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

描述（由申请人提供）：这个拟议的II期项目将导致能够转染单个靶细胞的商业产品。这种新的生物工具使用了一种被称为纳米山探针（NFP）的专利微制造芯片，通过单细胞电穿孔将分子输送到活的单个靶细胞中，通过在NFP芯片上的微流体悬臂顶端施加电场，在细胞膜上诱导出临时的纳米孔。这种新的转染技术被称为纳米山探针电穿孔（NFP-E），它已经显示出不可思议的前景，可以转化为一种新的商业生物工具。现有的单细胞转染技术，如微注射和微管电穿孔，需要大量的操作人员培训，高度依赖于用户和劳动密集型，并且经常因细胞膜上过大的机械力而损伤细胞。NFP悬臂减少了细胞膜上的机械应力，并且可以在制造过程中进行定制。该项目的长期目标是开发第一个易于使用、对细胞温和、自动化的单细胞转染系统，以提供相对高的通量并消除用户依赖的可变性。本方案的具体目标是：(1)优化产品组件的设计，使其易于即插即用组装和溶液加载和回收；(2)开发用于图像处理和分析的软件，通过自动化定位目标细胞、检测NPF尖端与细胞膜的接触以及施加电场的过程，简化NPF电穿孔；(3)进行三个关键转染实验，以证明NFP-E在研发中的潜力，建立实验方案，收集效率/可行性统计数据，验证规范

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.