Carbon Nanopipette Cellular Probe for Microinjections into Mammalian Cells

描述(申请人提供)：该第一阶段SBIR项目的主要目标是证明使用新型碳基纳米管(CNP)精确和可控地将大分子注射到单细胞中的可行性，并允许对穿透细胞时膜电位的变化进行电测量。最终目标是开发一种独特的、多功能的纳米管装置，用于同时进行微量注射和电学测量细胞膜渗透时细胞膜电位的变化。CNP的制造不需要任何繁琐的组装。CNP由一个拉动的玻璃毛细管组成，末端是一个暴露的碳尖，直径从几十纳米到数百纳米不等。在CNP的玻璃毛细管内涂覆的一层薄碳膜提供了一条导电路径，可用作测量细胞内膜电位变化的电极。此外，这些设备可以向单个细胞注入化学物质和大分子，并从细胞内部提取液体和特定蛋白质。与常用的拉制玻璃吸管相比，CNP具有显著的优势，例如尺寸更小(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(它们不容易破裂或堵塞)、自动化的潜力(可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提供了比玻璃同类产品更高的效率和更低的成本(以每个电池为基础)。将测试CNP注射到细胞质和细胞核中具有更高细胞存活率的可行性，并与商用微管进行比较，以证明CNP在显微注射中的优势。此外，碳纳米管作为纳米电极的独特特性将通过测量细胞穿透时电信号的变化来展示。将进行可行性研究，使用内径在~100 nm范围内的CNPs，以实现在细胞穿透时对细胞膜的较小损害。 公共卫生相关性：拟议的CNP设备将在单个细胞的操作和研究中应用，包括促进蛋白质和核酸的受控注射、药物输送、生物医学诊断和细胞工程研究。使用CNP的微注射与商业上可获得的玻璃微吸管的直接比较将通过用CNP取代传统的玻璃微吸管，从而在单细胞微注射应用中取得进步。

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.