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，需要可控制地和可靠地将试剂注入足够多的细胞中，以得出关于细胞反应的具有统计学意义的数据。尽管微吸管能够实现高度控制的细胞注射，但它们目前的使用需要熟练的操作员和缓慢、乏味的手动操作。包括转基因、电穿孔、光复制或病毒感染在内的批处理过程不能保证整体中的所有细胞都被注射，并且进入细胞的化合物保持其预期的成分。缺乏高吞吐量、可靠的注入方法仍然是许多重要项目的瓶颈。为了满足这一需求，一个在纳米技术、生物化学和医学方面拥有专业知识的跨学科团队提议为自动细胞注射系统开发组件。该系统将利用Bau研究小组发明的可批量生产的碳基纳米管(CNP)。CNP由直径从几十到数百纳米的纳米碳管组成，无缝连接到与标准细胞电生理设备兼容的宏观玻璃毛细管柄。玻璃吸管的整个内表面都衬有一层碳膜。因此，CNP提供了一条通过管子中空注入试剂的路径，以及一条通过导电碳衬里进行电测量的独立路径。与传统的拉制玻璃微吸管相比，CNP具有许多优势。它们具有良好的机械性能，具有生物相容性，不容易破裂或堵塞，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。CNP将被用来将试剂注入到以规则阵列放置在带有电极图案的表面上的预定位置的细胞中。细胞的定位将使用电极化力(介电泳法)来完成。CNP将通过其碳衬里的交流阻抗测量来感知细胞渗透，以向注射器提供信号。该系统的性能将通过自动向细胞中注入供体和受体荧光tRNA，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定位速率来测试。所提出的自动化细胞注射系统具有广泛的实用性，将促进许多医学和生物学领域的进步。

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.