Design, fabrication and characterization of micro/nano electroporation devices for drug/gene delivery.

摘要

Micro/nano fabrication technology plays a significant role in biological applications by miniaturizing an existing system or creating a new system. There are several advantage of the technology such as miniaturization of the current system, mass production, low cost and so on. Miniatured devices require very small amount of sample volume resulted in enhancement of their sensitivity and less consumption of reagent. Micro/nano electroporation is one of the main benefits by the technology because the scaling down of sizes to the micro/nano regime provides many advantages such as reducing Joule heating effect, stabilization of pH change and control of electrical parameters compared to conventional electroporation.;In this thesis, we present to facilitate novel micro/nanofabrication to fabricate micro/nano electroporation devices can perform localized electroporation for higher transfection and dynamic and impedance measurement of the cell to investigate mechanism of electroporation.;We developed a poly e-caprolacton (PCL) membrane with well defined micro-pore arrays fabricated by soft lithography for cell immobilization and uniform gene delivery. The fluorescence images of NIH 3T3 cells immobilized on PCL membrane showed uniform gene delivery after plasmid DNA delivery by electroporation. The device for local electroporation with well-defined micropore array structures have demonstrated great improvement in uniformity compared to a device with random pores in a track-etch membrane.;Next, we developed a single micro-pore providing localized electroporation by femtosecond laser for cell immobilization and gene delivery to single cell. Numerical simulations were demonstrated to show the electric field distribution around and across the cell. In this work an accurate equivalent model of the microfluidic device/cell system had been developed. Based on an equivalent circuit of the PCL membrane device, its various electronic components such as capacitance and resistance of the cells, capacitance of the double layer charge, and the charge transfer resistance were extracted. The impedance of cells before and after electroporation and accurate electrical modeling of the device were in good agreements.;Ease-to-use PDMS microfluidic devices with the cell trapping channel and locally electroporate the cell for gene/drug delivery were fabricated. Numerical simulations were demonstrated to show transmembrane potential around the cell during electroporation for pore formation studies by solving electrostatic problems. Dynamic current and impedance measurement were performed to understand mechanism of pore formation and electrical characteristic of the cell before and after EP.;Furthermore, we have developed PDMS chips having different channel sizes (5 mum, 1 mum, and 500 nm) that can selectively immobilize and locally electroporate single cell for device scaling down studies. We employed an optical tweezer to facilitate the control of the cell position at micro-channels instead of using hydrodynamic force and applied the electric field across the cell for localized drug/gene delivery. We focus on the gene/drug transfer mechanism of the cell electroporation at natural state in devices with different scales.