Research On Gene Transfection By Ultrasound Method Under The Microfluidic Environment

Gene transfection by ultrasound is a technology developed in recent years. Ultrasound don’t act on cells directly, instead, the DNA fragment are pushed into the cells through micro holes on cell membrane induced by cavitation effect. Gene delivery is an important research area in bioengineering, and has a significance prospect of application in clinical as a tool of gene therapy.Traditional gene delivery methods such as viral transfection, requires virus as a tool, so it is not suitable for human beings due to its low security. Conventional physical methods such as electroporation, microinjection usually causes mechanical damage on cell membrane, and can only be operated on some large cells. Ultrasonic gene delivery can overcome these shortcomings. The technology has been considered as an ideal means of gene transfection.The Research on ultrasonic mediated gene delivery usually need a large volume of samples. In contrast, in microfluidic, the gene delivery process can be executed in microchannel which has similar size as cells. Accurate, rapid and effective gene transfection is viable by combining microfluidic channel with ultrasonic transducers. This thesis aims to design a new gene delivery method in microfluidic. Chemical methods are used to study the cavitation yield of different transducer shape and frequency. The influence of ultrasound on the shape and survive rates of cells are also investigated. A self-focused ultrasonic transducer developed in house by MEMS technology were integrated to a microchannel to study transfection in microfluidic environment. Foundation work were laid for future development of microfluidic gene transfection devices.The main contents are as follows:1. A quantitative method is developed to determine the strength of cavitation and to investigate the key parameters influencing cavitation, which provides strong support for the development of a micro system that consists of a MEMS focused ultrasonic transducer arrays and microfluidic channel. We first measured the cavitation yield of bulk ultrasonic transducer of high and low frequency. The cavitation yield of ultrasound horn, ultrasonic cleaning tank, bowl-focused ultrasound transducer were measured. The factors influencing cavitation were discussed.2. Focused ultrasound transducers of smaller size were used to explore the impact of ultrasonic cavitation yield on human renal epithelial cell line-derived cells (293T cells). The 293T cells’ morphology were studied after ultrasonic irradiation and abnormal morphology of 293T cells were statistically profiled. Relationship between the percentages of abnormal cells with the yield of ultrasonic cavitation changing with ultrasonic irradiation time were investigated. The uptake of propidium iodide (PI) after ultrasound irradiation were measured to determine cell membrane permeability.3. Experiments of the in house developed microchannel ultrasonic gene delivery modules were performed, gene transfection in micro environment were realized. The viability and effectiveness of gene transfection in microfluidic environment is validated.