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

1404080邱培瑜题目：利用光热整合平台将感染性细菌导入BSL 3环境中的哺乳动物细胞中进行高通量筛选意义：细胞生物学和微生物致病性研究的一个主要障碍是缺乏任何有效高通量将微米级货物直接递送到细胞胞质溶胶中的方法。 该提案旨在开发一个能够克服这一障碍的紧凑型平台。 我们设计了一种新的策略，使用激光来产生微米大小的气泡，这些气泡瞬时破坏细胞膜并将细菌大小的货物驱动到细胞胞质溶胶中。 我们提出的平台的一个独特优势是，其紧凑的尺寸将使其能够安装在生物安全柜中，从而能够与目前任何可用的输送方法都无法安全研究的高度传染性细菌一起工作。 除了细菌，我们的平台还将能够直接将亚细胞器（如线粒体、染色体和微米大小的“纳米机器”）输送到细胞质中。 该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。技术说明：该平台是通过微机械加工制造的基于硅芯片的大规模并行光热纳米刀片实现的。将宿主细胞接种在纳米芯片上，将被注射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，可在大面积上提供空化激发。在激光脉冲之后，将立即触发压力驱动流以主动驱动微米大小的细菌进入细胞胞质溶胶。 大规模并行递送到100，000个细胞中仅需要10秒，这比当前基于移液管的递送方法快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是开发一个紧凑的光热平台，用于在BSL 3环境中高通量递送感染性样品。项目2是开发一种全光学驱动的纳米叶片平台，用于高通量大货物递送，项目3是证明大规模并行光热纳米叶片在高通量筛选中的实用性，以鉴定细胞溶质生长所需的细菌基因和限制细菌细胞溶质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.