Microfluidic electroporation for gene delivery and cellular analysis
Abstract
Electroporation based on microfluidic technique has demonstrated to be widely used for genomics and proteomics study. Exogenous molecules can be delivered into cells by transiently permeabilizing cell membrane. On the other hand, by permanently disrupting cell membrane, electroporation could also be used for extracting cell contents for analysis. In this thesis, I explored using novel microfluidic devices for both gene delivery and cellular analysis. One important advantage of our flow-through electroporation technique is that the sequence of the electric field variations (equivalent to the pulse pattern in electropulsation) can be conveniently adjusted by altering the geometry of the channel. To improve the transfection efficiency, the effects of the low field in the wide sections on transfection has been systematically investigated by varying the residence time (0.18 s~3.22 s) and field intensity (27 V/cm~ 160 V/cm) in wide sections. Besides dc electroporation, the use of continuous 10-10k Hz ac field (based on either sine waves or square waves) has also been explored for electroporation. The results reveal that electropermeabilization becomes weaker with increased frequency. In contrast, transfection efficiency reaches its maximum (~71%) at medium frequencies. It is postulated that the relationship between the transfection efficiency and the ac frequency is determined by electrophoretic movement of DNA in the ac field and variation of transfection under different electropermeabilization intensities. A droplet-based electroporation method has also been demonstrated for gene delivery. Cells are encapsulated into aqueous droplets and then electroporated when the cell-containing droplets flow through a pair of microelectrodes with a constant voltage established in between. The parameters and characteristics of the electroporation have been investigated. The delivering of enhanced green fluorescent protein (EGFP) plasmid into CHO cells has been deomonstrated. We envision the application of this technique to high throughput functional genomics studies. Conventional biochemical analysis mainly focused on the expression level of cellular proteins from entire cells. However, it has been increasingly acknowledged that the subcellular location of proteins often carries important information. Here, microfluidic flow-through electroporation has been applied to breach cell membranes and extract cytosolic proteins selectively in a single step. This approach has been demonstrated to allow monitoring the translocation of the transcription factor NF-κB from the cytosol to the nucleus upon stimulation without subcellular fractionation. In order to extend the selective extraction of protein method to cells in adherent status, we also do the electroporation extraction to the cells cultured on the microfluidic channel. Laser induced fluorescence detection method has been used to quantify the amount of released proteins. Integrating both the electroporation and detection steps on one chip facilitates high throughput operation and detection Our method has been demonstrated effectively in detect the difference of the protein release resulted from different subcellular location, which has been also applied to detect the translocation of NF-κB upon stimulation. Besides gene delivery and proteomics study, to illustrate microfluidic device as diagnostic tool, the rapid lysis of erythrocytes by hydrodynamic focus in microfluidic channel has been conducted to study the biomechanics. Our method has been demonstrated to effectively detect the erythrocytes with different rigidities and thus be promising in application of disease diagnosis involving erythrocyte rigidity.
Degree
Ph.D.
Advisors
Lu, Purdue University.
Subject Area
Genetics|Cellular biology|Engineering|Biomedical engineering
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