Carbon Nanopipe-Based Automated Cell Injection System

基于碳纳米管的自动细胞注射系统

• 批准号: 8700631
• 负责人: Haim H Bau
• 金额: $ 23.35万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-04-01 至 2016-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8700631
• 关键词: Acute; Address; Area; Beds; Biochemical; Biochemistry; Biocompatible; Biology; Blood capillaries; Businesses; Caliber; Carbon; Cell Count; Cell Nucleus; Cell membrane; Cells; Chinese Hamster Ovary Cell; Chronic Disease; Communities; Cytoplasm; Data; Dendrites; Detection; Electric Conductivity; Electrodes; Electrophysiology (science); Electroporation; Equipment; Feedback; Film; Fluorescence Resonance Energy Transfer; Future; Glass; Image; Injection of therapeutic agent; Label; Location; Mammalian Cell; Manuals; Measurement; Measures; Mechanics; Medicine; Methodology; Methods; Monitor; Nanostructures; Nanotechnology; Neurons; Pattern; Penetration; Pennsylvania; Performance; Positioning Attribute; Process; Property; Protein Biosynthesis; Reagent; Research; Signal Transduction; Specific qualifier value; Surface; System; Technology; Testing; Time; Transfection; Transfer RNA; Tube; Universities; Viral; Virus Diseases; Work; base; capillary; combinatorial; design; drug discovery; electric impedance; electrical measurement; micromanipulator; migration; minimally invasive; nanometer; operation; programs; public health relevance; research study; response; success; therapeutic development; therapy development; trafficking; vaccine development

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.

描述（由申请人提供）：在药物发现、疫苗开发、细胞治疗剂开发、基础生物学研究和组合生物化学中，需要可控地和可靠地将试剂注射到足够大量的细胞中，以确保关于细胞应答的统计学显著数据。尽管微量移液器能够以高水平的控制进行细胞注射，但它们目前的使用需要熟练的操作员和缓慢、繁琐的手动操作。涉及转染、电穿孔、纯化或病毒感染的分批方法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留其预期组成。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，一个拥有纳米技术、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移液管（CNP）。CNP由直径范围从数十纳米到数百纳米的纳米级碳管组成，该碳管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手柄。玻璃移液管的整个内表面衬有碳膜。因此，CNP提供了通过管的中空部进行试剂注入的路径和通过导电碳衬里进行电测量的独立路径。与传统的拉丝玻璃微量移液器相比，CNP具有许多优点。它们具有良好的机械性能，是生物相容的，不容易破裂或堵塞，足够坚硬以穿透细胞膜，并且由于它们的小尺寸，是微创的。CNP将用于将试剂注射到以规则阵列定位在用电极图案化的表面上的预定位置处的细胞中。细胞定位将通过使用电极化力（介电泳）来完成。CNP将通过其碳衬里的AC阻抗测量来感测细胞渗透，以向注射器提供信号。该系统的性能将通过自动注射供体和受体荧光tRNA到细胞中，然后通过FRET信号的强度在真实的时间内测量整体和特定蛋白质合成的局部速率来测试。所提出的自动化细胞注射系统具有广泛的实用性，并将促进医学和生物学的许多领域的进步。