Carbon Nanopipe-Based Automated Cell Injection System

描述（由申请人提供）：在药物发现、疫苗开发、细胞疗法开发、基础生物学研究和组合生物化学中，需要可控和可靠地将试剂注射到足够多的细胞中，以获得关于细胞反应的统计显著数据。虽然微移液器使细胞注射具有高水平的控制，但它们目前的使用需要熟练的操作人员和缓慢、繁琐的手动操作。涉及转染、电穿孔、光穿孔或病毒感染的批处理不能保证整体中的所有细胞都被注射，也不能保证进入细胞的化合物保持其预期组成。缺乏高通量、可靠的注入方法仍然是许多重大项目的瓶颈。为了满足这一需求，一个具有纳米技术、生物化学和医学专业知识的跨学科团队提议开发一种自动细胞注射系统的组件。该系统将利用Bau研究小组发明的可批量生产的碳基纳米吸管（CNPs）。CNP由一个直径从几十纳米到几百纳米的纳米级碳管组成，无缝连接到一个宏观玻璃毛细管手柄，该手柄与标准的细胞电生理设备兼容。玻璃移液管的整个内表面衬有一层碳膜。因此，CNP提供了通过管的空心注入试剂的路径和通过导电碳衬里进行电测量的独立路径。CNPs比传统的拉式玻璃微移液管有很多优点。它们具有良好的机械性能，具有生物相容性，不易破裂或堵塞，硬度足以穿透细胞膜，并且由于其体积小，具有最小的侵入性。CNPs将用于将试剂注入位于电极图案表面上预定位置的规则阵列的细胞中。利用电极化力（电介质电泳）来完成细胞定位。CNP将通过碳衬里的交流阻抗测量来感知细胞的穿透，并向注入器提供信号。该系统的性能将通过自动将供体和受体荧光trna注射到细胞中，然后通过FRET信号的强度实时测量整体和特定蛋白质合成的局部速率来测试。提出的自动细胞注射系统具有广泛的用途，并将促进医学和生物学许多领域的进步。

DESCRIPTION (provided by applicant): In drug discovery, vaccine development, cellular therapeutics development, basic biology research, and combinatorial biochemistry, there is a need to controllably and reliably inject reagents into a sufficiently large number of cells to assue statistically significant data about cellular responses. Although micropipettes enable cell injection with a high level of control, their current use requires skillful operators and slow, tedious, manual operation. Batch processes involving transfection, electroporation, photoporation or viral infection, cannot guarantee that all the cells in the ensemble are injected and that the compound that enters the cells retains its intended composition. The lack of a high throughput, reliable, injection methodology remains the bottleneck in many significant projects. To address this need, an interdisciplinary team with expertise in nanotechnology, biochemistry, and medicine proposes to develop components for an automated cell injection system. The system will utilize mass-producible, carbon-based nanopipettes (CNPs) invented by the Bau research group. The CNP consists of a nanoscopic carbon pipe of a diameter ranging from tens to hundreds of nanometers connected seamlessly to a macroscopic glass capillary handle that is compatible with standard cell electrophysiology equipment. The entire inner surface of the glass pipette is lined with a carbon film. Thus, the CNP provides a path for reagent injection through the hollow of the tube and an independent path for electrical measurements through the conductive carbon lining. The CNPs have many advantages over conventional, pulled glass micropipettes. They have good mechanical properties, are biocompatible, do not break or clog easily, are stiff enough to penetrate cell membranes, and, due to their small size, are minimally invasive. The CNPs will be used to inject reagents into cells positioned in a regular array at predetermined locations on a surface patterned with electrodes. The cell positioning will be accomplished with the use of electrical polarization forces (dielectrophoresis). The CNP will sense cell penetration via an AC impedance measurement through its carbon lining to provide a signal to an injector. The system's performance will be tested by automatically injecting donor and acceptor fluorescent tRNAs into cells and then measuring the localized rates of both overall and specific protein synthesis in real time by the intensity of a FRET signal. The proposed automated cell injection system has broad utility and will facilitate advances in many areas of medicine and biology.