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

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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.