Microfluidic Advantage: Novel Techniques for Protein Folding and Oxygen Control in Cell Cultures.

摘要

The young field of microfluidics has been growing due to its utility in chemical and biological applications. Microfluidic devices can be rapidly and inexpensively fabricated from silicone elastomers, making them ideal for prototyping and subsequent production. Further, the behavior of fluid flows in micrometer-diameter channels can be accurately predicted -- due to the properties of laminar flow and purely diffusive mixing -- decreasing experimental uncertainties, while allowing access to a wide range of experiments impossible with traditional methods. The projects presented here fall into two separate areas of biophysics, although they are all facilitated by microfluidics.;Chapter 2 deals with the control of the gas content in the medium of cell cultures. This is an important consideration, as the oxygen concentration, [O2], available to cells has been shown to affect their metabolism, growth, and gene expression. The first project is a microfluidic chemostat supplying nine different [O2] to bacteria growing in chambers beneath the gas channels. Here, we compared the growth rates of E. coli growing at nine different [O2] simultaneously. Section 2.2 introduces a multi-channel, computer-controlled gas mixer that can provide up to ten arbitrary gas mixtures to a microfluidic device. Finally, Section 2.3 describes gas control strips for use with mammalian cell cultures in standard multiwell culture plates. These gas control strips allow cell culture media in different rows of wells to contain different [O2].;Chapter 3 describes a novel system to rapidly heat and cool a small volume of solution of biological macromolecules using time-controlled deposition of heat into a small volume with a focused infrared laser beam. By fluorescently labeling the molecules, their conformational changes due to temperature shifts can be observed. This system improves the time resolution of the cooling transition over traditional methods by at least two orders of magnitude, down to one microsecond. Further, the temperature change from the laser heating pulse is several times larger than with other techniques. We used this system to measure the kinetics of fast DNA hairpin folding and unfolding under varying salt concentrations.