具有显著的优势，例如更小的尖端尺寸(对细胞的损害最小，能够探测细胞器)，更好的机械性能，更高的耐用性(尖端不容易破裂或堵塞)，自动化的潜力(可以通过电信号检测细胞的渗透)，在注射的同时进行电生理测量的潜力，以及多功能分析功能，同时在价格上与玻璃微吸管具有竞争力。此外，考虑到它们的耐用性，CNP电子探头比玻璃探头提供了更高的效率和更低的成本(以每个电池为基础)。与玻璃不同，CNP不需要毛细管内的电解液，因为碳尖与细胞介质接触。除了优化制备工艺以细化探针尖端外，在本方案中，将通过测量膜电位的变化或使用CNP纳米电极在单个细胞内记录的离子电流的变化来展示CNP作为纳米电极的独特特性。 与公众健康相关：CNP电极可以穿透单个细胞，对细胞膜的损害最小，并允许对膜电位或总离子电流的变化进行可靠和可重复性的测量，这将通过用CNP电子探针取代传统的玻璃微吸管电极，促进单个细胞的电生理应用。所提出的CNP设备将在生物学、生理学、生物医学诊断和药物输送领域的单个细胞的操作和研究中得到应用。

DESCRIPTION (provided by applicant): The main goal of this Phase I SBIR project is to optimize the fabrication process and demonstrate the feasibility of using a novel carbon-based nanopipette (CNP electroprobe) for penetration of cell membrane in single cells with minimal cell intrusion and allow measurement of changes in potential or ionic currents across the cell membrane. CNP fabrication does not require any cumbersome assembly. The CNP consists of a pulled glass capillary terminating with an exposed carbon pipe with a diameter ranging from tens to hundreds of nanometers. The entire inner lumen of the glass capillary is coated with a carbon film that provides an electrically conducting path from the distal end of the capillary to the exposed carbon tip. Conducting films can be patterned on the glass capillary's outer surface to form counter/reference electrodes. These devices can inject chemicals and macromolecules into single cells, extract fluids and specific proteins from cell's interior, and generate electrical signals upon cell penetration. The CNPs offer significant advantages over the commonly used pulled glass pipette electrodes such as smaller tip size (minimal damage to cells and the ability to probe organelles), better mechanical properties, higher durability (tips do not break or clog easily), potential for automation (the cell's penetration can be sensed through an electric signal), potential to carry out electrophysiological measurements concurrently with injection, and multifunctional analytic capabilities while being competitive in price with the glass micropipettes. Moreover, given their durability, the CNP electroprobe offers higher efficiency and lower cost (on a per cell basis) than their glass counterparts. Unlike their glass counterparts, the CNPs do not require electrolyte inside the capillary tube due to the carbon tip is in contact with cellular medium. In addition to optimizing fabrication process to refine probe tips, in this proposal, the unique characteristic of CNPs as nanoelectrodes will be demonstrated by measuring the variations in change in membrane potential, or change in ionic currents by using CNP nanoelectrodes for recording inside a single cell. PUBLIC HEALTH RELEVANCE: Demonstration of CNP electrodes to penetrate single cells with minimum damage to cell membrane and allow reliable and reproducible measurements of changes in membrane potential or total ionic currents will allow advancement in electrophysiology applications of single cells by replacing the conventional glass micropipette electrodes with CNP electroprobes. The proposed CNP devices will have applications in manipulation and study of individual cells in fields of biology, physiology, biomedical diagnostics, and in drug delivery.