Microfluidics for genetic and epigenetic analysis of cells.

摘要

Genetic and epigenetic analyses of cells are powerful tools broadly applicable to clinical diagnosis, drug screening, forensic identification, food safety inspection, environmental monitoring and biowarfare testing. The translation of conventional analytical techniques using macroscale apparatus to microfluidic chip format offers the advantages of enhanced sensitivity and speed, decreased the potential sample cross-contamination and product loss, reduced sample and reagent consumption, favorable fluidic properties, low cost, and disposability. This dissertation presents the development of several microfluidic chips that are capable of analyzing and manipulating cells for genetic or epigenetic studies.;Histone modifications are important epigenetic mechanisms involved in eukaryotic gene regulation. Chromatin immunoprecipitation (ChIP) assay serves as the prime technique to characterize the genomic locations associated with histone modifications. However, traditional tube-based ChIP assays rely on large numbers of cells as well as laborious and time-consuming procedures. I first demonstrate a novel microfluidics-based ChIP assay which dramatically reduced the required cell number and the assay time by conducting cell collection, cell lysis, chromatin fragmentation, immunoprecipitation, and washing on a microchip. Coupled with real-time polymerase chain reaction (PCR), the assay permitted the analysis of histone modifications from as few as ∼50 cells within 8.5 h. The results indicate that the method will provide a new approach for analysis of epigenetic regulations and protein-DNA interactions in general based on scarce cell samples.;Electrical lysis based on irreversible electroporation is a promising technique for genetic analysis due to its fast speed and reagentless procedure. However, knowledge on DNA extraction from electrically lysed samples is still lacking. To address this challenge, I constructed a novel integrated microfluidic chip capable of performing sample preparation for genetic analysis. The microchip was able to physically trap a given amount of cells, lyse the cells in 1.5 min using 10 square direct current (DC) electrical pulses, as well as purify and concentrate genomic DNA. Efficient integration of the three key steps successfully generated sufficient products amenable to off-chip real-time PCR assay from as few as ∼30 mammalian Chinese hamster ovary (CHO-K1) cells and 102 colony-forming units (CFU) of Gram-negative bacteria Salmonella typhimurium. DNA yield exhibited a great dependency on electrical field intensity and a good linearity with respect to the amount of bacterial cells. I envision that the device will be fully integrated with PCR assays and quantitative detection techniques to implement a total analysis system with sample-in-answer-out capability.;In addition, I present a strategy for active modulation of DNA adsorption and desorption on silica beads in an electrically actuated microfluidic chip by varying the buffer pH through electrolysis of water. This technique provides a novel method for DNA purification and concentration in a low ionic strength buffer, which eliminates the utilization of harsh chemicals typically involved in traditional DNA extraction procedure.;Genetic modification of cells is a critical step involved in functional gene studies. However, the throughput of current transfection methods is limited. In the final chapter, I present a novel flow-through electroporation method for delivery of genes into cells at high flow rates (up to ∼20 ml/min) based on disposable microfluidic chips, a syringe pump, and a low-cost DC power supply that provides a constant voltage. By eliminating pulse generators used in conventional electroporation, the method dramatically lowered the cost of the apparatus and improved the stability and consistency of the electroporation field for long-time operation. I tested the delivery of pEGFP-C1 plasmids encoding enhanced green fluorescent protein into CHO-K1 cells in the devices of various dimensions and geometries. Cells were mixed with plasmids and then flowed through a fluidic channel continuously while a constant voltage was established across the device. Together with the applied voltage, the geometry and dimensions of the fluidic channel determined the electrical parameters of the electroporation. With the optimal design, ∼75% of the viable CHO-K1 cells were transfected after the procedure. I also generalize the guidelines for scaling up these flow-through electroporation devices. This technique will serve as a generic and low-cost tool for a variety of biological applications requiring large volume of transfected cells.