Integrated Photothermal Platform for Delivering Infectious Bacteria into Mammalian Cells in BSL 3 Environment for High Throughput Screening

1404080 Chiou，Pei-Yu标题：在BSL 3环境中将感染细菌输送到哺乳动物细胞的集成光热平台用于高通量筛选意义：细胞生物学和微生物发病机制研究的一个主要障碍是缺乏任何有效的高通量输送微米级货物直接进入细胞胞浆的方法。这项提议旨在开发一个紧凑的平台，以克服这一障碍。我们设计了一种新的策略，使用激光创造微米大小的气泡，瞬时破坏细胞膜，并将细菌大小的货物驱动到细胞胞浆中。我们提议的平台的一个独特优势是，它的紧凑尺寸将使其能够放在生物安全柜中，从而能够与高传染性细菌一起工作，而这些细菌无法用目前任何可用的输送方法进行安全研究。除了细菌，我们的平台还将能够直接将线粒体、染色体和微米大小的“纳米机器”等亚细胞细胞器送入细胞胞浆。我们项目的成功将使研究人员能够提出迄今为止无法回答的实验问题，并在微生物发病机制、细胞生物学、细胞工程和再生医学等多个领域取得进展。技术描述：该平台是通过微机械加工制造的硅芯片大规模并行光热纳米片实现的。将宿主细胞种植在纳米级芯片上，然后将注入的细菌装载在定制的流体室中。手持式光纤激光器与定制的光学扫描仪相结合，可在大范围内提供空化激发。激光脉冲后，立即触发压力驱动流，将微米级的细菌主动驱动到细胞胞质中。大规模并行输送到100,000个细胞只需10秒，比目前基于吸管的输送方法快5个数量级。为了实现我们的目标，我们计划开展以下项目。项目1是开发一个紧凑型光热平台，用于在BSL 3环境中高通量运送感染样本。项目2是开发用于超高通量大件货物运输的全光驱动纳米刀片平台，项目3是展示大规模并行光热纳米刀片在高通量筛选中的应用，以确定胞浆生长所需的细菌基因和限制细菌胞浆生长的宿主细胞途径。

1404080Chiou, Pei-YuTitle: Integrated Photothermal Platform for Delivering Infectious Bacteria into Mammalian Cells in BSL 3 Environment for High Throughput ScreeningSignificance:A major obstacle in cell biology and microbial pathogenesis research is the absence of any method for efficient high-throughput delivery of micron-sized cargo directly into the cytosol of cells. This proposal aims to develop a compact platform that will overcome this obstacle. We have devised a novel strategy that uses a laser to create micron-sized bubbles that transiently disrupt the cell membrane and drive bacteria-sized cargo into the cell cytosol. A unique advantage of our proposed platform is that its compact size will allow it to fit in a biosafety cabinet, thus enabling work with highly infectious bacteria that could not be studied safely with any currently available delivery method. In addition to bacteria, our platform will also be able to deliver directly into the cell cytosol subcellular organelles such as mitochondria, chromosomes, and micron sized "nano-machines". Success of our project will enable researchers to ask experimental questions that hitherto could not be answered and to make advances in diverse fields, including microbial pathogenesis, cell biology, cell engineering, and regenerative medicine.Technical Description:This platform is realized by a silicon chip-based massively parallel photothermal nanoblade fabricated by micromachining. A nanoblade chip is seeded with the host cells, and the bacteria that will be injected are loaded in a custom-built fluid chamber. A handheld size fiber laser coupled with a custom built optical scanner to provide cavitation excitation over a large area. Immediately after laser pulsing, pressure driven flows will be triggered to actively drive micron-sized bacteria into cell cytosol. Massively parallel delivery into 100,000 cells takes only 10 seconds, which is 5 orders of magnitude faster than the current pipette based delivery approaches. To accomplish our goal, we plan to conduct the following projects. Project 1 is to develop a compact photothermal platform for high-throughput delivery of infectious samples in BSL 3 environment. Project 2 is to develop an all-optical driven nanoblade platform for ultrahigh throughput large cargo delivery and project 3 is to demonstrate the utility of massively parallel photothermal nanoblade in high-throughput screening to identify bacterial genes required for cytosolic growth and host cell pathways that restrict bacterial cytosolic growth.