Carbon Nanopipette ElectroProbe in Cell-Electrophysiology Applications

描述（由申请人提供）：这个第一阶段SBIR项目的主要目标是优化制造过程，并证明使用新型碳基纳米吸管（CNP电探针）在单个细胞中穿透细胞膜的可行性，同时最小程度地侵入细胞，并允许测量跨细胞膜的电位或离子电流的变化。CNP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，直径从几十到几百纳米不等。玻璃毛细管的整个内腔涂有碳膜，该碳膜提供从毛细管远端到暴露的碳尖端的导电路径。导电膜可以在玻璃毛细管的外表面形成图案，以形成计数/参比电极。这些装置可以将化学物质和大分子注入单个细胞，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。

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.