Carbon Nanopipette Cellular Probe for Microinjections into Mammalian Cells

描述（由申请人提供）：该I期SBIR项目的主要目标是证明使用新型碳基纳米移液管（CNP）将大分子精确和受控微量注射到单细胞中的可行性，并允许在穿透细胞时对膜电位变化进行电学测量。最终目标是开发一种独特的多功能纳米移液管装置，用于同时进行显微注射和细胞渗透后细胞膜电位变化的电测量。CNP制造不需要任何繁琐的组装。CNP由一根拉伸玻璃毛细管组成，末端为直径范围为数十至数百纳米的暴露碳尖端。CNP的玻璃毛细管内涂覆的薄碳膜提供了导电路径，可用作测量细胞膜电位变化的电极。此外，这些设备可以将化学物质和大分子注入单个细胞，并从细胞内部提取液体和特定蛋白质。与常用的拉丝玻璃移液器相比，CNP具有显著的优势，例如尺寸更小（对细胞的损伤最小，探测细胞器的能力），更好的机械性能，更高的耐久性（它们不容易破裂或堵塞），自动化潜力（可以通过电信号感测细胞的穿透），与注射同时进行电生理学测量的潜力，和多功能分析功能，同时在价格上与玻璃微量移液器具有竞争力。此外，考虑到其耐用性，CNP比其玻璃对应物提供更高的效率和更低的成本（以每个电池为基础）。将检测CNP注射到细胞质中和注射到细胞核中的可行性，并将其与市售微量移液器进行比较，以证明CNP在显微注射中的优势。此外，将通过测量细胞穿透后电信号的变化来证明CNP作为纳米电极的独特特性。将使用内径在~100 nm范围内的CNP进行可行性研究，以减少细胞渗透时对细胞膜的损伤。 公共卫生相关性：所提出的CNP装置将应用于单个细胞的操作和研究，包括促进蛋白质和核酸的受控注射、药物递送、生物医学诊断和细胞工程研究。使用CNP的显微注射与市售玻璃微量移液器的直接比较将允许通过用CNP替代常规玻璃微量移液器来推进单细胞显微注射应用。

DESCRIPTION (provided by applicant): The main goal of this Phase I SBIR project is to demonstrate the feasibility of using a novel carbon-based nanopipette (CNP) for precise and controlled microinjection of macromolecules into single cells and allow electrical measurement of variations in membrane potential upon penetrating a cell. The ultimate goal is to develop a unique and versatile nanopipette device for concurrent microinjection and electrical measurements of changes in cell membrane potential upon penetration of a cell. CNP fabrication does not require any cumbersome assembly. The CNP consists of a pulled glass capillary terminating with an exposed carbon tip with a diameter ranging from tens to hundreds of nanometers. A thin carbon film coated inside the glass capillary of the CNP provides an electrically conducting path that can be used as an electrode for measurement of changes in membrane potential in cells. In addition, these devices can inject chemicals and macromolecules into single cells and extract fluids and specific proteins from cell's interior. The CNPs offer significant advantages over the commonly used pulled glass pipettes such as smaller size (minimal damage to cells and the ability to probe organelles), better mechanical properties, higher durability (they 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 CNPs offer higher efficiency and lower cost (on a per cell basis) than their glass counterparts. The feasibility of the CNP to inject into the cytoplasm and inject into the nucleus with higher cell survival rates will be tested and compared to that of commercially available micropipettes to demonstrate the advantages of CNPs in microinjection. Furthermore, the unique characteristic of CNPs as nanoelectrodes will be demonstrated by measuring the change in electrical signal upon cell penetration. Feasibility studies will be performed using CNPs with inner diameter in the ~100 nm range to achieve less damage to cell membrane upon cell penetration. PUBLIC HEALTH RELEVANCE: The proposed CNP devices will have applications in manipulation and study of individual cells including facilitating controlled injection of proteins and nucleic acids, drug delivery, biomedical diagnostics, and cellular engineering research. Direct comparison of microinjection using CNP to commercially available glass micropipettes will allow advancement in single cell microinjection applications by replacing the conventional glass micropipettes with CNPs.