A Force-controlled Probe Based Platform for Single-Cell Biomolecular Delivery

基于力控探针的单细胞生物分子传递平台

• 批准号: 8780634
• 负责人: Ruiguo Yang
• 金额: $ 50万
• 依托单位: INFINITESIMAL, LLC
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-05-01 至 2017-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8780634
• 关键词: Algorithms; Automation; Biological; Cell Communication; Cell Line; Cell Survival; Cell membrane; Cells; Computer software; Detection; Development; Diagnostic; Electroporation; Feedback; Gap Junctions; Gene Expression; Gene Proteins; Genes; Goals; Image; Image Analysis; Individual; Lead; Legal patent; Life; Mechanical Stress; Mechanics; Methods; Microfluidics; Microinjections; Molecular; Monitor; Pharmaceutical Preparations; Phase; Play; Positioning Attribute; Process; Protocols documentation; RNA; Recovery; Research Personnel; Small Business Technology Transfer Research; Solutions; Standardization; Stem cells; System; Techniques; Testing; Therapeutic; Therapeutic Human Experimentation; TimeLine; Training; Transfection; Translations; Vision; Work; base; biological research; cantilever; cell injury; design; drug discovery; electric field; image processing; intercellular communication; nanopore; novel; operation; personalized medicine; plasmid DNA; prototype; public health relevance; research and development; research study; response; software development; statistics; tool

DESCRIPTION (provided by applicant): This proposed Phase II project will lead to a commercial product capable of transfecting individual target cells. This new biotool uses a patented microfabricated chip, called a nanofountain probe (NFP), to deliver molecules into live individual target cells by single-cell electroporation, which induces temporary nanopores in the cell membrane via the application of an electrical field that is localized at the tip of a microfludic cantilever on the NFP chip. This novel transfection technique is called nanofountain probe electroporation (NFP-E), and it has shown incredible promise for translation into a novel commercial biotool. Existing techniques for single-cell transfection, such as microinjection and electroporation by micropipette, require extensive operator training, are highly user-dependent and labor intensive, and routinely damage cells from excessive mechanical force on the cell membrane. The NFP cantilever reduces the mechanical stress on the cell membrane and can be tailored during fabrication. The long-term objective of this project is to develop the first sinle-cell transfection system that is easy to use, gentle on cells, and automated to offer relatively high throughput and eliminate user-dependent variability. The specific aims for this proposal are the following: (1) to optimize the design of product components for plug-and-play assembly and easy solution loading and recovery; (2) to develop software for image processing and analysis to simplify NFP-Electroporation by automating the process of locating target cells, detecting contact of the NPF tip with a cell membrane, and applying the electric field; and (3) to perform three key transfection experiments to demonstrate the potential of the NFP-E in R&D, to establish experimental protocols, collect statistics of efficiency/viability, verify specifications (efficiency, viability, throughput), and refine the image recognition software for a number of cell lines and primary cells. Accomplishing these aims will produce a product that could enable new capabilities for single-cell research and therapeutics including cell reprogramming/differentiation, cell-cell signaling, gene expression and protein interaction, cell-to-cell variability, drug discovery, personalized drug response diagnostics, and personalized medicine.

描述（由申请方提供）：该拟定II期项目将产生能够分离单个靶细胞的商业产品。这种新的生物工具使用一种专利的微加工芯片，称为纳米峰探针（NFP），通过单细胞电穿孔将分子递送到活的单个靶细胞中，通过施加位于NFP芯片上微流体悬臂尖端的电场，在细胞膜中诱导临时纳米孔。这种新的转染技术被称为纳米峰探针电穿孔（NFP-E），它已经显示出令人难以置信的前景，转化为一种新的商业生物工具。用于单细胞转染的现有技术，例如显微注射和通过微量移液管的电穿孔，需要广泛的操作者培训，高度依赖于用户和劳动密集型，并且常规地由于细胞膜上的过度机械力而损伤细胞。NFP悬臂降低了细胞膜上的机械应力，并且可以在制造期间进行定制。该项目的长期目标是开发第一个单细胞转染系统，该系统易于使用，对细胞温和，并且自动化，以提供相对高的通量并消除用户依赖的可变性。该提案的具体目标如下：（1）优化产品组件的设计，以实现即插即用组装和易于溶液加载和回收;（2）开发图像处理和分析软件，通过自动化定位靶细胞、检测NPF尖端与细胞膜的接触以及施加电场的过程来简化NFP-电穿孔;以及（3）进行三个关键的转染实验以证明NFP-E在研发中的潜力，建立实验方案，收集效率/活力的统计数据，验证规格 （效率，活力，通量），并完善图像识别软件的一些细胞 细胞和原代细胞。实现这些目标将产生一种产品，可以实现单细胞研究和治疗的新能力，包括细胞重编程/分化，细胞间信号传导，基因表达和蛋白质相互作用，细胞间变异性，药物发现，个性化药物反应诊断和个性化医疗。